Image analysis and measurement of biological samples

Image analysis and measurement of biological samples

IMAGE ANALYSIS AND MEASUREMENT OF BIOLOGICAL SAMPLES

Refer to related applications

This case claims priority for the following cases: U.S. Patent Application No. 61/675,811, application date July 25, 2012; U.S. Patent Application No. 61/676,178, application date July 26, 2012; U.S. Patent Application No. Application date of No. 61/766,116 on February 18, 2013; and U.S. Patent Application No. 61/802,194 on application date of March 15, 2012; the full content of the disclosure of these patent applications is hereby quoted and incorporated into this Disclosure of instructions.

The present invention relates to image analysis and measurement of biological samples.

Background of the invention

The analysis of biological samples obtained from an individual may be very important for the diagnosis, monitoring, and/or treatment of an individual's health. Various methods are known for the analysis of biological samples. However, in order to provide better diagnosis, monitoring and/or treatment of individuals, it is desirable to improve the analysis of biological samples.

Introduce into the invention

All the announcements, patents, and patent applications mentioned in this specification are cited here and incorporated into the disclosure of this specification as if each individual announcement, patent, and patent application are specifically and individually indicated It is quoted here and integrated into the same degree as the disclosure of this manual.

Summary of the invention

The methods, devices, systems, and equipment described herein are useful for optical and image analysis and/or measurement of biological samples.

In one embodiment, a method for measuring a component of interest in cells of a cell group in a sample is provided, including: a) obtaining a label of one of the cells of the cell group present in the sample A quantitative measure; b) According to the measure of part a), determine the approximate amount of one of the cells of the cell population present in the sample with the aid of a computer; c) According to the result of part b), choose to add to the sample The amount of reagent, wherein the reagent is specifically bound to the component of interest in the cells of the cell population, and is assembled to facilitate detection; d) According to the result of part c), add a selected amount of reagent to the sample E) assay and analyze the reagents bound to the component of interest in the cells in the sample; and f) determine the amount of the component of interest in the cells of the cell group of the sample based on the amount of the reagent bound to the component of interest. In an embodiment of this method, the reagent of part c) is an antibody.

The applicant further discloses here a method for measuring a component of interest in cells of a cell population in a sample, comprising: a) obtaining a label of one of the cells of the cell population present in the sample A quantitative measure; b) according to the measure of part a), determine the approximate amount of one of the cells of the cell group present in the sample with the aid of a computer; c) add a quantitative one of the cell markers to the sample, in the sample The amount of the cell marker added is based on the results of part b), and the cell marker system specifically binds to The component of interest in the cells of the cell group is assembled to facilitate detection; d) the label of the cell in the analysis sample bound to the component of interest; and e) according to the amount of the label bound to the component of interest And determine the amount of the component of interest in the cells of the cell group of the sample.

In another embodiment, a method of focusing a microscope is proposed, which includes: a) mixing a sample containing an object for microscopy analysis with a reference particle of a known size to effectively generate a sample containing the sample and the reference particle A mixture; b) placing the mixture of step a) in an optical path of a microscope; c) exposing the mixture of step a) to a light beam, which is configured to make the reference particle visible; And d) focusing the microscope based on the position of the reference particle in the mixture.

In yet another embodiment, a method for identifying a cell in a sample containing a plurality of cells is provided here, which includes: (a) assaying and analyzing one cell in the plurality of cells for at least one of the following : (I) the presence of a cell surface antigen; (ii) the amount of a cell surface antigen; or (iii) the cell size; (b) the cell in the assay (a) is related to at least one of the following: (i) Nuclear size; or (ii) nuclear shape; and (c) the quantitative cell light scattering of the cell in the assay (a) and (b), where the information obtained from steps (a), (b), and (c) The combination of is used to identify the cell in the sample containing a plurality of cells.

In yet another embodiment, a system is proposed here, which includes a detector assembly for holding a sample container of a sample to be tested. In a non-limiting embodiment, the sample container is an optical cuvette with features and/or materials that permit the optical cuvette to engage and move from a position to the detector assembly. In some embodiments, the detector assembly has a first A surface that is configured to join a surface of the sample container so that the interface between the two does not cause light interference on the optical path from the detector assembly to the sample in the sample container. In one embodiment, there may be more than one location on the detector assembly for one or more of the sample containers. Several embodiments have the same sample container for each location. Optionally, some embodiments aim at different sample containers in at least some of the positions where the detector assembly is connected.

In an embodiment disclosed herein, a sample container such as, but not limited to, an optical test tube with optical properties, dimensions, materials, and/or physical characteristics is proposed here, which is permitted to contain the sample to be analyzed by the detector assembly The sample, while keeping the sample physically separated from the detector assembly without direct contact. This type of sample container is particularly useful for sample fluids containing shaped parts.

In an embodiment disclosed herein, the detector assembly may be a multi-channel microscopy unit, which is configured to detect, obtain, or measure one or more cells in a sample The shape and physical, optical, and biochemical properties are all in the same device. Can provide quantitative information and descriptive information. An embodiment of the detector assembly may use multiple labels of the same color or the same wavelength, where the detector assembly is configured to deconvolute these labels derived from the sample (for example, bound to It is possible to reduce the number of channels and light sources required in the assembly.

It should be understood that several embodiments herein may include a container such as, but not limited to, a light test tube with a physical characteristic in the shape of a light test tube material to increase dark field illumination, where some characteristic parts are assembled to provide light. Reflectance (including but not limited to the light reflectance inside the light test tube), and some The characteristic elements can be selectively assembled for mechanical support; in the embodiment, some characteristic elements can provide mechanical support and also provide light reflectance. In the embodiment, the sample container is assembled to provide the sample penetrating illumination by the light reflection inside the sample container. In the embodiment, the sample container is configured to provide the sample penetrating illumination by the light reflection inside the sample container; this reflectance may include partial internal reflection (PIR), and this reflectance may include Total internal reflection (TIR). In the embodiment, the sample container is configured to provide sample penetrating illumination by light reflection inside the sample container, wherein when the optical elements are used to detect or measure the light, the reflected light The source is arranged on the same side of the sample container (that is, the light source is a surface illumination light source).

Here the system can simultaneously use surface (direct) and penetrating (reflected) illumination for imaging in dark fields. This is different from traditional dark field imaging, which mainly uses surface illumination instead of penetrating illumination. As such, the combination of surface-and penetration-illumination as disclosed herein is different from known systems, where the penetration-illumination system is derived from the same light source as the surface-illumination. Optionally, the use of shaped sample containers such as light test tubes can be used to provide surface-illumination. In an embodiment, the shaped sample container is assembled to provide surface-illumination through light reflection. In an embodiment, the shaped sample container is assembled to provide surface-illumination by light reflection in the sample container. In an embodiment, one or more of the size, shape, surface, material, or other characteristics of a shaped sample container is effective to provide internal reflection of light inside the shaped sample container. In an embodiment, one or more of the size, shape, surface, material, or other characteristics of a shaped sample container is effective to provide light in the internal portion of the shaped sample container Reflection (PIR). In an embodiment, one or more of the size, shape, surface, material, or other characteristics of a shaped sample container effectively provides total internal reflection (TIR) of light inside the shaped sample container. Optionally, the intensity of penetrating illumination is not negligible. In an embodiment, a shaped sample container may include a reflective surface to effectively increase the intensity of the transmitted illumination light. The dark field light source can be a light emitting diode (LED), a laser, or other illumination source that can provide the desired illumination and/or laser wavelength.

In one embodiment, the combination of a microscope objective lens and a light source such as, but not limited to, a ring light (used in dark field microscopy), the physical distance between them allows the detector assembly to have a reduced size. In one embodiment, only light at the desired wavelength or within the desired wavelength range is directed to the sample. In one embodiment, the light is unpolarized light. In another embodiment, the light is polarized light.

In yet another embodiment, the information obtained from the cytometric assay includes the information obtained from the sample preparation period and/or the analysis period is used to guide and/or trigger the secondary process. Such a secondary procedure can provide an alert directly for inspection. In an embodiment, this secondary procedure may use the estimated cell number or other information obtained during the sample preparation step of a procedure to guide the performance of a test analysis, where the test analysis may be one of the subsequent procedures of the procedure The verification analysis of the step may be the verification analysis of another procedure.

The technique of counting cells can also provide a way to deal with sample containers with irregular shapes and/or uneven chamber surfaces. One method involves the use of: a) Volume metering channel technology to introduce a sample of a known volume into an analysis area, such as a channel in a sample container. The method may include calculating Count all the cells in the sample container. Since the sample volume is known, the cell concentration of the volume is also known (this can be done in a water-repellent container or a light test tube or a sample container with a chamber containing such a surface). Another method includes: b) a ratio-based metering technique to mix the sample with a known amount of beads, which is used to determine the cell concentration in the sample based on the number of beads observed.

In yet another embodiment disclosed herein, a method is proposed to include measuring the formed blood components, such as but not limited to measuring the volume of red blood cells (RBC) in a blood sample. The method is achieved by swelling and obtaining RBCs. It is substantially spherical and uses dark field microscopy to measure the RBC volume.

In yet another embodiment disclosed herein, a method including measuring platelet volume is provided. The method may include labeling platelets with fluorescent dyes and measuring the observed platelet size; adding beads of known size to the sample; and comparing the observed image size of the beads with the observed platelet image size, using beads As a calibration to determine the size of platelets in the sample and determine the volume of platelets.

Accordingly, the applicant discloses here: a system for analyzing a sample, the system comprising: a sample container including a sample chamber configured to contain the sample, and at least a part of the sample container includes a light transmission The light-transmitting material includes a light-transmitting surface and a reflective surface; and an illumination source is configured to provide light, and the light system illuminates and passes through the light-transmitting surface; wherein the sample holder is configured to effectively let The light from the illumination source simultaneously provides both surface illumination and penetrating illumination to a sample in the sample holder, where the surface illumination system includes The light containing light travels from the illumination source to the sample without being reflected on a surface of the light-transmitting material of the sample holder, and the penetrating illumination system includes light traveling inside the light-transmitting material, and from the light At least one surface of the transmissive material travels to the sample after at least one reflection. In an embodiment, the sample container having one of the characteristic components disclosed herein may include an optical test tube with a long channel configured to hold the sample. In an embodiment, the sample container may have one or more light non-transmissive surfaces.

In the embodiment of the system disclosed herein, the penetrating illumination can be provided at least in part by the internal reflection of light from a surface, and can be provided at least in part by the total internal reflection of light inside the light test tube. In the embodiment of the system disclosed herein, the penetrating illumination can be provided at least in part by partial internal reflection of light from a surface, and can be provided at least in part by partial internal reflection of light inside the light test tube.

In an embodiment, the same container may have two or more sample chambers for holding samples. A sample container having the characteristics disclosed herein, for example, a light test tube can have a rectangular cross-sectional shape; can have a circular cross-sectional shape; can have a zigzag longitudinal cross-sectional shape; can have a stepped longitudinal cross-sectional shape ; Or can have other shapes.

In the embodiment, the sample container can move relative to an illumination source and can move relative to a plurality of positions, wherein a light-transmitting surface of the sample container can be illuminated by the illumination source at various positions.

In an embodiment, an illumination source may include a ring light. In an embodiment, a ring light can be selected from a light emitting diode (LED)-based ring light and a laser-based ring light.

In an embodiment, a system as disclosed herein may include a support structure having a light-transmitting surface shaped to engage with a light-transmitting surface of the sample container.

In an embodiment, a system as disclosed herein may have a compression device configured to keep the sample container in a desired position for illumination by the illumination source.

In an embodiment, a system as disclosed herein may include a detector configured to image at least a portion of a channel in the sample container.

In an embodiment, as disclosed herein, the container may include a long channel configured to contain at least a portion of the sample, and one of the detectors is configured to image an entire long channel in the sample container road.

In an embodiment, the container as disclosed herein can be configured to hold the sample in a static non-flowing manner during imaging; in an embodiment, the container can be configured in a static non-flowing manner as disclosed herein Hold a part of the sample and hold the other part in a flowing manner.

In an embodiment, an illumination source as disclosed herein can move relative to the sample container.

In an embodiment, as disclosed herein, the container can be configured to contain the sample in a fluid manner during imaging.

In an embodiment, the container as disclosed herein may include a fluid circuit completely confined in the sample holder, and the sample is located in the fluid circuit, effectively keeping the sample separate from the detector.

In the embodiment, the container is movable relative to the detector as disclosed herein. In the embodiment, a detector phase as disclosed herein The sample container is removable.

In an embodiment, the sample container and an illumination source as disclosed herein include at least a part of an optical analysis unit, and the system further includes a clinical analysis unit configured to perform clinical analysis on the sample.

In the embodiment, a system as disclosed herein is configured to provide a single sample of a liquid to the optical analysis unit and the clinical analysis unit respectively, effectively allowing the clinical analysis unit and the optical analysis unit to be used at the same time Perform optical analysis and clinical analysis on the sample part. In the embodiment, such clinical analysis can be selected from general chemical analysis, nucleic acid analysis, and enzyme-linked binding analysis.

In an embodiment, a system as disclosed herein may include a plurality of clinical analysis units, wherein each clinical analysis unit of the plurality of clinical analysis units is configured to provide selected from general chemical analysis, nucleic acid analysis, and One of the clinical analysis of enzyme binding analysis.

The applicant further proposes an optical test tube comprising a sample chamber assembled to contain a sample, at least a part of the optical test tube includes a light-transmitting material, the light-transmitting material includes a light-transmitting surface and a reflective surface, wherein the light-transmitting The surface and the reflective surface are assembled to effectively allow light to pass through the light-transmitting surface at the same time, providing both surface illumination and penetrating illumination to the sample in the sample chamber, where the surface illumination includes light from the illumination The source travels to the sample without being reflected on a surface of the light-transmitting material, and the penetrating illumination system there includes light traveling inside the light-transmitting material, and at least one reflection from at least one surface of the light-transmitting material Then travel to this sample.

In an embodiment, an optical test tube as disclosed herein has a sample chamber including a long channel. In an embodiment, an optical test tube as disclosed herein has two or more sample chambers for holding samples.

In an embodiment, a light cuvette as disclosed herein may have one or more light non-transmissive surfaces.

In an embodiment, the penetrating illumination may be provided in an optical test tube as disclosed herein, at least partly by internal reflection of light inside the optical test tube. In an embodiment, the penetrating illumination may be provided in a light test tube as disclosed herein, at least partly by reflection within a part of the light on a surface of the light test tube. In an embodiment, the transillumination may be provided in an optical test tube as disclosed herein, at least in part by total internal reflection of light on a surface of the optical test tube.

In an embodiment, an optical test tube as disclosed herein may have a rectangular cross-sectional shape; in an embodiment, an optical test tube as disclosed herein may have a circular cross-sectional shape. In an embodiment, an optical test tube as disclosed herein may have a zigzag longitudinal cross-sectional shape; in an embodiment, an optical test tube as disclosed herein may have a stepped longitudinal cross-sectional shape.

The applicant reveals the method here. For example, the applicant here discloses a method for identifying a cell in a sample containing a plurality of cells, including: (a) placing the sample in a sample seat, which contains a sample chamber assembled with A sample is contained, at least a part of the sample holder includes a light-transmitting material, the light-transmitting material includes a light-transmitting surface and a reflective surface, wherein the light-transmitting surface and the reflective surface are configured to effectively allow light to pass through at the same time The light-transmitting surface provides both surface illumination and penetrating illumination For the sample in the sample chamber, the surface illumination system includes light traveling from the illumination source to the sample without being reflected on a surface of the light-transmitting material, and the penetrating illumination system includes the light on the surface of the light-transmitting material. The light-transmitting material travels inside, and travels to the sample after at least one reflection from at least one surface of the light-transmitting material; (b) illuminating the sample holder to effectively provide both surface illumination and penetrating illumination of the sample at the same time; And (c) identify a cell in the sample. In an embodiment, the method disclosed herein includes where the identifying includes identifying the cell using a detector configured to image at least a portion of the sample chamber. In the embodiments disclosed herein, one of the sample chambers used in these methods may include a long channel.

The applicant further discloses here a method of focusing a microscope, including: a) mixing a sample containing an object for microscope analysis with a reference particle of a known size to effectively generate one of the sample and the reference particle Mixture; b) placing the mixture of step a) in a light path of a microscope; c) exposing the mixture of step a) to a beam of light, which is configured to make the reference particle visible; and d) Focusing the microscope based on the position of the reference particle in the mixture.

The applicant discloses here a method for identifying a cell in a sample containing a plurality of cells, comprising: (a) assaying and analyzing one of the cells in relation to at least one of the following: (i) a The presence of cell surface antigen; (ii) the amount of a cell surface antigen; or (iii) the cell size; (b) the cell in the assay (a) is related to at least one of the following: (i) nuclear size; or ( ii) nuclear shape; and (c) the quantitative cell light scattering of the cell of the assay (a) and (b), wherein the combination of the information obtained from steps (a), (b), and (c) is used To identify the cell in the sample containing multiple cells.

It should be understood that the embodiments in this disclosure can be adapted to one or more of the characteristics described in this disclosure.

The summary of the present invention is provided to introduce the choice of concepts in a simplified form, which will be further described in the detailed description section as follows. The summary of the present invention is not intended to identify the key features or main features of the subject matter of this case, nor is it intended to limit the scope of the subject matter of this case.

<p>7Arrow</p><p>600Light test tube, sample container</p><p>602Open</p><p>604,610Structure</p><p>606Support structure, base</p><p>608Analysis Area</p><p>612Cover</p><p>613Controlled thickness area</p><p>614Upper surface</p><p>618surface</p><p>620Base Bracket</p><p>621-626Arrow</p><p>621Distance</p><p>622,623Height</p><p>624Thickness</p><p>625,626Width</p><p>650, 654, 660Illumination source, optics, ring light</p><p>652Toroidal reflector</p><p>670Objective lens</p><p>674,676Outer surface</p><p>678Inner surface</p><p>680, 682, 684Laser source</p><p>690Two-color components</p><p>692Filter wheel</p><p>694Porosity</p><p>696Extra lens</p><p>698Fuliye lens</p><p>700Detector</p><p>701-706Analysis Module</p><p>707Cell Surgery Unit</p><p>708Sample Disposal System</p><p>709 Bracket</p><p>710Programmable processor</p><p>711Extraction type burette</p><p>712Positive Displacement Burette</p><p>713Centrifuge</p><p>714Spectrophotometer</p><p>715Chemical Analysis Unit</p><p>716Optical Multiplier Tube (PMT)</p><p>717Cassette</p><p>800Light transmission layer</p><p>802Optical features</p><p>820, 825, 830path</p><p>DWidth, optical detector</p><p>PIRPartial internal reflection</p><p>TIRTotal internal reflection</p><p>x,y,zcoordinate axis</p>

Figure 1 shows: (A) a plot of the side scatter intensity (x-axis) of a mixture of cells containing natural killer cells and neutrophils labeled with a fluorescent binder that recognizes CD16 versus fluorescence intensity; (B) A bar graph shows the ratio of the nuclear area of natural killer cells (NK) and neutrophils (Neu) to the total cell area; (C) stained with anti-CD16 antibody (left column) and nuclear stain (right column) Natural killer cells; (D) Neutrophils stained with anti-CD16 antibody (left column) and nuclear stain (right column).

Figure 2 shows: (A) platelets labeled with fluorescent conjugated CD41 and CD61 antibodies (bright spots); (B) 10 times (left) and 20 times (right) magnified image intensity distribution of fluorescent labeled platelets; (C) ) The image intensity distribution of fluorescently labeled platelets shows the measured intensity (light gray) and the curve that matches the measured intensity (dark gray).

Figure 3 shows: a graph showing the relationship between the nominal diameter of a standard particle, in micrometers (x-axis) and the size measurement based on fluorescence intensity, in au (y-axis). The figure also shows representative beads at different points along the curve.

Figure 4 shows: (A) only surface lighting, and (B) surface-and Spherical red blood cells and platelets imaged by dark field microscopy in a mixed light test tube of penetration-illumination.

Figure 5 shows: (A) putative banded neutrophils stained with anti-CD16 antibody and nuclear stain; (B) putative segmented neutrophils stained with anti-CD16 antibody and nuclear stain.

FIG. 6A shows an embodiment of an optical system suitable as a component of the device or system as disclosed herein, and an embodiment of an optical system suitable for the method as disclosed herein, including embodiments of optical elements (such as a light source shown as a ring light and an objective lens) , Optical test tube, and a supporting structure assembled to fix and position an optical test tube for imaging. In this embodiment, the optical test tube has a rectangular cross-sectional shape.

FIG. 6B shows an embodiment of an optical system suitable for use as a component of the device or system as disclosed herein, and an embodiment of an optical system suitable for the method as disclosed herein, including embodiments of optical elements (for example, a light source shown as a ring light, and an objective lens) , Optical test tube, and a supporting structure assembled to fix and position an optical test tube for imaging. In this embodiment, the optical test tube has a circular cross-sectional shape.

FIG. 7A shows an embodiment of elements suitable for a device or system as disclosed herein and an optical system suitable for the method disclosed herein.

Fig. 7B shows an embodiment of elements suitable for an apparatus or system as disclosed herein and an optical system suitable for the method disclosed herein, including another lens and a slit adapted to limit the angle of the scattered light reaching a detector scope.

Figure 8A presents a view of the elements of an optical system including a support structure for fixing a light tube for imaging of a sample, in which the light from the ring light illumination system directly falls on the sample (surface illumination), and the light is also From that The characteristic reflection of the light test tube thus also provides penetrating illumination. In this embodiment, the optical test tube has a stepped longitudinal cross-sectional shape.

Fig. 8B presents a view of the elements of an optical system including a supporting structure for fixing a light test tube for imaging of a sample, in which the light from the ring light illumination system directly falls on the sample (surface illumination), and the light is also The reflection from the characteristic piece of the light test tube thus also provides penetrating illumination. As shown in the figure, the incident light can be completely reflected on a surface (total internal reflection, TIR) or only part of the incident light can be reflected on a surface (partial internal reflection, PIR). In this embodiment, the optical test tube has a zigzag longitudinal cross-sectional shape.

FIG. 8C shows an embodiment of an optical system suitable for use as a component of the device or system as disclosed herein, and an embodiment of an optical system suitable for the method as disclosed herein, including embodiments of optical elements (for example, a light source shown as a ring light, and an objective lens) , Optical test tube, and a supporting structure assembled to fix and position an optical test tube for imaging. In this embodiment, the optical test tube includes a characteristic component that affects the light path for illuminating the optical test tube and the sample inside the optical test tube.

FIG. 8D shows an embodiment of an optical system suitable for use as a component of the device or system as disclosed herein, and an embodiment of an optical system suitable for the method as disclosed herein, including embodiments of optical elements (for example, a light source directed from a lateral direction), a light test tube, And it is assembled with a supporting structure for fixing and positioning a light test tube for imaging. In this embodiment, the optical test tube includes a characteristic component that affects the light path for illuminating the optical test tube and the sample inside the optical test tube.

Figure 8E provides a schematic representation of a light test tube being transferred from a sample preparation position to a sample observation position close to a light detector (labeled D).

Figure 8F provides a transfer mechanism for removing a light test tube from the sample This is a schematic representation of another detail of the system for transferring the preparation position to the sample observation position close to a photodetector.

Figure 9 is a composite image showing images of blood cells taken from whole blood using different imaging techniques and dyes. Figure 9A is a dark field image; Figure 9B is an image showing fluorescence from a tagged anti-CD14 antibody attached to monocytes; Figure 9C is an image showing an image from tagged anti-CD123 attached to basophils Fluorescence of antibodies; Figure 9D is an image showing fluorescence from tagged anti-CD16 antibodies attached to neutrophils; Figure 9E is an image showing fluorescence from tagged anti-CD45 antibodies attached to white blood cells ; Figure 9F is an image showing white blood cells and platelet cells stained with nuclear stain Dige (DRAQ5®) (red blood cells are deficient in nucleus so they are not stained by Dige).

Figure 10 is a composite image showing a representative image of blood cells taken from white blood cells, showing monocytes, lymphocytes, eosinophils, and neutrophils.

Figure 11 shows a mapping of the fluorescence detected on cells labeled with different labels (tagged antibodies directed to different cell surfaces or other labels); such multiple labeling is useful for identifying cell lines. Figure 11A identifies monocytes by plotting FL-17 intensity versus FL-9 intensity. Figure 11B identifies basophils by plotting the intensity of FL-19 versus the intensity of FL-15. Figure 11C identifies lymphocytes by plotting the intensity of FL-15 versus the intensity of FL-11. Figure 11D identifies neutrophils and eosinophils by plotting the intensity of FL-15 versus the intensity of FL-9.

Figure 12 shows a comparison of the cell count obtained by this method (measured from the same amount of blood sample) and other methods (using a commercial hematology analyzer). Figure 12A plots the white blood cell count obtained by this method compared to the white blood cell count obtained by a commercial blood analyzer. Figure 12B plots the red blood cell count obtained by this method compared to the red blood cell count obtained by a commercial blood analyzer. Figure 12C plots the platelet count obtained by this method compared to the platelet count obtained by a commercial blood analyzer. Figure 12D plots the neutrophil count obtained by this method compared to the neutrophil count obtained by a commercial blood analyzer. Figure 12E plots the monocyte count obtained by this method compared to the monocyte count obtained by a commercial hematology analyzer. Figure 12F plots the lymphocyte count obtained by this method compared to the lymphocyte count obtained by a commercial hematology analyzer.

Detailed description

For the description and disclosure of the full scope and advantages of the devices, systems, and methods disclosed herein, for example, please refer to US Patent No. 8,380,541; US Patent Application No. 13/769,798, dated February 2013 18; U.S. Patent Application No. 61/802,194, application date March 15, 2013; U.S. Patent Application No. 13/769,779, application date February 18, 2013; U.S. Patent Application No. 13/244,947 , Application date September 26, 2011; PCT/US2012/57155, application date September 25, 2012; U.S. Patent Application No. 13/244,946, application date September 26, 2011; U.S. Patent Application No. 13 /244,949, application date September 26, 2011; and U.S. Patent Application No. 61/673,245, application date September 26, 2011, the full text of the disclosures of these patent cases and patent applications are hereby quoted here. And integrated into the disclosure of this manual.

It is necessary to understand the summary description part of the previous article and the detailed description part of the following article It is illustrative and only for illustrative purposes, and does not limit the invention as claimed in the patent. Unless the context clearly indicates otherwise, it may be noted that if used in this specification and the appended claims, the singular forms "onea", "onean", and "the" include plural forms. For example, the reference to "a material" can include a mixture of materials; the reference to "a compound" can include multiple compounds and their classes. Unless it conflicts with the teachings explicitly stated in this specification, the references cited here are incorporated into the disclosure of this specification in their entirety.

In this specification and in the scope of the subsequent patent applications, multiple terms will be mentioned, which must be defined as having the following definitions: "selective" or "selectively" means that the circumstances described later may or may not occur , So the description includes what happened in the situation and what didn't happen in the situation. For example, if a device optionally contains a feature for the sample collection unit, it means that the sample collection unit may or may not exist , and as such, the description includes that one of the devices has the structure of the sample collection unit and There are neither the structure of the sample collection unit.

As used herein, "substantial" means more than the minimum or invalid amount; and "substantially" means more than the minimum or invalid. So, for example, as used here, the term "substantially different" means that there is a high enough degree of difference between the two values, so that those skilled in the art will consider that the two values are within the context of the characteristics measured by these values. The difference is statistically significant. Therefore, the difference between two values that are substantially different from each other is typically greater than about 10%, and may be greater than about 20%, greater than about 30%, greater than about 40%, or greater. At about 50%, it changes as a function of the reference value or the comparator value.

As used herein, "internal reflection" refers to the reflection of light inside a material (first material) at the boundary between the first material and another material (second material). For example, a first material can be a solid, such as glass or plastic, and the second material can be, for example, air. The internally reflected light travels inside the first material before the light is reflected. The internal reflection can be partial (partial internal reflection: PIR) or all (total internal reflection: TIR). In this way, the internal reflection system where all light incident on a surface is reflected back inside the first material is TIR, and the internal reflection system where all light incident on a surface is not reflected back inside the first material is PIR. (In PIR, some light can pass through the boundary, and some light is reflected back into the material on the surface). The angle of incidence is an important factor that determines the degree of internal reflection; the angle of incidence is a measure of the angle of the incident light with respect to a vertical line of the boundary surface. Whether TIR occurs depends on the incident angle of light relative to the boundary surface between the first and second materials; the refractive index of the first material; the refractive index of the second material; and other factors (such as the wavelength of light that may affect TIR, The reason is that the refractive index typically varies with wavelength). The angle at which light is totally internally reflected is named the critical angle; incident light with an incident angle greater than the critical angle will be totally internally reflected (will stay inside the material: TIR). However, in PIR, part of the incident light with an incident angle smaller than the critical angle will also be internally reflected (the rest of the light will be refracted and sent out from the first material into the second material).

As used here, "sample" can be, but is not limited to, blood samples, urine samples, or other biological samples. The sample can be, for example, a blood sample (e.g., a sample can be obtained from a finger puncture, or a vein puncture, or an arterial blood sample, and can be whole blood, serum, plasma, or other blood samples), a urine sample, a living body Section samples, tissue sections, stool samples, or other biological samples; water samples, soil samples, food samples, air samples; or other samples (such as nasal swabs or nasopharyngeal washes, saliva, urine, tears, gastric juice, spinal cord Fluid, mucus, earwax, oil, glandular secretion, cerebrospinal fluid, tissue, semen, and vaginal fluid, throat swab, exhalation, hair, nails, skin, biopsy, fetal fluid, amniotic fluid, umbilical cord blood, lymph , Body cavity fluid, sputum, mucus, pus, microbiota samples, meconium, milk and/or other secretions).

Thus, as used herein, "sample" includes a portion of blood, urine, or other biological samples, which can have any suitable size or volume, and is preferably a small size or volume. In some embodiments of the system, assay and method disclosed herein, a small volume of blood sample or a small volume of blood sample can be used for measurement, where the small volume contains no more than about 5 ml; or contains no more than about 3 ml; or not more than about 1 ml; or not more than about 1 ml; or not more than about 500 microliters; or not more than about 250 microliters; or not more than about 100 microliters; or not more than about 100 microliters About 75 microliters; or not more than about 50 microliters; or not more than about 35 microliters; or not more than about 25 microliters; or not more than about 20 microliters; or not more than about 15 microliters; Or contain no more than about 10 microliters; or contain no more than about 8 microliters; or contain no more than about 6 microliters; or contain no more than about 4 microliters; or contain no more than about 3 microliters; or contain no more than about 2 microliters; or not more than about 1 microliter; or not more than about 0.8 microliter; or not more than about 0.5 microliter; or not more than about 0.3 microliter; or not more than about 0.2 microliter; or Contains no more than about 0.1 microliter; or contains no more than about 0.05 microliter; or contains no more than about 0.01 microliter.

In an embodiment, the volume of the sample collected through finger puncture may be, for example, about 250 microliters or less, or about 200 microliters or less, or about 150 microliters or less, or about 100 microliters or less, or about 50 microliters. Liters or less, or about 25 microliters or less, or about 15 microliters or less, or about 10 microliters or less, or about 10 microliters or less, or about 5 microliters or less, or about 3 microliters or Below, or about 1 microliter or less.

As used here, the term "service location point" can include a location where services are physically acceptable (such as testing, monitoring, processing, diagnosis, guidance, sample collection, ID authentication, medical services, non-medical services, etc.), and can include However, it is not limited to individual homes, individual offices, locations of health care providers (e.g. doctors), hospitals, emergency rooms, operating rooms, clinics, health care specialist offices, laboratories, retailers (e.g. pharmacies (e.g. retail pharmacies) , Clinic pharmacies, hospital pharmacies), pharmacies, supermarkets, grocery stores, etc.), transportation (e.g. cars, ships, trucks, buses, planes, motorcycles, ambulances, action units, fire trucks/trucks, emergency vehicles) , Law enforcement vehicles, police cars, or other vehicles equipped to transport individuals from one point to another, etc.), mobile medical care units, action units, health-care residences, government offices, office buildings, tents, and locations where body fluid samples were obtained ( For example, a blood donation center), at or near the entrance of the individual's desired approach, at or near the location of the individual's desired access device (for example, a computer location, if the individual wants to approach the computer), the sample processing device receiving sample location, or elsewhere in the text Any other point of the device location described.

The term "cell" used in the context of biological samples covers samples that are usually similar in size to individual cells, including but not limited to vesicles (E.g. liposomes), cells, virus particles, and substances bound to small particles such as beads, nanoparticles, or microspheres.

As used herein, the term "binding agent" generally refers to any compound or macromolecule, such as an antibody, that binds tightly or specifically to a target. Binders include, but are not limited to, antibodies (single or multi-strain antibodies, antibody fragments, immunoadhesins, and other such antibody variants and mimics), natural binding proteins (such as endogenous factors specific for vitamin B12) Proteins), ligands that bind to their target receptors, enzyme substrates that bind to specific enzymes, binding pairs such as avidin and biotin, small molecules that bind tightly or specifically to a target molecule, etc. A binding agent may be, or may contain, or may be linked to a label such as a dye, or a fluorescent group, or other detectable moiety.

As used here, the terms "coloring" and "staining" are interchangeable and refer to elements, compounds, and macromolecules that make the components of an object or sample easier to detect compared to not using the coloring or dyeing process. For example, the treatment of blood samples with DNA dyes such as propanium iodide makes the progesterone cells more visible, making the detection and quantification of these cells easier than when they are not used, even in non-progesterone cells (e.g. This is true even in the presence of red blood cells.

As used herein, the term "ploidy" means the DNA content in a cell, and the assay and measurement of the DNA content of the cells in the sample. The ploidy measurement provides a measure of whether a cell or a population of cells has a normal or abnormal amount of DNA, or whether an abnormal number of cells in a population are proliferating due to DNA replication during cell division and proliferation. Ploidy measurement can be imaged after staining gestational nuclei with DNA-specific dyes in a sample Technical progress.

Quantitative microscopy

In some embodiments, methods, systems, and devices for quantitative microscopy are presented here. Quantitative microscopy may involve one or more of quantitative fluorescence microscopy, quantitative dark field microscopy, quantitative bright field microscopy, and quantitative contrast microscopy methods to measure one or more cell attributes . Any of these methods can provide morphological information about the cells. This information can be measured quantitatively. In some embodiments, it is used for quantitative microscopy, as this is two or more of quantitative fluorescence microscopy, quantitative dark field microscopy, quantitative bright field microscopy, and quantitative relative microscopy analyze. Quantitative microscopy may include the use of image analysis techniques and/or statistical learning and classification methods to process images taken by microscopy.

A number of different cell attributes can be measured during quantitative microscopy. Measurable cell properties include, but are not limited to: physical properties: such as cell size, volume, conductivity, low and high angle scattering, and density.

Morphological attributes: such as cell shape, area, size, and internal structure; cell nucleus shape, area, size, and internal structure; mitochondrial shape, area, size, and internal structure; and the ratio of nuclear volume to cell volume.

Intramolecular properties: such as nuclear centroid/cell centroid distance (that is, the distance between the nuclear center and the cell center), nuclear leaf centroid distance (that is, the distance between the centers of the different leaves of the nucleus), protein distribution inside the cell (such as muscle Kinesin, tubulin, etc.), and the distribution of intracellular organelles (such as lysosomes, mitochondria, etc.).

Biochemical properties: such as cell protein, cell surface protein, cytoplasmic protein, nuclear protein, cell nucleic acid, cell surface nucleic acid, cytoplasmic nucleic acid, nuclear nucleic acid, cell carbohydrate, cell surface carbohydrate, cytoplasmic carbohydrate, and nuclear carbohydrate The degree of performance.

In some embodiments, methods, systems, and devices for quantitative measurement of 2, 3, 4, 5 or more attributes of cells in a sample are provided here, wherein the attributes are selected from the group consisting of physical attributes, Morphological attributes, intracellular attributes, and biochemical attributes. In some embodiments, methods, systems, and devices for quantitative measurement of 2, 3, 4, 5 or more attributes of cells in a sample are provided here, wherein the attributes are selected from: cells Size, cell volume, cell conductivity, cell low-angle scattering, cell high-angle scattering, cell density, cell shape, cell area, cell internal structure, cell nucleus shape, cell nucleus area, cell nucleus size, cell nucleus internal structure, mitochondrial shape, granule Mitosome area, mitochondrial size, mitochondrial internal structure, nuclear volume to cell volume ratio, nuclear centroid/cell centroid distance (that is, the distance between nuclear center and cell center), nuclear leaf centroid distance (also The distance between the centers of the different leaves of the cell nucleus), the protein distribution inside the cell (such as actin, tubulin, etc.), the distribution of intracellular organelles (such as lysosomes, mitochondria, etc.), and the degree of expression of cellular proteins , The degree of expression of cell surface protein, the degree of expression of cytoplasmic protein, the degree of expression of nuclear protein, the degree of expression of cell nucleic acid, the degree of expression of cell surface nucleic acid, the degree of expression of cytoplasmic nucleic acid, the degree of expression of nuclear nucleic acid, cell carbohydrates The degree of expression of carbohydrates, the degree of expression of cell surface carbohydrates, the degree of expression of cytoplasmic carbohydrates, and the degree of expression of nuclear carbohydrates.

In some embodiments, a method for quantitatively measuring 2, 3, 4, 5 or more attributes of cells in a biological sample by microscopy is provided, wherein the method may include one of the following steps or entities Or more. The quantitatively measured cell properties can be selected from the properties listed in the previous paragraph. Biological samples can be pre-processed before microscopy. Pre-processing can include any procedure that assists in the analysis of samples by microscopy, including: sample processing to enrich the cells of interest in microscopy; sample processing to reduce components in the sample that may interfere with microscopy; addition of materials to the sample Assist in analyzing samples by microscopy (such as diluents, blocking molecules to reduce non-specific binding of dyes to cells, etc.). Optionally, prior to microscopy, the sample may be exposed to one or more binding agents that specifically bind to cellular components. The binding agent can be directly linked to the dye or other particles for the visualization of the binding agent. The sample may also be exposed to a secondary binding agent, which is bound to the binding agent that binds to cellular components. The secondary binding agent can be directly linked to the dye or other particles for the visualization of the binding agent. Before microscopy, samples can be verified and analyzed in a spectrophotometer. For microscopy, a biological sample containing or suspected of containing an object for microscopic analysis can be introduced into a sample container, such as a glass slide or a light test tube. The sample container containing the sample can be introduced into a device that is assembled to perform quantitative microscopy of the sample. The microscope can be coupled with an image sensor to capture images generated by the microscope objective lens. In this device, multiple images of the sample can be taken by microscopy. Any one or more of quantitative fluorescence microscopy, quantitative dark field microscopy, quantitative bright field microscopy, and quantitative contrast microscopy can be used to obtain an image of the sample. Optionally, the image of the entire sample in the sample container can be taken by microscopy. Multiple fields of view of the microscope may be required to capture images of the entire sample in the sample container. sample The container can be moved relative to the microscope, or the microscope can be moved relative to the sample container to produce different fields of view to inspect different parts of the sample in the sample container. Multiple images of the same field of view of the sample in the sample container can be captured. Optionally, multiple filters can be used for the same type of microscopy and the same field of view of the sample to capture different images of the same sample containing different information about the sample. The filters that can be used include, but are not limited to, band pass filters and long wave pass filters. The filter allows certain wavelengths of light to pass and blocks other wavelengths of light to pass. Alternatively, multi-type microscopy (such as fluorescence, dark field, bright field, etc.) can be used to capture images of the same field of view of the sample to capture different images of the same sample containing different information about the sample. Optionally, video can be used to collect microscopy images. Optionally, microscopy images can be collected in 3-D. A microscopy device or system implemented as described herein can be configured to link information of a cell in one image of the sample to information of the same cell in a different image of the sample. According to different images of the same sample and/or the same cell, the multiple attributes of the cells in the sample can be determined. In certain aspects, the combination of multiple attributes/multiple pieces of information about the cells of the sample can be used to achieve clinical decisions and/or conclusions about the cells that are impossible to achieve based on the information obtained from the single attributes of the cells.

In some embodiments, devices and systems are provided for quantitatively measuring 2, 3, 4, 5, or more attributes of cells in a biological sample by microscopy. In some embodiments, the device or system includes a microscope or cytometer and a spectrophotometer. The device or system may further include a fluid processing equipment, which is configured to move between the spectrophotometer and the microscope or cytometer Dynamic sample. In some embodiments, the devices and systems used to implement the methods disclosed herein are assembled as described in U.S. Patent Application No. 13/244,947 and U.S. Patent Application No. 13/769,779. The full text of both cases is Yan is quoted here and incorporated into the disclosure of this manual. Although the foregoing description is described in terms of cell veins, it must be understood that some or all of the foregoing description can also be applied to the crystals, particles, filaments, or other cell-sized objects that are visible in the sample.

Dynamic dilution

In some embodiments, methods, systems, and devices are provided here for dynamic dilution of cell-containing samples.

As a non-limiting example, the dynamic dilution method of the sample may include one or more of the following steps or elements, so that the desired number or concentration of cells or objects in the sample is determined, and this information is used to adjust the downstream sample A factor of processing. In this non-limiting embodiment, one or more stains or dyes can be added to the biological sample containing cells. The mixture of stain and sample can be incubated. The cells in the mixture of stain and sample can be washed to remove excess (unbound) stain. The stained, washed cells can be prepared into the desired volume for further analysis. The stained, washed cells can be analyzed to determine the approximate number or concentration of cells in the sample or part thereof. According to the number or concentration of stained cells in the sample or part thereof, a certain volume of the sample can be obtained for further analysis, thereby obtaining the desired number or concentration of cells for further analysis. In some embodiments, the sample can be diluted as described in US Patent Application No. 13/355,458, the full text of which is hereby incorporated into the disclosure of this specification.

In an embodiment as described here, it is desirable to provide another Detection techniques, such as but not limited to counting cells based on fluorescence instead of using cell counters to estimate cell concentration. The reason for describing this estimate is that in order to obtain accurate and reproducible staining of patient samples, it is often expected that the staining agent (DNA dye/antibody/binding agent/etc.) will make the best price determination for a specific number/concentration of cells. . For example, a stain of a known concentration will be applied to a specific number of cells (e.g., 0.2 micrograms of stain per thousand white blood cells (WBC)). After the incubation period, the sample will be washed to remove excess (unbound) dye, prepared to an appropriate cell density, and imaged.

In this non-limiting example, in order to obtain an estimate of the cell concentration for a target cell type, the sample is non-destructively measured using different modes for cytometry, such as but not limited to a spectrophotometer, In order to obtain sample processing information for cell counting assays. The method can include selecting another marker that is unique to the cell population of interest. In a non-limiting example, CD20 can be selected for B cells. The method involves labeling the sample with an anti-CD20 binding agent conjugated to a colored fluorescent group different from CD5. Then use a device such as but not limited to a fluorescence spectrophotometer to non-destructively and quickly measure the fluorescence signal of the sample. Using calibration, it may be possible to predict B cell concentration with limited accuracy and provide an estimate. In a non-limiting embodiment, calibration can correlate signal intensity with the number of cells for this type of signal. The generation of these calibration curves can be used to estimate the number of cells or objects. However, other techniques for estimating the number of cells based on the total signal intensity, such as optical and electrical, are not excluded. Based on the approximate concentration of B cells, the system can estimate the appropriate amount and concentration of anti-CD5 binding agent, thus maintaining the proportional relationship between CD5 performance and CD5 fluorescence. In this way, stains and staining procedures can be targeted to specific cells The number is optimized/standardized.

In order to maximize the use of patient samples (which can be low-volume samples, such as blood samples obtained from a finger puncture, with a volume equal to or less than about 120 microliters), it is desirable to develop a method that can count the number of WBCs contained in a given volume of blood (For example, determine WBC concentration/μl). This allows the number of WBCs to be determined or at least estimated before adding the colorant. Once the decision is made, the desired number of cells can be allocated for incubation with a known concentration of dye to obtain the best resolution of the cell subpopulation.

In applications where it is desired to measure the ploidy of cells, the cells in the sample can be stained with a DNA dye, and then the staining intensity can be quantified (where "staining intensity" is the intensity of the light signal caused by the dye). Therefore, the intensity of the dyeing signal caused by this kind of dyeing depends on the ratio of DNA/dye (the ratio of the amount of DNA dyed by the dye to the amount of dye added). If a preset amount of dye is added to each sample, samples with extremely high cell concentrations will be less bright than samples with low cell concentrations. This situation will confuse the quantification of DNA content in each cell. As disclosed here, before adding the dye, an estimate of the number of nucleus cells in the sample is obtained, and the amount of dye is allowed to be adjusted so that the quantification of DNA and the DNA content of each cell in the sample can be performed. Thus, for example, a sample or a sample can be treated with a stain or dye that indicates the cell surface markers of the cells to be quantified, and the surface markers are used to non-destructively estimate the cell concentration in the sample. The estimated concentration can then be used to calculate the amount of dye that must be added to the sample, thus maintaining a consistent DNA:dye ratio (mole to mole) frequently for subsequent measurements.

The first implementation of the fluorescence-based method for counting cells In an example, a method may involve cell ploidy (for example, counting cells by staining with a fluorescent group conjugated antibody). In this non-limiting example, it is desirable to count the number of WBCs in the blood sample, so that a predetermined number of WBCs can be stained with a predetermined concentration of DNA dye (for example, 4',6-diamidino-2-phenylindole (dapkin) (DAPI)), or 1,5-II{[2-(Di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (Dige (DRAQ5® )), or propanenium iodide, or other DNA dyes). The method of this example includes using a fluorescent group conjugated antibody and a spectrophotometer to count WBC. It must be understood that this method is helpful for staining cells with DNA dyes and determining ploidy, where the ratio of cell number to DNA dye concentration (cell#: [DNA dye]) is required to produce comparable and consistent data. In view of the variation in the number of blood cells per microliter of blood within a healthy population, it is typically desirable to determine the number of WBCs per microliter before attempting to chromoploid.

In one embodiment, the procedure involves the use of cells, which are first stained with a fluorescent group-conjugated antibody (where the antibody is preferably directed against a ubiquitously expressed antigen, such as CD45, or against a subset of T cells). Group-specific antigens such as CD3), or stained with fluorescent dyes that label all cells (for example, cell membrane or cytoplasmic stains such as eosin, or lectins or other stains or dyes), where it is derived from the fluorescent group The fluorescence wavelength of is spectrally separated from the emission wavelength of the DNA dye (and preferably far away). After an incubation period, the sample is washed to remove excess (unbound) dye, prepared into an appropriate volume, and analyzed by a spectrophotometer. The information obtained allows to determine the number of WBCs in the blood sample, so a specific volume of blood (to obtain a specific/desired number of WBCs) can be allocated and stained with DNA dyes. The information obtained is used as a basis for use as described The number of WBCs determined by the aforementioned fluorescent group conjugated antibody is useful to calculate and adjust the amount of DNA dye to be used to stain the sample.

Another embodiment includes determining the number of cells (through DNA staining) before staining the surface of the cells. Additional details can be obtained in the section on cell counting later in the article. Occasionally, it is desirable to count the number of WBCs in the blood sample so that a specific number of WBCs can be stained with the optimal concentration of antibody. In one embodiment, the method includes the use of DNA dyes and spectrophotometer to count WBC, as discussed above, for example.

In addition, if the number of cells per microliter is determined before staining, the known number of cells can be divided equally and stained for each sample, regardless of the following: (i) the degree of variability in the healthy population, and (ii) Disease state. In order to determine the number of cells per microliter of blood, it may be possible to use DNA dyes such as Dapi (DAPI), Dige (DRAQ5®), or propanenium iodide. Optionally, unbound dye can be washed away. The spectrophotometer can be used to determine the number of gestational nucleus (eg Diege positive) cells per microliter of blood.

The number and concentration of white blood cells (WBC) in the same amount of blood can vary from person to person. However, in order to properly analyze the WBC in the blood sample, a sufficient amount of reagents (such as antibodies targeting specific WBC specific antigens) can be added, and the sufficient number depends on the number and concentration of white blood cells (WBC) in the blood sample. A procedure named "Dynamic Dilution" can be used to ensure that sufficient antibody reagents are added to the sample. In a non-limiting embodiment, the procedure processes the blood cells to obtain a temporary cell number to calibrate the appropriate amount of reagents to be used in the sample (for example, a mixture of antibodies for staining white blood cells (WBC)) to provide complete staining of the blood cells. In this procedure, the cell line is dyed with DNA Staining, which is spectrally separated/far away from the luminescence of the fluorescent group-conjugated antibody that will be used in subsequent steps or assays. Optionally, after an incubation period, the sample can be washed to remove excess (unbound) DNA dye. After an incubation period, the sample can be prepared into an appropriate volume, and imaged or measured using a spectrophotometer. As a result, the obtained data allows the number of WBCs in a known amount of sample to be counted/determined, so that a specific volume of blood can be divided equally (to obtain a specific/desired number of WBCs) and stained with an appropriate amount of antibody (that is, determined according to the use of DNA dyes) The estimated number of WBCs can determine the amount of antibody required to provide the desired saturation of antibody staining). In this way, an appropriate amount of antibody dye required for the number of WBCs in the sample solution is calculated and added with the permission of the estimate provided by the DNA dye.

Dynamic dilution scheme:

In one embodiment, the dynamic dilution scheme involves taking a blood sample containing white blood cells to estimate the amount of reagent containing antibodies that target the WBC required for analysis of the sample.

In this non-limiting example, a blood sample of a known volume is taken. A known amount of nuclear dye (for example, a DNA staining dye such as propanenium iodide, Dapi, or Digg) is added to this known volume of blood sample. The mixture is then incubated at 25°C to 40°C for a period of 2 to 10 minutes.

Second, add red blood cell (RBC) lysis buffer. In this non-limiting example, the mixture is then incubated at 25°C to 40°C for a period of 2 to 10 minutes at a temperature of 25°C to 40°C. An appropriate dissolution buffer may be, for example, hypotonic saline solution; hypotonic sucrose solution; isotonic ammonium chloride solution; isotonic solution containing mild surfactants such as saponin; or other solutions in which RBC will dissolve It's a buffer. In the embodiment, these dissolution buffers will contain a fixative such as paraformaldehyde to help stabilize the WBC. Surfactants such as saponin cause a large number of pores in the cell membrane. Because of its unique cellular properties, red blood cells are particularly sensitive to the formation of this hole and completely dissolve, and the contents of RBC leak into the surrounding fluid. The presence of the fixative prevents inadvertent dissolution of white blood cells. Platelets also remain insoluble. The purpose of this step is to remove red blood cells (RBC) from the mixture, because the number of red blood cells exceeds the number of white blood cells by about 1000:1. Platelets do not interfere with imaging and are therefore not considered in this process. In an embodiment, the lysis buffer may also contain non-fluorescent beads of known concentration; these beads can be used as size and/or concentration markers. The dissolution of RBC along with the subsequent steps of this protocol essentially removes any RBC interference with WBC imaging or optical metrics.

Secondly, the processed sample is separated, where the separation can be performed by any suitable method, such as, but not limited to, centrifuging the processed sample in a centrifuge at 1200xg for 3 minutes.

After separation (e.g. centrifugation), the supernatant is removed; then the remaining pellets are resuspended. In the examples, the pellets are resuspended in part or all of the surfactant. In this step, a solution of known volume containing resuspended pellets is obtained.

If necessary, another separation step and another resuspension step can be performed. These steps provide a concentrated sample containing approximately 10-fold concentrated cells (neglect the cells that may be lost in each step).

Then measure the amount of DNA staining dye in the resuspended concentrated sample. For example, it can be measured spectrophotometrically from fluorescent DNA Dyeing dyes such as Diege's fluorescence. In an embodiment, the sample can be illuminated by light with a wavelength of 632 nanometers (Diege's laser wavelength), and the light emitted by the cell suspension can be filtered by a 650 nanometer long-pass filter, and then can be used in a spectrophotometer Measure the emitted light. This luminescence metric is then correlated with a previously generated calibration curve to estimate the rough concentration of white blood cells in the cell suspension. Typically, the cell concentration is in the range of about 1,000 cells/microliter to about 100,000 cells/microliter. The estimated number of WBCs obtained in this way can be used to calculate an appropriate dilution factor to ensure that the number of cells in the sample when used in subsequent quantitative measurements is limited to the range surrounding the predetermined target concentration (such as double or other range) ). Then, the sample is diluted according to the calculated dilution factor to provide a sample with a WBC concentration in the desired concentration range.

The purpose of this "dynamic dilution" step is to ensure that WBC in the sample does not exist at too high or too low concentrations. If the cell concentration is too high, the accuracy of the image processing algorithm is impaired; and if the cell concentration is too low, the number of sampled cells is insufficient. The dilution of the concentrated sample as disclosed herein provides the WBC concentration within the desired range and ensures that the signal obtained from the sample during analysis will fall within the optimal range for detection and analysis.

In addition, estimating the number of WBCs in this way permits calculation of the required amount of reagents (within a small range) for further analysis and method steps applied to the sample. The reason is that the number of WBCs in the sample can be changed, but each test analysis requires The amount of reagents may depend on the number of WBCs in the sample to be analyzed. For example, after estimating the number of WBCs by a dynamic dilution scheme, the reagents to be added include antibodies that target specific antigens seen on different types of WBCs, or if these antigens appear on multiple types of WBCs, they are not The same amount exists on different types of WBC. In the absence of such an estimate of the number of WBCs in the sample, a predetermined amount of dyes and other reagents must be used in the subsequent analysis of the sample, resulting in incorrect reagent amount and inaccurate or incomplete analysis results . As such, this dynamic dilution scheme is used as an important and useful initial step for the complete evaluation of blood samples obtained from patients, and compared to other possibilities, permits more precise and accurate measurements to be made.

Dynamic dyeing

In some embodiments, methods, systems, and devices for dynamic staining of samples containing cells are presented here.

A measure of the component of interest in the cells of a cell population

In one embodiment, a method of dynamically staining a cell sample relates to a measurement method of a component of interest in cells of a cell group in a sample.

As used herein, "component of interest" refers to any type of molecule that can be present in a cell. "Concerns of concern" include protein, carbohydrates, and nucleic acids. Typically, the "component of interest" is a specific class of molecules such as a specific antigen. Non-limiting examples of the "component of interest" of a cell include: CD5 protein, CD3 protein, and the like.

As used herein, "cell population" refers to any grouping of cells based on one or more common characteristics. The "cell population" can have any extent and can include a large number of cells or only a few cells. Non-limiting examples of "cell population" include: red blood cells (RBC), white blood cells, B cells, CD34+B cells, and the like.

In some embodiments, it may be desirable to obtain a quantitative measurement from an individual The attention component in the cells of a certain cell group in the same book. For example, it may be desirable to measure the degree of CD5 ("component of concern") expression in B cells ("cell population") in a cell sample obtained from an individual suffering from chronic lymphocytic leukemia. The detection and/or measurement of the content of the component of interest may involve the use of binding agent molecules with affinity for the specific component of interest, such as antibodies or single chain variable fragments (scFv). In order to accurately measure the content of a specific component of interest in a cell in a method involving the use of a binding agent molecule, the cell can be exposed to the binding agent molecule at a specific ratio or range of the binding agent molecule to the target component of interest. For example, it may be desirable to propose that the amount of binding agent to the cell assembly is such that there is a linear relationship between the content of the component of interest in the cell and the binding dose of the component of interest bound to the cell. For example, it may be undesirable to have too little binding agent (thus not enough binding agent to bind to all components of interest in the cell) or too much binding agent (thus binding agent non-specifically binds to the cell).

Using traditional methods, it may be difficult to provide an appropriate concentration of binding agent to the sample to accurately measure the amount of the component of interest in a cell group of the sample, because the size of the cell group and/or component of interest in the sample can be There is significant variation among different samples. On the contrary, methods, systems, and devices for dynamically staining cell samples are proposed here to respond to samples containing a wide range of cell populations and components of interest.

In one embodiment, a method for measuring components of interest in cells of a cell group in a sample is provided. The method is not limited but can include one or more of the following steps.

First, one of the intracellular markers existing in the cell group can be obtained Quantitative or semi-quantitative measurement of recording. The marker may be any marker that exists in the cell population of interest, and may be a marker that exists exclusively in the cell population of interest (that is, any other cell types that are not present in the sample). Any method can be used to measure the label, but the limitation is that the method will not damage the sample and any system or device can be used. A binding agent that recognizes the label can be mixed with the sample. The binding agent may be attached with a molecule (such as a fluorescent label) that assists in the detection of the binding agent. In one embodiment, the label can be detected and/or measured by fluorescence spectroscopy. In the embodiment where the binding agent has a fluorescent label and the label is measured by fluorescence spectroscopy, the fluorescence spectroscopy can be used to measure the bulk fluorescence from the sample or part thereof instead of measuring Fluorescence from individual cells.

Second, it is based on the quantitative or semi-quantitative measurement of the markers present in the cells of the cell population. The approximate amount or concentration of cells in the cell population of interest present in the sample can be determined. For example, the approximate amount or concentration of cells in the cell population of interest present in the sample can be determined through the use of a calibration curve. The calibration curve can be prepared and/or can be derived from different label/binding agent combinations. The calibration curve can be developed, for example, by measuring the signal obtained from a known number of cells with a certain label and bound to a certain binding agent. In some embodiments, a computer can be used to determine the approximate amount or concentration of cells in the cell population of interest that are present in the sample. In certain aspects, it is possible to determine the approximate amount or concentration of cells in the cell population of interest in the sample, and this determination does not deviate from the true concentration by more than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400 or 500%.

Third, according to the determined amount or concentration of cells in the cell group of interest in the sample, the amount of reagent added to the sample can be selected, wherein the reagent is specifically bound to the component of interest in the cells of the cell group. The agent can be or can include any molecule that specifically binds to the ingredient of interest. For example, the reagent may be a binding agent such as an antibody. The reagents can be configured to facilitate detection (for example, by fluorescence or by luminescence) and/or to produce a detectable signal in at least some cases. In some embodiments, the reagent can be attached to a molecule to assist in the detection of the reagent. The amount of reagent added to the sample can be any amount. In some embodiments, the amount of reagent that can be added to the sample is such that there is an approximately linear relationship between the content of the component of interest in individual cells of the cell group and the signal generated by the reagent bound to the component of interest in the individual cells of the cell group .

Fourth, after selecting the amount of reagent added to the sample, the selected reagent can be added to the sample.

Fifth, the cells in the sample can be tested against reagents that bind to the components of interest.

Sixth, according to the amount of the reagent bound to the component of interest, the amount of the component of interest of the cells in the cell population present in the sample can be determined.

In some embodiments, the fifth and sixth steps can be performed together, so that the metric system of the amount of the reagent bound to the component of interest is sufficient to identify the amount of the component of interest in the cells of the cell population of the sample.

In other embodiments, the providers here are systems and devices for dynamic staining of samples. Without limitation, these systems and devices may include spectrophotometers and fluorescent microscopes. In one embodiment, the dynamic dyeing system and method The method can be combined as described in US Patent Application No. 13/244,947 or 13/355,458, and the full text of the two cases are both quoted here and incorporated into the disclosure of this specification. In one embodiment, the systems and devices can be automated to determine the amount of a reagent added to the sample to determine the cells present in the sample based on the measurement of the amount of label present in the cells of the cell population The amount of concern for the cells in the population. In another embodiment, the systems and devices can be automated to determine the amount of a reagent added to a sample to determine the amount of reagent present in the sample based on the measurement of the second amount present in the cells of the cell population The first component amount of cells in the cell population.

Context-based autofocus

In some embodiments, methods, systems, and devices are provided here for context-based automatic focusing of microscopes.

The length of many clinically relevant objects in biological samples spans a wide range. For example, common cells are about 1 micrometer in length, common red blood cells are about 6-8 micrometers in length, common white blood cells are about 10-12 micrometers in length, common epithelial cells are about 100 micrometers in length, and cylinders and crystals can be about 200-300 micrometers in length. In addition, there are many amorphous bodies such as urine mucus, which are in the form of cords or filaments in the range of about 10-400 microns.

One of the challenges of microscopy is to obtain images of the field of view of any unknown composition containing objects of various sizes, such as those described above. Due to the limited depth of focus of many microscope objectives (typically about 1-10 microns), for a given field of view containing elements of various sizes, it may be necessary to obtain accurate and sharp images of each element of the given field of view. The problem with many traditional auto-focusing methods is that they are designed to focus on the main feature in a field of view, so that the sharpness of the feature can be Be maximized. This method may not be effective when shooting different size elements in the same book.

In one embodiment, a method for context-based automatic focusing of microscopes is proposed, which includes mixing reference particles of known sizes with microscopy samples. In an embodiment, more than one reference particle is added to the sample; preferably all or substantially all of these reference particles have the same known size. In an embodiment, the number of reference particles added to a specific volume of sample is known. These reference particles can be detected during microscopy and used to achieve focus. By using reference particles to achieve focus, the focal plane can be selected independently of the overall image composition. In one aspect, this method can be used to focus on a sample with unknown composition. In another aspect, this method can support the generation of precise focal planes regardless of microscope accuracy or microscopy-related hardware independence. For example, when a focal plane is selected according to the feedback obtained from the sharpness of the reference particles in a field of view, it can achieve precise focusing of each element inside a sample and the focusing hardware [such as microscope objective lens, sample container The accuracy or precision of the shape (such as a light test tube or a glass slide) or the non-uniformity of the sample container is irrelevant.

In one embodiment, the reference particle may contain or or be labeled with a molecule to assist the detection of the particle during microscopy. In one embodiment, a reference particle can be labeled with or contain fluorescent molecules. Fluorescent molecules can absorb light at a first wavelength, and can emit light at a second wavelength in response to light absorbed at the first wavelength. In one embodiment, a sample mixed with reference particles can be exposed to a light wavelength at which a reference particle of interest can excite fluorescent molecules, and the luminescence from the fluorescent molecules can be measured. Specific firefly from reference particles Light can be used to detect reference particles, and information from the detected reference particles in the sample can be used for autofocus.

The reference particle can be any shape, such as a sphere or a rectangular parallelepiped. Reference particles include but are not limited to beads and microspheres. The reference particles can be of any size, such as having about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 microns in diameter or length. The reference particles may be made of a suitable material or may contain any suitable material, such as polystyrene, polyethylene, latex, acrylic, or glass. For example, the reference particles may be polystyrene beads, such as polystyrene beads having a diameter of about 0.1 micrometer to about 50 micrometers; or about 1 micrometer to about 20 micrometers; or about 5 micrometers to about 15 micrometers; or about 10 micrometers in diameter grain.

In one embodiment, a method of focusing a microscope is provided, which may include one or more of the following steps. First, a sample containing an object for microscopic analysis (such as bacteria, red blood cells, etc.) can be mixed with a reference particle. The reference particle may contain or be labeled with a molecule to assist the detection of the particle, such as a fluorescent group. Second, the mixture containing the reference particles and the sample can be placed on the optical path of the microscope, such as a light test tube or a glass slide. Optionally, the reference particle can sink to the bottom of the sample in the light test tube or glass slide, so that the reference particle is parked on the lowest surface of the light test tube or glass slide to contact the sample. The microscope can be of any type, including a fluorescent microscope. Third, the mixture can be exposed to a light beam that is configured to visualize the reference particles. The beam can be of any type, and can be any type relative to the reference particle. Directionality. For example, the light beam may be a wavelength capable of lasering inside the reference particle or a fluorescent group attached to the reference particle. Exposure of the reference particle to the light beam may result in, for example, the generation and emission of light at a specific wavelength from the reference particle and/or the scattering of light from the reference particle. Fourth, the emitted or scattered light from the reference particles can be detected by a microscope, and this information can be used to determine the position of the reference particles inside the mixture and/or focus the microscope. Optionally, the microscope can focus on a focal plane suitable for an object having a size similar to the reference particle. The image from the microscope can be taken with an image sensor. The image can be stored and/or used for image analysis.

In some embodiments, a plurality of reference particles can be added to the sample. The reference particles may all have the same size or different sizes. In some embodiments, reference particles of different sizes contain different fluorescent groups. Different fluorescent groups can have different absorption wavelengths, different emission wavelengths, or both.

In one embodiment, a method for focusing a microscope is proposed, which includes mixing more than one reference particle of known size with a microscopy sample, wherein at least two of the reference particles have different sizes and contain different fluorescent bases. group. The method may include one or more of the following steps. First, the sample containing the microscopic analysis object can be mixed with two or more reference particles, wherein at least two of the reference particles have different sizes and contain different fluorescent groups (that is, the "first reference particle" and " The second reference particle"). Second, the mixture containing the reference particles and the sample can be placed on the optical path of the microscope. The microscope can be of any type including a fluorescent microscope. Third, the mixture can be exposed to a light beam configured to make the first reference particles visible. The beam can be any One type, and can be any direction relative to the first reference particle. For example, the light beam can be a wavelength of a fluorescent group that can be lasered inside or attached to the first reference particle. Exposure of the first reference particle to the light beam may result in the generation and emission or scattering of light of a specific wavelength from the first reference particle. Fourth, the emitted or scattered light from the first reference particle can be detected by a microscope, and this information can be used to determine the position of the first reference particle inside the mixture and/or focus the microscope. Optionally, the image of the first focal plane can be captured by an image sensor. The image can be stored and/or used for image analysis. Fifth, the mixture can be exposed to a light beam configured to make the second reference particles visible. The light beam can be of any type and can be in any direction relative to the second reference particle. Exposure of the second reference particle to the light beam can result in the generation and emission or scattering of light of a specific wavelength from the second reference particle. Sixth, the emitted or scattered light from the second reference particle can be detected by a microscope. This information can be used to determine the position of the second reference particle in the mixture and/or focus the microscope to a size similar to that of the second reference particle One of the second focal planes of the object. Optionally, the image of the second focal plane can be captured by an image sensor. The image can be stored and/or used for image analysis.

In other embodiments, the providers here are systems and devices for context-based automatic focusing of microscopes. These systems and devices may include a fluorescent microscope, but are not limiting. In one embodiment, the systems and devices can be automated to add reference particles of known size to the microanalysis sample to form a mixture, place the mixture on the optical path of the microscope, and expose the mixture to the assembled To visualize a beam of the parameter, determine the position of the reference particle inside the mixture and/or according to the reference inside the mixture The position of the particles is focused on the microscope. In one embodiment, a context-based microscope auto-focusing system and device can be configured as described in US Patent Application No. 13/244,947 or 13/355,458. The full text of the two cases is hereby quoted and incorporated into this text. Disclosure of instructions.

Positioning the same container

In some embodiments, methods, systems, and devices for determining the position of a stamp on a sample container, or a part thereof, or a sample container are presented here. Such a decision is preferably a precise decision. Even after the sample container has been removed, or the field of view has been changed (for example, by changing the focus, or by inspecting different areas of the sample container), it can still be used to identify a field of view inside a sample container Cells, particles, or other objects in it.

In an embodiment, the image-based feedback mechanism can be used to accurately and precisely determine a position in the optical test tube, such as a channel or other area containing a sample (for example, refer to the analysis area 608 in FIGS. 7 and 8). Especially when the sample container is removed and then returned to the previous position, this decision is very important for the comparison of the images and optical metrics taken before and after the movement. The variability from multiple sources may affect the position of the sample relative to the axis of the imaging system; for example, the variability of the optical cuvette components, the variability of the optical cuvette assembly, the variability of the optical cuvette position on the imaging system, and other variability. The possible source may affect the position of the sample relative to the imaging system, even if the sample remains in the same position on the sample container. The method of identifying the position of the sample container relative to the imaging system and characterizing it is disclosed here. For example, in order to accurately and reproducibly image an image of a region of interest in a light test tube, a light test tube registration program can be run. In the embodiment, this kind of program starts by analyzing a An image taken at a predetermined position in the sample container, the predetermined position is close to a registered feature or fiducial mark inside the field of view, or otherwise can be detected by the program. An optical test tube registration program includes an image processing program that searches for the existence of a fiducial mark in the image, and returns a yes/no answer (about whether the fiducial mark appears in the viewing area) or the mark is in the image The probability of the image. When the fiducial mark does not appear in the viewing area, a search algorithm is used to move the viewing area to a different position on the sample container or in the seat, and repeatedly shoot the image. This process is repeated until the program finds the fiducial mark (that is, the question about whether the fiducial mark appears in the viewing area is obtained, or the probability of the mark in the area is maximized). Once the fiducial mark position has been identified, all other positions in or on the sample container can be determined because the dimensions and layout of the sample container are known. In this way, after identifying the position of the fiducial mark, any imaging point of interest can be found and imaged. The reason is that the position of the point of interest is also known (that is, the distance and directionality from the fiducial mark are known, and because of the fiducial marks The location is known, so the point of interest is also known). In an embodiment, the fiducial mark may be or may include a characteristic piece specially made on the optical test tube itself (for example, it may be a hole, a convex portion, a printed or molded pattern, or other characteristic pieces), which may be specific to each The parts are made in the same position to any desired tolerance. In an embodiment, a fiducial mark may be or include a characteristic piece of an optical test tube (for example, a channel edge), which is often at a fixed distance from the point of interest (for example, when the fiducial mark is the channel edge, the fiducial mark Often a fixed distance from the central axis of the channel).

Cell count / count the number of cells

In some embodiments, here is provided for calculating the internal details of the sample. Methods, systems, and devices for the number of cells.

Some traditional methods of staining samples containing cells involve staining a specific volume of sample (such as blood) with a specific concentration or amount of staining agent. It can be called "volume dyeing". Volume staining has many disadvantages including: (i) it fails to resolve the normal variation in cell subpopulations between different individuals (for example, different healthy individuals may have a wide and different number of cell subpopulations, such as CD3+ T cells (here "CD3+" indicates These T cells show CD3 markers)); and (ii) the significant difference in cell composition between the diseased samples and the normal samples has not been resolved (for example, the percentage and number of CD3+ T cells in the blood of patients with T-cell leukemia are usually higher than normal Individual percentages and numbers have increased significantly).

In order to stain cell-containing samples accurately and reproducibly, it may be desirable to add a specific amount of cell staining agent (eg, DNA dye, antibody, binding agent, etc.) to a specific number or concentration of cells. For example, it may be desirable to add a specific white blood cell stain relative to 0.2 micrograms per 1000 white blood cells in the sample. After the incubation period of the dye and cells, the sample can be washed to remove excess (unbound) dye, ready for proper cell density for microscopy, and imaging. In this way, stains and staining procedures can be optimized or quantified for specific cell numbers.

In one embodiment, a method for counting the number of cells of interest in a sample is provided. The method may include one or more of the following steps or elements. The first stain that will bind to the cells of interest in the sample can be added to the sample. The mixture of the first stain and the sample can be cultivated. The cells in the mixture of the first stain and the sample can be washed to remove excess (unbound) stain. Washed cells stained with the first stain can be prepared into a stage Hope the volume is for further analysis. The washed cells stained with the first stain can be analyzed by a spectrophotometer. The information obtained from the spectrophotometer can be used to count the approximate number of cells in the sample. For example, the first staining agent may be a fluorescent dye that binds to nucleic acid, and the spectrophotometer may include a light source that emits light at the laser wavelength of the fluorescent dye, and a light sensor that can detect the emission wavelength light of the fluorescent dye Device. In this embodiment, the approximate amount of nucleic acid in the sample can be obtained based on the fluorescent signal from the dye, and from the approximate amount of nucleic acid in the sample, the approximate number of cells in the sample can be determined. Based on the approximate number of cells in the sample, a second stain that will bind to the cells of interest in the sample can be added to the sample. In the embodiment, in view of the approximate number of cells determined by using the first stain, the second stain added to the sample can be determined. In the embodiment, the second staining dose added to the sample can be calculated by using the number of cells determined by the first staining agent to obtain the desired second staining agent ratio for each cell. A mixture of the second stain and the sample can be cultivated. The cells in the mixture of the second stain and the sample can be washed to remove excess stain. The washed cells stained with the second stain can be prepared into a desired volume for further analysis. The washed cells stained with the second stain can be analyzed by microscopy.

Count the number of cells in the sample before determining the ploidy of the cells

In one embodiment, a method for counting the number of cells in a sample before determining the ploidy of the cells is provided, wherein the method includes one or more of the following steps or metabolites. A first stain that binds to the cell of interest in the sample and is spectrally different from the DNA dye can be added to the sample. The cell of interest may be, for example, white blood cells. The white blood cell may be, for example, a fluorescent group-conjugated antibody. Fluorescent group-conjugated antibodies, for example, can bind to a wide range of antigens (e.g. CD45), or can bind to an antigen expressed by a specific subpopulation of cells (eg CD3 of T cells). The mixture of the first stain and the sample can be cultivated. The cells in the mixture of the first stain and the sample can be washed to remove excess (unbound) stain. The washed cells stained with the first stain can be prepared into a desired volume for further analysis. The washed cells stained with the first stain can be analyzed by a spectrophotometer. The information obtained from the spectrophotometer can be used to count the approximate number of cells in the sample. Based on the approximate number of cells in the sample, a second stain that will bind to the cells of interest in the sample can be added to the sample. The second stain may be a DNA dye, such as propanenium iodide or 4,,6-diamidino-2-phenylindole (DAPI). In the embodiment, in view of the approximate number of cells determined by using the first stain, the second stain added to the sample can be determined. In the embodiment, the second staining dose added to the sample can be obtained by using the number of cells determined by the first staining agent to obtain the desired second staining agent ratio for each cell. A mixture of the second stain and the sample can be cultivated. The cells in the mixture of the second stain and the sample can be washed to remove excess stain. The washed cells stained with the second stain can be prepared into a desired volume for further analysis. The washed cells stained with the second stain can be analyzed for ploidy by microscopy.

In the method of determining cell ploidy, it is necessary to combine a given number of cells used for ploidy analysis with a certain amount or concentration of DNA stain to produce accurate and consistent data about cell ploidy . In one embodiment, the number of white blood cells per volume of blood in the healthy population may vary, and before attempting to stain white blood cells for ploidy analysis, it may be desirable to determine the number of white blood cells in a certain volume of blood.

The method for determining the ploidy of a cell provided above can also be performed for any method, in which the number of cells in the sample needs to be counted before determining the attributes related to the nucleic acid content of a cell. For example, the aforementioned method can be used for methods involving counting the number of cells in a sample before determining the morphology of a cell nucleus, the size of the nucleus, the ratio of the nuclear area to the total cell area, etc.

Count the number of cells in the sample before staining the cell surface

In one embodiment, a method for counting the number of cells in a sample before staining the cell surface is provided, wherein the method includes one or more of the following steps or metabolites. A first stain that is bound to the cell of interest in the sample and is spectrally separated from the luminescence of a dye to be used to stain the surface of the cell of interest can be added to the sample. The cell of interest may be, for example, the number of white blood cells. The first staining agent may be, for example, a DNA dye (e.g., propanenium iodide, Dig or Dapi). The mixture of the first stain and the sample can be cultivated. The cells in the mixture of the first stain and the sample can be washed to remove excess (unbound) stain. The washed cells stained with the first stain can be prepared into a desired volume for further analysis. The washed cells stained with the first stain can be analyzed by a spectrophotometer. The information obtained from the spectrophotometer can be used to count the approximate number of cells in the sample. In the embodiment, in view of the approximate number of cells determined by using the first stain, the second stain added to the sample can be determined. In the embodiment, the second staining dose added to the sample can be obtained by using the number of cells determined by the first staining agent to obtain the desired second staining agent ratio for each cell. The second staining agent may be, for example, a fluorescent group-conjugated antibody. Fluorescent group-conjugated antibodies, for example, can bind to a broadly expressed antigen (such as CD45), or can bind to an antigen expressed by a specific subset of cells (such as T cell CD3). A mixture of the second stain and the sample can be cultivated. The cells in the mixture of the second stain and the sample can be washed to remove excess stain. The washed cells stained with the second stain can be prepared into a desired volume for further analysis. The washed cells stained with the second stain can be analyzed for cell surface antigens by microscopy.

In the cell surface antigen staining method of cells, it is necessary to combine a given amount of cells for analysis with a given amount or concentration of a cell surface antigen staining agent to produce accurate and consistent data about the cell surface content. In one embodiment, the number of white blood cells per volume of blood varies within the healthy population. Therefore, before attempting to stain white blood cells for cell surface antigens, it may be desirable to determine the number of white blood cells in a certain volume of blood. In another embodiment, the number of white blood cells per volume of blood may vary between healthy and sick individuals. Thus, before attempting to stain white blood cells for cell surface antigens, it may be desirable to determine the number of white blood cells in a certain volume of blood. As for the theoretical example, a healthy individual has 100 blood cells per microliter of blood, 10 of which are CD3+ T cells; and lymphoma patients have 1,000 blood cells per microliter of blood, of which 900 are CD3+ T cells. Traditionally, if 100 microliters of blood are stained, a sample obtained from a healthy individual contains about 10,000 blood cells, of which 1,000 are CD3+ T cells. A 100 microliter blood sample from a patient with lymphoma contains 100,000 blood cells, of which about 90,000 are CD3+ T cells. In this theoretical embodiment, comparing samples obtained from healthy individuals, the diseased samples contain ten times the total cell number and ninety times the CD3+ Number of T cells. If the lesion samples are stained with the traditional "volume staining" method optimized for healthy individuals, the samples from lymphoma individuals may be understained. For this reason, for example, in the sample The previous estimate of the number of cells is used to adjust the amount of dye applied to the sample. This method provides advantages over traditional volume staining.

Accordingly, the method proposed here can be used to count the number of cells in a sample before cell staining to generate accurate and/or consistent data about the sample.

Method speed

The method, system, and device proposed here can support rapid acquisition of sample analysis results. The method proposed here can provide analysis results less than, for example, about 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes from the start of the method .

The rapid analysis results can be used to provide real-time information about the patient's treatment, diagnosis, or monitoring. For example, rapid analysis results can be used to guide surgeons in the patient's treatment decisions. During the operation, the surgeon can obtain biological samples from the patient for analysis. By using the methods provided here to receive rapid analysis of samples, surgeons can make treatment decisions during the operation.

In another embodiment, the rapid analysis results provided by the method, system, and device proposed here can support the patient at the service point where the patient provides biological samples at the same visit, and receive relevant information from the patient at the service point. Information on the biological samples provided.

For example, the applicant describes the rapid test analysis here, preparing a whole blood sample to analyze whether there are multiple markers and cell types in white blood cells. This type of verification analysis can be used to prepare whole blood samples for imaging analysis; the sample is ready for imaging in less than 0 minutes.

Rapid White Blood Cell Assay Analysis from Whole Blood

This assay analyzes a whole blood sample that is ready for leukocyte cytometry analysis in less than about 15 minutes or less than about 20 minutes. Such cytometry analysis of prepared cells can also be performed quickly, so cytometry WBC analysis can be completed from whole blood in about half an hour. In addition, this type of verification analysis only uses a small amount of blood samples, so it can free up resources, and the verification analysis that requires a larger amount of blood is less inconvenient or uncomfortable for individuals.

The reagents used in this assay include: phosphate buffered saline, Lyse Fix buffer, beads, resuspension buffer, and reagent mixture containing dyes and dye-conjugated antibodies. These anti-systems target specific WBC markers.

Phosphate buffered saline: 137mM NaCl, 3mM KCl, 8mM Na₂HPO₄, 1.5mM KH₂PO₄, Adjust the pH to pH 7.2 to 7.4 (using HCl).

Resuspension buffer (RSB): 5% bovine serum albumin in PBS.

Lyse Fix buffer: 0.0266% saponin in PBS containing 10% paraformaldehyde (PFA), where "%" indicates g/100ml (the final ratio is about 13:1 saponin PBS: PFA ).

Reagent mixture 1: Dige, conjugated to Pacific Blue^TM) Dye anti-CD14 antibody, Fc block (for example, immunoglobulin such as mouse IgG) in 0.2% BSA in PBS.

Reagent mixture 2: anti-CD16 antibody conjugated to phycoerythrin (PE) dye, anti-CD45 antibody conjugated to Alexa Fluor® 647 dye, conjugated to PECy5 Dye anti-CD123 antibody, Fc block (for example immunoglobulin such as mouse IgG) in 0.2% BSA in PBS.

The verification and analysis steps include: obtaining whole blood from an individual.

Fifty microliters of whole blood is placed in a test tube. If necessary, the blood sample can be obtained directly from a test tube. When 50 microliters is taken from the individual's total blood volume, all the samples are added or taken to a test tube; when more than 50 microliters are taken from the individual, then 50 microliters is one part of the sample.

Centrifuge the sample at 1200xg for 3 minutes.

Remove 20 microliters of plasma from the tube.

Place the test tube on the hot section (to raise the temperature to 37°C), add 20 microliters of RSB and mix thoroughly.

Add Mix 1 (approximately 5 microliters).

The samples were incubated at 37°C for 2 minutes.

Add dissolution and fixation buffer (the ratio of (dissolution and fixation buffer) to (stained blood) is about 6:1; about 300-350 microliters). Bed particles of known concentration can be included in the dissolving fixation buffer to provide a target (reference particle) for focusing and to provide calibration for sample concentration (for example, as described in the section titled "Context-based autofocus"). Polystyrene or other beads having a diameter of about 1 micrometer to about 30 micrometers can be used. For example, 10 micron polystyrene beads with a concentration of about 100 to about 2000 beads/μl can be used.

Dissolve the fixation buffer and incubate for 3 minutes at 37°C; about 1.5 minutes after adding the buffer, pipette the solution up and down five times to mix.

Centrifuge the sample mixture at 1200xg for 3 minutes.

Remove the supernatant (about 350 microliters). Store the supernatant and adjust the volume in a later step if necessary.

Mix 2 (approximately 15 microliters) was added to provide the final mixture.

Load the final mixture into a pre-warmed thermographic light test tube (37°C).

Before imaging, the light test tube was incubated at 37°C for 5 minutes.

Image the sample.

As such, the sample is ready for imaging in less than about 15 minutes. In the embodiment, several steps can be shortened (for example, in an alternative embodiment, the centrifugation step or the cultivation step can be shortened). Because the method disclosed above uses a mixed solution containing multiple dyes to prepare samples, it is possible to perform analysis on whether these samples have several cytotype markers within a single field of view, and provide effective imaging of the samples with minimal replication efforts. The light scattering image of the same field of view provides yet another analysis aspect, which can be effectively applied without requiring separate samples or separate fields of view for the image analysis mode of several samples. Contains reference particles of known size, with permission to use autofocus to further assist imaging, and since the concentration of reference particles is known, it provides an independent measure of sample dilution and cell concentration in each image.

Imaging of prepared samples can also be completed quickly; for example, such imaging can be completed in about 10 minutes (typically about 2 minutes to 12 minutes) by an automated device that has the characteristics described here and, for example, In U.S. Patent Application No. 13/244,947, U.S. Patent Application No. 13/769,779 and related applications. In this way, in the embodiment, the preparation of the whole analysis of the blood sample and the imaging of the prepared sample can be completed in about 30 minutes or less.

Images and image analysis obtained from samples prepared according to the methods discussed above (and similar methods discussed below) are suitable for identifying WBCs derived from whole blood Different ethnic groups. Such identification and quantification can be quickly accomplished on the same sample by illuminating the sample with light of different wavelengths (for example, sequentially) and recording and analyzing the resulting image and light intensity. These methods are suitable for providing images and mapping, such as shown in Figures 9, 10 and 11, which are prepared using the methods disclosed herein (such as those discussed above and below). The comparison shown in Figure 12 confirms that these methods are accurate and reliable, and have good correlation with other methods (for example, using Abbott's Cell-DYN Ruby System (CELL-DYN Ruby System) (Aberdeen, Illinois, USA) Special diagnostic company) analysis, the reference analyzer shown in Figure 12 for comparison).

Analysis of lesion samples

Any of the methods provided here can be used to analyze cell-containing lesion samples. If a lesion sample is a tissue sample, the sample can be processed to separate the tissue cells into individual analysis cells by the methods provided herein.

Analyzing lesion samples by any of the methods provided here can support rapid lesion analysis, and the results of lesion analysis can be quickly integrated into a patient's treatment decision.

Additional procedures in response to analysis results

In some embodiments, the devices and systems provided here can be configured to trigger additional procedures in response to the results obtained by the analysis methods provided here.

In one embodiment, a device or system can be programmed to warn the user when the result is outside the expected range. The warning can prompt the user or medical staff to manually analyze the sample, check whether the device or system is operating properly, etc.

In another embodiment, a device or system can be programmed to automatically run one or more additional tests when the result falls within or outside a certain range. In some embodiments, the device and system provided herein can perform multiple different verification analyses, and the device or system can run an additional test to verify or further study the results produced by the method provided herein.

Analysis using non-specific dyes

A non-limiting example of accelerated imaging uses a "high brightness" condition, where the cell line is labeled with a very high concentration of dye. In this embodiment, non-specific dyes that mark DNA, cell membranes, or other parts of cells are used. This example does not use antibody dyes that target specific rare proteins or other labels.

Using non-specific dyes, it is possible to obtain cell information without the need for separation steps (for example, centrifugal analysis or physical separation). Without this separation step, it can be moved directly to the imaging sample more quickly, such as but not limited to large area cells, which can include the following two: a) non-target cells such as red blood cells (RBC); and b) focusing on target cells or Objects such as white blood cells (WBC). Thus, in a non-limiting example of imaging a blood sample, 5 million RBCs and 5,000 or other numbers of WBCs can be imaged. Targeted cells can differentiate based on the inside of the cell, such as, but not limited to, the shape of the nucleus. In one embodiment, the nuclear stain is used to stain the cell nucleus in the sample, and according to the type and quantity of staining of a specific cell (for example, whether there is nuclear staining, or the shape of the stained nucleus, or other characteristics), it can be based on this Staining determines the cell type, even if the dye is non-specific. In other embodiments, other cell internal shapes (for example, cytoplasm with particles or other objects) can be indicative or characteristic, and can be used To identify and quantify the cells in the sample. For urine samples, any existing cells and crystal shapes in the sample can be used to identify the sample and determine whether there is an abnormality. In this way, non-specific dyes can be used to quickly image cells in a way that determines cells as needed.

Analysis using multiple lasers and/or inspection channels

In situations where even smaller samples are used for cytometry, in the case of advanced cytometry assays, additional lasers and/or detection wavelengths can be used. For example, in the lymphocyte subset assay analysis, various cells such as T cells, B cells, K cells, and other cells are counted for the classification of WBC. In this case, only two markers are used to identify the cell as a lymphocyte. In order to further isolate the cells in the blood sample, for example, two markers can be used again. Thus, if there is a system that only detects two colors at a time, the number of wavelengths used for analysis is insufficient.

In one embodiment, the sample can be distributed to make two separate sample parts. Then, using different parts of the sample, one component of the system can be imaged with one combination and another component of the system can be imaged with another combination. Unfortunately, this doubles the size and time. The more independent channels are built into the system, the fewer the number or volume of these sample parts will be used.

Example

Cell processing

In embodiments, it is often useful to process biological samples for imaging, testing, and analysis. For example, it is often useful to process biological samples containing cells for imaging, testing, and analysis.

The processing of biological samples may include pre-processing (e.g. sample preparation) Used for subsequent processing or measurement), processing (such as changing the sample so that it is different from its original state or previous state), and post-processing (such as discarding all or part of the sample after the measurement or use of the sample). Biological samples can be divided into multiple parts, such as blood or urine samples, or such as slicing, mincing, or dividing the tissue sample into two or more pieces. The processing of a biological sample, such as a blood sample, may include mixing, stirring, sonication, homogenization, or other processing of the sample or part of the sample. The processing of biological samples, such as blood samples, may include centrifugation of samples or parts thereof. The processing of a biological sample, such as a blood sample, may include time for the components of the sample to separate or settle, and may include filtration (e.g., passing the sample or part of it through a filter). The processing of a biological sample, such as a blood sample, may include permitting or causing agglutination of the blood sample. The processing of a biological sample, such as a blood sample, may include the concentration of the sample or part of the sample (for example, precipitation or concentration of a blood sample or a solution containing a tissue homogenate obtained from a tissue sample) to provide pellets and supernatant. The processing of a biological sample, such as a blood sample, may include the dilution of a portion of the sample. The dilution can be a dilution of a sample or a single part, including a dilution of pellets or supernatant from the sample. The biological sample can be diluted with water or with a saline solution such as a buffered saline solution. The biological sample may be diluted with a solution that may or may not contain a fixative (such as formaldehyde, paraformaldehyde, or other agents that cross-link proteins). The biological sample can effectively generate an osmotic pressure gradient solution between the surrounding solution and the interior or internal chamber of these cells, and dilute the solution that effectively changes the cell volume. For example, when the concentration of the solution obtained after dilution is lower than the effective concentration inside the cell or the internal cell chamber, the volume of such cells will increase (that is, the cells will swell). Biological samples can be diluted with a solution and the solution may or may not contain osmolality agents (e.g. glucose, sucrose, or other sugars; salts such as sodium, potassium, ammonium or other Salt; or other osmotic active compound or ingredient). In embodiments, the osmotic pressure agent can effectively maintain the integrity of the cells in the sample by stabilizing or reducing the possible osmotic pressure gradient between the surrounding solution and the interior or internal chamber of these cells, for example. In embodiments, the osmotic pressure agent can effectively provide or increase the possible osmotic pressure gradient between the surrounding solution and the internal or internal chambers of these cells and effectively make the cells at least partially collapse (when the concentration of the internal or internal chambers of the cells) When the concentration is lower than that of the surrounding solution), or effectively swelling the cells (when the concentration of the cell's interior or internal chamber is higher than the concentration of the surrounding solution).

The biological sample can be stained, or a label can be added to the sample, or the sample can be prepared in other ways for the detection, visualization, or quantification of the sample, part of the sample, component parts of the sample, or cells or structural parts inside the sample. For example, a biological sample can be exposed to a dye-containing solution. The dye can stain or otherwise make visible a cell, a cell part, or a material or molecule associated with the cell in the sample. The dye can be bound to an element, compound, or other component of the sample, or modified by it; for example, the dye can change the color, or in other ways, change its color in response to a change or difference in the pH of a solution in which the dye exists One or more of the properties, including its optical properties; the dye can change the color, or in other ways, respond to an element or compound of a solution in which the dye exists (such as sodium, calcium, carbon dioxide, glucose, or other ions, Element or compound) concentration changes or differences change one or more of its properties, including its optical properties. For example, a biological sample can be contacted with a solution containing antibodies or antibody fragments. For example, a biological sample can be exposed to a solution containing particles. The particles added to the biological sample can be used as a standard product (for example, it can be used as a size standard product, where the size of the particle Small or size distribution is known; or it can be used as a concentration standard, where the number, content, or concentration of particles is known), or it can be used as a marker (for example, particles bind or adhere to specific cells or cell types, specific cells Label or cell compartment, or where the particle binds to all cells in the sample).

Cytometry includes the observation and measurement of cells such as red blood cells, platelets, and white blood cells, including qualitative and quantitative observation and measurement of cell number, cell type, cell surface markers, internal cell markers, and other characteristics of the cells. When a biological sample includes or is a blood sample, the sample can be divided into multiple parts and can be diluted (for example, to provide a larger volume for convenient disposal, to change the density or concentration of cell components in the sample to provide the desired dilution density, concentration, or cell Number Yu its scope, etc.). The sample can be treated with an agent that affects agglutination, or it can be processed or disposed to concentrate or precipitate sample components (for example, ethylenediaminetetraacetic acid (EDTA) or heparin can be added to the sample, or the sample can be centrifuged or allowed to settle ). The sample or this part can be treated by adding dyes or other reagents that can react with specific cells or specific cell components or add labels. For example, dyes that mark the nucleus (e.g., hematoxylin dyes, anthocyanin dyes, digesol dyes such as Dige and others); dyes that mark cytoplasm (e.g., eosin dyes, luciferin-containing dyes, and others) can be separated Ground or together are used to assist the visualization, identification, and quantification of cells. More specific markers include specific antibodies and antibody fragments for cell targets, such as cell surface proteins, intracellular proteins and chambers, and other targets that are also useful in cytometry.

Biological samples can be measured and analyzed by cytometry using the following optical devices, including, for example, photodiode detectors, photomultipliers, charge-coupled devices, laser diodes, spectrophotometers, cameras, microscopes, or measurement Light intensity (single wavelength, multiple wavelengths, or one wavelength range or multiple ranges of light), other devices that form an image, or both. A field of view containing a sample or a sample can be imaged using these detectors, or scanned, or both. Before processing, dilution, separation, centrifugation, agglutination, or other changes, biological samples can be measured and analyzed by cytometry. During or after the processing, dilution, separation, centrifugation, agglutination, or other changes of the biological sample, the biological sample can be measured and analyzed by cytometry. For example, just after receiving the sample, the biological sample can be measured and analyzed by cytometry. In other embodiments, the biological sample can be measured and analyzed by cytometry during or after the processing, dilution, separation, centrifugation, agglutination, or other changes of the biological sample.

For example, the sample or sample part can be prepared for cytometry by deposition or centrifugation. Before cytometric analysis, the deposited part or pellet part of such a sample can be resuspended in the preferred buffer (for example, by suction, stirring, sonication, or other treatments). Prior to cytometric analysis, the biological sample can be diluted or resuspended in water or a saline solution such as a buffered saline solution. The solution used for such dilution or resuspension may or may not contain a fixative (such as formaldehyde, paraformaldehyde, or other agents that cross-link proteins). The solution used for such dilution or resuspension can effectively generate an osmotic pressure gradient between the surrounding solution and the interior or internal chamber of the cells in the sample, effectively changing the cell volume of some or all of the cells in the sample. For example, when the concentration of the solution obtained after dilution is lower than the effective concentration inside the cell or the internal cell chamber, the volume of such cells will increase (that is, the cells will swell). The biological sample can be diluted with a solution and the solution may or may not contain an osmotic agent (for example, glucose, sucrose, or other sugars; salts such as sodium, potassium, ammonium, or other salts; or other osmotic activation Compound or ingredient). In an embodiment, the osmotic pressure agent can effectively maintain the integrity of the cells in the sample by stabilizing or reducing the possible osmotic pressure gradient between the surrounding solution and the interior or internal chamber of these cells, for example. In embodiments, the osmotic pressure agent can effectively provide or increase the possible osmotic pressure gradient between the surrounding solution and the internal or internal chambers of these cells, and effectively make the cells at least partially collapse (when the concentration of the internal or internal chambers of the cells) When the concentration is lower than that of the surrounding solution), or effectively swelling the cells (when the concentration of the cell's interior or internal chamber is higher than the concentration of the surrounding solution).

For example, after a part of the sample is diluted with a dye-containing solution, a biological sample can be measured or analyzed. For example, after a part of the sample is diluted with a solution containing antibodies and antibody fragments, a biological sample can be measured or analyzed. For example, after a part of the sample is diluted with a solution containing particles, a biological sample can be measured or analyzed. The particles added to the biological sample can be used as a standard product (for example, it can be used as a size standard product where the particle size or size distribution is known; or it can be used as a concentration standard product where the number, content, or concentration of particles is Known), or can be used as a label (for example, particles bind or adhere to specific cells or cell types, specific cell markers or cell compartments, or where particles bind to all cells in the sample).

For example, a biological sample can be measured or analyzed after the following treatments, which can separate one or more types of cells from another type or types of cells. This separation can be done by gravity (for example, deposition); centrifugation; filtration; , Or other ingredients); or other means to complete. Separation can be accomplished by means of or by changing cell types. For example, The solution can be added to a biological sample such as a blood sample, which causes some or all of the cells in the sample to swell. When one type or multiple types of cells swell faster than another type or multiple types of cells, the cell types can be distinguished by observing or measuring samples after adding the solution. These observations and measurements can be performed one or more times, and are selected to emphasize response differences (such as size, volume, internal concentration, or other properties affected by this swelling), and thus increase the sensitivity of these observations and measurements And accuracy. In some cases, in response to this swelling, one or more types of cells may burst, allowing the observation and measurement of other cell types in the sample.

The observation, measurement, and analysis of a biological sample by cytometry may include photometric measurements. For example, the use of photodiode detectors, photomultipliers, charge-coupled devices, laser diodes, spectrophotometers, cameras, microscopes, or Other means or devices. Cytometry can include preparing and analyzing cell images (such as two-dimensional images) in a biological sample, where the cell lines are labeled (such as using fluorescence, chemiluminescence, enzymes, or other labels) and plating (such as Allowed to settle on the substrate) and use the camera to image. The camera may include a lens, which may be attached to or used in conjunction with a microscope. The cell can be identified in the two-dimensional image by the tag attached to it (for example, the light emitted from the tag).

The cell image prepared and analyzed by the cytometer as disclosed herein may include no cells, one cell, or multiple cells. As previously disclosed, a cell or a cell in the image of the cytometer as disclosed herein can be tagged. As previously disclosed, a cell or a cell in an image of the cytometer as disclosed herein can be tagged to effectively identify the image and the individual from which the sample was obtained.

In some embodiments, a verification analysis system is configured to perform cytometric verification analysis. Cytometry analysis is typically used for optical, Electrical or acoustic methods to measure the characteristics of individual cells. For the purposes disclosed herein, "cells" can encompass non-cellular samples, which usually have a similar size to individual cells, including but not limited to cysts (such as liposomes), small groups of cells, virus particles, cells, Protozoa, crystal particles, small bodies formed from lipids and/or proteins, and substances bound to small particles such as beads or microspheres. These characteristics include, but are not limited to, size; shape; granularity; light scattering pattern (or luminous flux); whether the cell membrane is intact; the concentration, morphology, and space-time distribution of the internal contents of the cell, including but not limited to protein content , Protein modification, nucleic acid content, nucleic acid modification, organelle content, nuclear structure, nuclear content, internal cell structure, internal cystic content (including pH), ion concentration, and whether there are other small molecules such as steroids or drugs; And cell surface (both cell membrane and cell wall) markers include proteins, lipids, carbohydrates, and their modifications. By using appropriate dyes, stains, or other label molecules that are in pure form, conjugated to other molecules, or immobilized or bound to nanoparticles or microparticles, cytometry can be used to determine specific proteins, nucleic acids, lipids, carbohydrates, Or the presence, quantity and/or modification of other molecules. The properties that can be measured by cytometry also include the measurement of cell function or activity, including but not limited to phagocytosis, antigen presentation, cytokine secretion, changes in the expression of internal and surface molecules, and binding to other molecules or cells or substrates , Active transport of small molecules, mitosis or meiosis; protein translation, gene transcription, DNA replication, DNA repair, protein secretion, apoptosis, chemotaxis, mobility, adhesion, antioxidant activity, RNAi, protein or Nucleic acid degradation, drug reaction, infection, and specific pathway or enzyme activity. Cytometry can also be used to determine information about a population of cells, including but It is not limited to cell count, percentage of total population, and variation in sample population for any of the foregoing characteristics. The assay described here can be used to measure one or more of the aforementioned characteristics for each cell, and it can excellently determine the interaction or other relationships between different characteristics. The assay described here can also be used to independently measure multiple populations of cells, such as using specific antibodies from different cell lines plus a mixed cell population for labeling. The microscopy module permits the use of the device to perform histological, pathological and/or morphological analysis, and also helps to evaluate objects based on both physical and chemical properties. The tissue can be homogenized, washed, deposited on a light test tube or glass slide, dried, stained (such as with antibodies), incubated, and then imaged. When combined with data transmission techniques as described elsewhere in this document, these invention-assisted images are transmitted from CMOS/CDD or similar detectors to, for example, a licensed pathologist for review, which is a traditional device that only performs flow cytometry. Impossible to achieve. The cytometer can measure surface antigens and cell morphology; compared with traditional hematology laboratory devices, surface antigens can be tested more sensitively and specifically. The interpretation of cell assay analysis can be automated by gating one or more metrics; gating thresholds can be set by experts and/or learned from training data based on statistical methods; gating rules can be for individual individuals and/or individual groups Specificity.

In some embodiments, the cytometer module is incorporated into a service device point to provide a measurement of cell attributes typically measured by common laboratory devices and laboratories, for interpretation and review by orthodox trained medical personnel, Improve the speed and/or quality of clinical decision-making. Therefore, a service device point can be configured for cytometry analysis.

Example 1

Obtain a cell sample containing white blood cells including natural killer cells and neutrophil cells. The sample is treated with a fluorescently labeled identity binding agent (anti-CD16 binding agent), which binds to both natural killer cells and neutrophils. The sample was also treated with nuclear dye (Dige). The sample was imaged by fluorescence microscopy and dark field microscopy. Record and analyze the fluorescence level and lateral light scattering of different cells in the sample. The segmented image containing the signal of the anti-CD16 binding agent provides quantitative information on the fluorescence intensity of each cell (corresponding to the degree of CD16 expression), and also the size of each cell. Dark field images provide quantitative information on the scattering properties of each cell. The image containing the DNA dye signal is segmented to determine the fluorescence intensity, the size and shape of the nucleus.

As shown in Figure 1A, two large groups of cells were identified based on the CD16 fluorescence and light scattering measurements of different cells. The group of cells with bright/high CD16 fluorescence signal and high scattering (Figure 1A, right circle) are neutrophils. The group of cells with moderate CD16 fluorescence signal and low scattering (Figure 1A, left circle) are natural killer cells. Although the measurement of fluorescence and light scattering of different cells provides sufficient information to classify most of the cells in the sample as natural killer cells or neutrophils, for some cells, the measurement of these attributes does not provide sufficient information Information to classify cells with a high degree of accuracy. For example, the measurement of fluorescence and light scattering of cells does not provide enough information to accurately classify the small group of cells in the smallest circle (that is, the middle circle) in FIG. 1A. In order to identify whether the cells in the smallest circle are natural killer cells or neutrophils, examine the stained images of their nuclei (Dige) and whole cells (anti-CD16). Obtain quantitative measurements of the nucleus and total cell area of these cells, and determine the ratio of the cell nucleus to the total cell area. As shown in Figure 1B, the nucleus area between natural killer cells (NK) and neutrophils (Neu) is compared There is a clear difference in the ratio of the total cell area. In this way, the use of quantitative microscopy to examine the multiple attributes of the cells in the sample is used to allow the cells to be clearly classified. Figure 1C shows an image of natural killer cells from the smallest circle in Figure 1A. All images have the same length scale. The image on the left is a cell stained for the whole cell area (anti-CD16), and the image on the right is the same cell with only the nucleus (Dige) stained. The images in the top and bottom columns are different examples of natural killer cells. Figure ID shows an image of the neutrophil from the smallest circle in Figure 1A. All images have the same length scale. The image on the left is a cell stained for the whole cell area, and the image on the right is the same cell with only the nucleus stained. The images in the top and bottom columns are different examples of natural killer cells.

In addition, the neutrophil nucleus has a unique leafy shape, while the nucleus of natural killer cells (and other lymphocytes) is round, uniform, and smooth. Based on the shape of the nucleus, image segmentation algorithms can be used to identify and classify cells.

Example 2

Obtain a sample containing platelets. Platelets are labeled with fluorescent conjugated anti-CD41 antibody and anti-CD61 antibody. Beads with a diameter of 3 microns are also added to the sample. The sample was imaged at 10x and 20x magnification (Figure 2A). The intensity of the fluorescence distribution of individual platelets was measured (derived from the secondary antibody), and it was determined to have a Gaussian distribution shape (Figure 2B). Fluorescence measurements of individual platelets were plotted and the intensity distribution matching was determined (Figure 2C). In Figure 2C, the gray line is the fluorescence intensity measured across individual platelet counts, and the black line is the match. Matching parameters such as Gaussian mean, variable, volume, width, and bottom area can be evaluated as predictors of platelet volume. The Gaussian volume and matching width have been determined to be closely related to the average platelet volume.

For the aforementioned measurement, the 3 micron beads are used as a reference and benchmark to control variables to accurately determine the optimal focal plane, and the effect of this variable on the volume measurement.

In addition, the estimated platelet size based on the matched 2D model can be calibrated to the equivalent range (Figure 3)

Example 3

Obtain a sample containing red blood cells (RBC). With a low concentration of surfactant (DDAPS or SDS), the red blood cells are processed to swell the red blood cells into a spherical shape. The red blood cells are imaged by dark field microscopy in two different light test tubes: (A) a light test tube only permits pure surface illumination (Figure 4A); and (B) a light test tube permits a mixture of surface and penetrating illumination (Figure 4B). Compared with the light test tube that only allows pure surface lighting, the red blood cells are much more clearly visible in the light test tube that permits a mixture of surface lighting and penetration lighting (Figure 4).

Example 4

Obtain a sample containing neutrophils. In neutrophils, the shape of the nucleus and the chromatin morphology can indicate whether it is an immature "banded" neutrophil or a mature "segmented" neutrophil. Banded neutrophils are immature neutrophils that have recently sprouted from bone marrow. An increase in the proportion of banded neutrophils can indicate an ongoing infection or inflammation.

The sample is mixed with a fluorescently labeled anti-CD16 antibody, which recognizes the cell surface receptor CD16 on neutrophils. The sample is also stained with fluorescent nuclear dye. The sample was imaged with a fluorescence microscope to obtain nuclear staining and CD16 staining data from the cells. Banded neutrophils usually have a degree of CD16 expression similar to that of mature segmented neutrophils, so they cannot be compared with those of mature segmented neutrophils. The fluorescence intensity difference of CD16 staining.

Image analysis with image segmentation is used to identify the staining and morphology of banded neutrophils and segmented neutrophils, thereby permitting cell classification. Check the size, shape, and fluorescence intensity of the nucleus. In addition, the cell nucleus is analyzed to determine the number of leaves (the intensity peak is in the nuclear area), the distance between the nuclear lobes, and the change in the curvature of the nuclear contour (second derivative). Figure 5A shows a representative image of banded neutrophils. In these images, the nucleus appears as light gray and the cytoplasm appears as darker gray. When neutrophils differentiate through bone marrow cell lines, they develop a characteristic U-shaped nucleus before reaching full maturity. Figure 5B shows representative images of segmented neutrophils. In these images, the nucleus appears as light gray and the cytoplasm appears as darker gray. Segmented neutrophil nuclei have multiple segments/lobes (typically about 3-5). Thus, this analysis confirms the identification and quantification of different neutrophil subpopulations in the blood.

Example 5

Obtain a sample of cells from individuals suffering from chronic lymphocytic leukemia (CLL). The objective is to quantify the degree of CD5 expression on B cells obtained from an individual. The anti-CD20 antibody system is selected as the B cell binding agent. Mix the sample with the first colored fluorescent tagged anti-CD20 antibody system. After an appropriate incubation time, the sample is washed to remove unbound anti-CD20 antibodies. The sample is exposed to a light source capable of exciting the first fluorescence, and the fluorescence signal is measured using a spectrophotometer. Based on the fluorescent signal, the approximate concentration of B cells in the sample is determined. The determined approximate concentration of B cells actually falls within 1.5 times the true concentration of B cells in the sample.

According to the approximate concentration of B cells in the sample, an appropriate amount of anti-CD5 binding agent is added to the sample, so the ratio between CD5 performance and CD5 fluorescence is maintained Tie. The anti-CD5 binding agent is coupled to a second fluorescent group and has a different peak laser wavelength from the first fluorescent group (attached to the anti-CD20 binding agent). The anti-CD5 antibody system is added to the sample, and then individual cells of the sample are exposed to a light source capable of exciting the second fluorescent group, and the fluorescent signal from the individual cells is measured. According to the fluorescent signal obtained from the cells, the average CD5 content of B cells in the sample is determined.

Although this embodiment is described with CD5 as an example, it should be understood that the concept of knowing the approximate number to guide the addition of the desired amount of material used in the subsequent steps is not limited to CD5, and does not rule out the application of this concept to other types Of cells, analytes, or objects.

Example 6

Blood cells can be imaged, identified, and quantified according to the methods disclosed herein. For example, a two-dimensional image of cells in a biological sample can be prepared and analyzed as described in this embodiment, where the cell lines are labeled (for example, using fluorescence, chemiluminescence, enzymes, or other labels) and plated (For example, allow to settle on the substrate) and use the camera to image. The camera can include a lens, and can be attached to or used in conjunction with a microscope. Cells can be identified in a two-dimensional image by the attached tag (for example, the light emitted by the tag).

80 microliters of whole blood obtained from finger puncture was loaded into a sample container with a lid pre-loaded with 2 mg/ml EDTA. In this case, use an enclosed sample container (with a movable lid or a pierceable lid); it must be understood that any suitable container for holding such a small amount of sample can be used, including but not limited to a container with a lid or a container without a lid. The sample container was centrifuged at 1200xg for 5 minutes to separate blood cells and plasma. The centrifugation of the sample container causes the blood sample in the sample container to separate into Two major components (from top to bottom of the sample container): 1) plasma and 2) accumulation of blood cells. This processing procedure ensures that no blood droplets remain isolated, but are integrated into the liquid body. In addition, this treatment separates the blood cells from the plasma components, thus reducing metabolism and permitting a longer storage period for the samples.

The centrifuged sample container is loaded into a cassette containing a plurality of fluid separation reagents, tube tips, and cytometry light test tubes. The cassette contains all the reagents required for the verification and analysis. The cassette is loaded into a device equipped with at least one centrifuge, a burette and a platform to load the optical test tube. The burette in the device has a plurality of nozzles, and the size of some nozzles is different from the size of several other nozzles.

Inside the device, a nozzle on the burette is lowered onto the light test tube carrier tool so as to engage a corresponding hole on the carrier tool. Then the tool moves to the cassette and drops onto the cytometry light test tube. Then the pin on the tool engages the corresponding hole on the light test tube and picks up the light test tube. The optical test tube is transferred to a load station elsewhere inside the device.

Secondly, inside the device, the larger nozzle of the dropper is lowered into the cassette to engage the tip of the dropper stored in the cassette. Then, by placing the tip of the burette in the sample container and repeatedly extracting and distributing materials into the tip of the tube, the burette and the tip are used to mix the cells and plasma in the sample container together. Once the cells are resuspended in the plasma and the whole blood sample is thoroughly mixed, 5 microliters of the mixed whole blood are drawn to provide an aliquot for measuring the properties of the blood sample. The 5 microliter aliquots are used to measure the red blood cells and platelets in the sample. As discussed later, the remaining portion of the sample after removing the 5 microliters is used to measure the white blood cells in the sample.

5 microliters of whole blood was dispensed into a container containing a mixture of phosphate buffered saline and 2% by weight of bovine serum albumin to dilute the whole blood 20 times (to obtain 100 microliters of diluted sample). After vigorous mixing, 5 microliters of the sample was transferred to another container containing the labeled antibody reagent mixture: anti-CD235a conjugated to Alexa Fluor® 647 (AF647) and anti-CD61 conjugated To phycoerythrin (PE). The mixture is incubated for 5 minutes. Subsequently, 10 microliters of this mixture was mixed with 90 microliters of buffer containing less than 0.1% by weight of zwitterionic surfactant. Surfactant molecules modify the curved nature of the red blood cell membrane, so that all cells obtain a stable spherical shape. Because the buffer system and cytoplasm used are isotonic, this transformation system is of constant volume; therefore, there is no osmotic pressure driving fluid exchange through the cell membrane. After incubating for another 2 minutes, 30 microliters of this solution was mixed with a solution containing glutaraldehyde, fixative, and 10 micron diameter non-fluorescent beads. The mixture has a final concentration of 0.1% glutaraldehyde and 1000 beads per microliter. Glutaraldehyde quickly fixes cells, thus preventing cell lysis and other active biological treatment procedures.

In this non-limiting example, a burette is then joined with a tip in the cassette, 7 microliters of the above mixture is sucked and 7 microliters are loaded into the light test tube placed on a platform with a carrier tool Inside of a channel. After the mixture is loaded into the light test tube, the burette draws 10 microliters of mineral oil from the container in the cassette, and places a drop of mineral oil on the two open ends of the loaded channel of the light test tube. Mineral oil is added to the end of the open channel to prevent the liquid from evaporating from the loaded channel of the light test tube. Secondly, the device-level sample processing equipment connects the optical cuvette carrier/optical tube combination, and transfers the optical cuvette carrier/optical tube combination from the cassette-containing module to the cytometry module of the device. In the cytometry module, the installation The hierarchical sample processing equipment places the optical test tube carrier/light test tube combination on the microscope stage of the cytometry module. In addition to the 2-minute waiting time, the time required for these operations may allow the swollen cells to settle to the bottom of the light test tube before imaging.

After the light test tube carrier/light test tube combination is placed on the microscope stage, the stage is moved to a predetermined position so that the optical system of the cytometer can view one end of the channel containing the sample. At this position, the optical system relays the sample image obtained from the ring light illuminating the dark field. These images are coupled to the action of the optical system on an axis perpendicular to the plane of the light test tube to find the best focal plane. Once focused, the optical system is used to obtain fluorescent images of the sample at different wavelengths that match the fluorescent groups used. For example, in order to make the anti-CD235 red blood cells that have been conjugated to the Elisa Fluorescent 647 visible, a red (630nm wavelength) light source is used for the laser sample, and a wavelength between 650nm and 700nm is used. Used to image samples. The combination of a dichroic mirror and a band-pass emission filter is used to filter out undesired wavelengths from the optical signal. Because the cells have settled to the bottom of the light test tube, the image on a single focal plane is enough to make all the cells in that area visible.

The data obtained from the image is processed by a controller connected to the sample processing device. The image processing algorithm used here uses a combination of adaptive threshold processing and edge detection, using fluorescent images of cells to detect them. According to the local intensity and intensity gradient, a region of interest (RoI) is generated around each cell. Using dark field images, the beads in the sample are also identified and an area of interest surrounding the beads is generated. The number of all regions of interest in each field of view is calculated, and the intensity of each image in that field of view is calculated. Calculated by image processing The information output by the method includes the shape or morphological measurement, fluorescence intensity and dark field intensity for each area of interest. This information is analyzed using statistical methods to classify objects as red blood cells (positive for CD235a, but negative for CD41/CD61), platelets (positive for CD41/CD61 and negative for CD235a, or beads. Shape description Symbols such as circumference, diameter, and roundness are used to calculate the volume of each red blood cell and platelet. Because the beads are added at a known concentration, the average ratio of beads to cells above the entire channel is used to calculate the cell concentration. Expressed in cells/microliter. According to the steps performed to process the sample, this concentration refers to the concentration of cells in the original whole blood sample since the dilution correction. Calculate the following numbers from the sample: 1) The number of red blood cells in the light test tube; 2 ) The average volume of red blood cells in the light test tube; 3) the red blood cell distribution width (RDW) of the red blood cells in the light test tube; 4) the number of platelets in the light test tube; and 5) the average volume of platelets in the light test tube. Based on these calculations, the following are calculated for the original blood sample.

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After taking 5 microliters of liquid for information analysis of red blood cells and platelets, the remaining 75 microliters of blood samples were used to analyze the white blood cell population of the whole blood sample. The remaining 75 microliters of whole blood is also repeatedly pumped and dispensed in the same container using a dropper The samples inside are mixed. About 40 microliters of the remaining 75 microliters of mixed whole blood are sucked into the tip of the dropper, and transferred to a centrifuge tube in the cassette through the dropper. The centrifuge tube containing the blood sample is connected to the burette, and transferred to and deposited in the swing bucket in a centrifuge in the module. The centrifuge provides 1200xg centrifugation for 3 minutes to separate the blood into EDTA-containing plasma as the supernatant and the accumulated blood cells in the pellets.

After centrifugation, the centrifuge tube is removed from the centrifuge and placed back into the cassette. The plasma supernatant is removed by a dropper and transferred to a separate reaction vessel in the cassette. From a reagent container in the cassette, draw 16 microliters of resuspension buffer with a dropper, and add it to the cell pellets in the centrifuge tube. Then, the burette resuspended the cell pellets in the resuspension buffer by repeatedly drawing and dispensing the mixture in the centrifuge tube. Next, draw 21 microliters of resuspended whole blood with a dropper and add it to another container containing 2 microliters of anti-CD14-Pacific Blue and Dige, mix, and incubate for 2 minutes. Then 20 microliters of this mixture is added to 80 microliters of lysis buffer. The dissolution buffer is a solution of a mild surfactant such as saponin in combination with a fixing agent such as paraformaldehyde. The cleaning agent causes a large number of holes in the cell membrane. Due to its unique cell membrane properties, red blood cells are particularly susceptible to the formation of holes and completely dissolve, and their contents leak out into the surrounding liquid. The presence of the fixative prevents accidental dissolution of white blood cells. Platelets also remain insoluble. The purpose of this step is to remove red blood cells from the mixture, because the number of red blood cells exceeds the number of white blood cells by approximately 1000:1. Platelets do not interfere with imaging, so it has nothing to do with this processing procedure. The lysis buffer also contains a known concentration of 10μM non-fluorescent beads.

After incubating for 5 minutes, the container was centrifuged again at 1200xg for 3 minutes. superior The clear liquid is drawn with the tip of a dropper to remove red blood cell fragments and other debris, and deposit in the waste area in the cassette. There is about 15 microliters of liquid containing accumulated white blood cells in the cell pellet.

In order to determine a rough approximation of the number of white blood cells present in the cell pellet, the burette first resuspends the white blood cells in the container, and then sucks the liquid for transfer and checks with a spectrophotometer. The white blood cell suspension is illuminated with 632 nanometer wavelength light, which is the Alissa fluorescence 647 and the laser wavelength of Digger. The light emitted by the cell suspension is filtered by a 650 nanometer long wave pass filter and measured in a spectrophotometer. This measurement is linked to the calibration curve previously generated to estimate the rough concentration of white blood cells in the cell suspension. Typically, the cell concentration is in the range of about 1,000 cells/microliter to about 100,000 cells/microliter. This estimate is used to calculate the approximate dilution factor to ensure that the cell concentration in the light test tube is limited to twice the predetermined target concentration. The purpose of this step is to ensure that the cells do not exist in the light test tube at too high or too low density. If the cell density is too high, the accuracy of the image processing algorithm is impaired; and if the cell density is too low, the number of sampled cells is insufficient.

Based on the dilution factor calculated in the above steps, a diluent containing labeled antibodies against CD45 (pan-leukocyte marker), CD16 (neutrophil marker) and CD123 (basophil marker) is added to the cell suspension and mixed .

Once the optical test tube of the composite optical test tube carrier is positioned on the optical test tube carrier section, 10 microliters of the mixture of leukocyte suspension cytometry buffer is loaded into each of the two channels in the optical test tube. After the mixture is loaded into the channel of the light test tube, the burette draws 10 microliters of mineral oil from the container in the cassette, And drop a drop of mineral oil on the two open ends of the two channels of the light test tube loaded with white blood cells.

Secondly, the device-level sample processing equipment connects the optical cuvette carrier/optical tube combination, and transfers the optical cuvette carrier/optical tube combination from the cassette-containing module to the cytometry module of the device. In the cytometry module, the device-level sample processing equipment places the optical test tube carrier/optical tube combination on the microscope stage of the cytometry module. After the light test tube carrier/light test tube combination is placed on the microscope stage of the cytometry module, the two channels of the light test tube containing white blood cells are imaged as described above for the RBC/platelet mixture.

The dark field image of white blood cells is used to count the number of cells in a field of view (as shown in Figure 9A). Cell surface markers are used to determine the cell type of individual white blood cells in an image; for example, CD14 is used to label monocytes; CD123 is used to label basophils; CD16 is used to label neutrophils; and CD45-AF647 is used to label all white blood cells (Figure 9B-9E). The nuclear staining agent Dige is used to mark the nucleus, and thus is used to distinguish pregnant nuclear cells (such as white blood cells) from mature red blood cells, which have no nucleus (Figure 9F).

The image processing algorithm used here uses fluorescent images of cells to detect them using a combination of adaptive threshold processing and edge detection. According to the local intensity and intensity gradient, a region of interest (RoI) is generated around each cell. Using dark field images, the beads in the sample are also identified and an area of interest surrounding the beads is generated. The number of all regions of interest in each field of view is calculated, and the intensity of each image in that field of view is calculated. The information output by the image processing algorithm includes the shape or morphological measurement for each area of interest, fluorescence intensity and dark field intensity. This information is analyzed using statistical methods to Bodies are classified as lymphocytes, monocytes, basophils, eosinophils, neutrophils, or beads. According to the cell count of different types, the corresponding bead count, and the dilution ratio reflected during sample processing, the absolute concentration of cells per microliter of the original whole blood is calculated. This point is calculated for all white blood cells and each subtype, and reported as both absolute concentration (cells/microliter) and ratio (%).

Examples of images and drawings of these measurement results are shown in FIGS. 9, 10, and 11.

Figure 9 shows representative images of blood cells obtained from a whole blood sample; these images were taken using different imaging techniques and dyes. The image shown in Figure 9A was taken using dark-field illumination of blood cells from whole blood. The image shown in Figure 9B was taken of blood cells from whole blood, showing fluorescence from an anti-CD14 antibody labeled with Pacific Blue dye; the fluorescent cells are monocytes. The image shown in Figure 9C was taken of blood cells from whole blood, showing fluorescence from anti-CD123 antibodies tagged with PECy5 dye; the fluorescent cells are basophils. The image shown in Figure 9D was taken of blood cells from whole blood, showing fluorescence from anti-CD16 antibodies tagged with PE dye; the fluorescent cells are neutrophils. The image shown in Figure 9E was taken of blood cells from whole blood, showing fluorescence from anti-CD45 antibodies tagged with AF647 dye; in this case, all white blood cells are fluorescing. The image shown in Figure 9F was taken of blood cells from whole blood stained with Dige to stain the nucleus. Thus, in these cases, white blood cells and platelets are stained and fluoresce, but red blood cells and platelets (deficient nucleus) are not stained and do not fluoresce.

Figure 10 shows a representative composite image of cell types in whole blood shown in images taken according to the method disclosed herein. Monocytes (tagged And seen in the upper left quadrant of the figure, the red center is surrounded by a blue-purple circle), lymphocytes (labeled and seen in the middle of the figure, the bright red center is surrounded by a dark red circle), eosinophils (labeled and seen in the lower left quadrant of the figure, green The image of the heart surrounded by a red border) and neutrophils (labeled and seen in the lower right quadrant of the figure, the green heart is surrounded by a yellow and green border) are shown in this figure.

The place of interest must be to identify and quantify the various cell types seen in these blood samples. There are multiple processing methods for this classification method, which can be regarded as a statistical problem of multi-dimensional classification in some embodiments. It must be understood that there are a wide variety of methods in this field that can be used to solve these types of classification problems. A specific example of this analysis is provided below.

Figure 11 shows the mapping of each cell type identified and quantified by the cytometry assay described in this embodiment. FIG. 11A shows a plot of the intensity points (cells) of labeled FL-17 (anti-CD14 antibody labeled with Pacific Blue dye) versus the intensity of FL-9 (dark field scattering signal) to identify monocytes. Figure 11B shows the intensity points (cells) of labeled FL-19 (anti-CD123 antibody labeled with PE-CY5 dye) relative to the intensity of FL-15 (anti-CD16 labeled with PE dye) to identify basophils A mapping of sex cells. Figure 11C shows a function of identifying lymphocytes by the intensity points (cells) of labeled FL-15 (anti-CD16 labeled with PE dye) relative to the intensity of FL-11 (anti-CD45 antibody labeled with AF647 dye) picture. Figure 11D shows the identification of neutrophils and eosinophils by the intensity points (cells) of labeled FL-15 (anti-CD16 labeled with PE dye) relative to the intensity of FL-9 (dark field scattering signal) picture.

The initial recognition of monocytes (9.6%, as shown in Figure 11A) is used to guide the subsequent recognition of basophils (0.68%, as shown in Figure 11B). like The recognition system of monocytes and basophils shown in Figures 11A and 11B is used to guide the subsequent recognition of neutrophils and eosinophils (68% neutrophils, 3.2% eosinophils, accounting for Figure 11B WBC). Finally, the lymphocyte lineage is identified as shown in Figure 11C (accounting for 93% of the WBC in Figure 11C and 18% of the cells in the original sample).

This method has a good correlation with other methods. The numbers of white blood cells, red blood cells, and platelets were counted using a complete blood sample that was anticoagulated with EDTA. Further count the white blood cells to determine the number of neutrophils, monocytes, and lymphocytes in the sample. In the measurement shown in Figure 12, the whole blood sample anti-coagulated with EDTA is divided into two, and a sample is run on the system disclosed here using the method disclosed herein. The other sample was run on a multi-parameter automated hematology analyzer commercially available from the Albert Cell Dynamics Rubi System (Sen Lake Albert Diagnostics, Illinois, USA). A comparison of the results obtained using the two methods is shown in Figure 12.

As shown in Figures 12A-12C, as shown in the analysis by this method, the number of white blood cells (WBC, Figure 12A), red blood cells (RBC, Figure 12B) and platelets (Figure 12C) measured by this method is the same as in the same sample The number of WBC, RBC, and platelet measured by other methods in the corresponding liquid fraction of the chromosome has a good correlation. As shown in Figures 12D-12F, the numbers of neutrophils, monocytes, and lymphocytes measured by either method are very similar and correlate well with each other.

In the multiple aspects of the term as used herein, "cytometry" refers to the observation, analysis, methods, and results of cells in a biological sample, where the cell line is essentially parked in the fluid or on the substrate. By cytometry The cells to be measured and analyzed can be detected and measured by any optical, electrical or acoustic detector. Cytometry may include preparation and analysis of cellular images in or derived from biological samples (e.g., two-dimensional images). Cells can be labeled (e.g., using fluorescence, chemiluminescence, enzymes, or other tags) and plated (e.g. permitted to settle on a substrate) and are typically imaged by a camera. Microscopes can be used for cell imaging in cytometry; for example, cells can be imaged by cameras and microscopes, such as cameras that use microscopes for imaging. An image used by cytometry and cytometry typically includes more than one cell.

Optical system

One embodiment of an optical system suitable for use herein will now be described with reference to FIGS. 6A and 6B. Although these embodiments of the system are described in terms of being able to perform cytometry, it must be understood that the embodiments of the system can also have uses and capabilities beyond cytometry. By way of example, but not limitation, the imaging and image processing capabilities of the system disclosed herein can be used in many applications, including applications other than cytometry. Because the image of the sample under analysis is taken, the image information in the system is typically linked or correlated to a quantitative measure, and the image linked to the quantitative information can be further analyzed to collect the clinical information in the image (otherwise it will not be reported).

The sample to be analyzed, for example, by cytometry or other optical or imaging means, can be placed on a sample container for analysis. For example, a light test tube can be used as such a sample container. The embodiment shown in FIG. 6A shows a perspective view of a light test tube 600 having a plurality of openings 602 for receiving samples or parts thereof for analysis. The horizontal cross-sectional shape of the embodiment of FIG. 6A is a rectangular horizontal cross-sectional shape. Although the system is described in terms of the context of the light test tube, it is necessary to understand other samples The holding device can also be used to replace or combine the light test tube 600.

As can be seen from the embodiment of FIG. 6A, the opening 602 allows a sample handling system (not shown in the figure) or other delivery systems to deposit samples into the opening 602. The opening 602 can be connected and lead to an analysis area 608 in the light test tube. Samples can be analyzed at the place. In a non-limiting embodiment, an analysis area 608 can be a chamber. In another non-limiting embodiment, an analysis area 608 may be a channel. In the embodiment, an analysis area 608 configured as a channel can be connected to two entry ports 602. In yet another non-limiting embodiment, an analysis area 608 can be a channel in which the sample is contained in a non-flowing manner. In any of the embodiments herein, the system can hold the sample in a non-flowing manner during the analysis. Optionally, some alternative embodiments may be configured to permit the sample to flow through the analysis area before, during, or after analysis. In some embodiments, after analysis, the sample is extracted from the optical test tube 600 and then delivered to another station (in a system with multiple stations) for further processing and/or for further processing and/or analysis. Some embodiments may use gates in the system to control the sample flow.

FIG. 6A shows that in some embodiments of the optical cuvette 600, an optical cuvette 600 may have a plurality of openings 602. The sample can be added to the sample container through the access port 602. An opening 602 is operatively connected (for example, fluidly continuous) to an analysis area 608. An analysis area 608 is operatively connected (for example, in fluid continuous) to a plurality of openings 602. It should be understood that several embodiments may have more or fewer openings 602 in the optical cuvette 600. Some embodiments may link certain openings 602 so that selected pairs or other sets of openings 602 can access the same channel (for example, an analysis area 608 assembled as a channel). As a non-limiting example, there may be an opening 602 at each end of the analysis area 608. Optionally, in the analysis area 608 There can be more than one opening 602 at one end of the.

The embodiment of the optical test tube 600 may have a structure 610 that allows the same treatment system to engage and transport the optical test tube 600. An optical cuvette 600 as illustrated in FIGS. 6A and 6B can be joined through an element 610 by the same treatment system, and the optical cuvette 600 can be efficiently transferred from one location to another. For example, before or after the optical cuvette 600 is transferred to a position (such as above the detector for optical imaging and analysis), an element 610 can also be used to fix an optical cuvette 600 at a desired position, and the optical cuvette 600 can be fixedly positioned by the element 610 Or by using a component 610 to fix and position a tool or device for fixing the optical test tube 60 in position. In a non-limiting embodiment, the structure 610 may have openings in the light test tube 600, and the openings allow a dropper or other elongated member to be joined to the light test tube 600 and transferred to a desired location. Optionally, instead of or in combination with the opening(s), the structure 610 can or can include a protrusion, hook, magnet, magnetizable element, metal element, and/or other features that can be used to engage an optical test tube transfer device . In an embodiment, a force (such as compressive force or other force) may be applied to an optical test tube 600; for example, a compressive force may be applied to an optical test tube 600 to press the optical test tube 600 onto a substrate or surface (for example, The surface of the base support 620), effectively configuring the optical test tube 600 to make effective optical contact with the surface. In embodiments, such force (such as compressive force) can assist in providing desired optical properties, such as providing good contact between the light test tube 600 and the base support 620, effectively allowing light to pass through without significant distortion at the interface, or without Significant reflection at the interface, or other desired optical properties. In an embodiment, such a force (such as a compressive force) can be applied at least partially through a structure 610 or a plurality of structures 610.

As shown in Figure 6B (perspective view), the light test tube 600 may have a round water Flat cross-sectional shape. An opening 602 (or multiple openings 602, which may exist in similar embodiments but not shown in the figure) may allow the sample handling system or other delivery system to deposit the sample into the opening 602, and then lead it to the analysis area 608 in the light test tube. Samples can be analyzed here. Non-limiting examples of suitable analysis areas 608 include an analysis area 608 with a chamber and an analysis area with a channel. In an embodiment, such analysis area 608 may be located inside a ring structure, such as the ring structure shown in FIG. 6B. In an embodiment, an opening 602 can be connected to an analysis area 608. In an embodiment, an analysis area 608 inside a structure 604 can form a continuous annular chamber, and an opening 608 is connected to effectively permit the flow inside the chamber to flow in either direction away from the opening 602. In an embodiment, an analysis area 608 inside a structure 604 can form an annular channel or chamber, one end is connected to an opening 608, and the other end is separated from the opening 602 or blocks the opening 602, effectively permitting the flow inside the chamber It flows only in a single direction away from the opening 602. In an embodiment, such a one-way annular channel or chamber may have a vent or other opening at a position selected from the opening 602. In yet another non-limiting embodiment, the analysis zone can be or can include a channel, wherein the sample is contained in a non-flowing manner; the sample can be contained in a non-flowing manner in an analysis zone 608 that includes an annular channel, The annular channel can be connected to an opening 602 from two directions, or the annular channel can be connected to an opening 602 from a single direction only. In any of the embodiments herein, during the analysis, the system can hold the sample in a non-flowing manner. Optionally, these alternative embodiments may be configured to permit the sample to flow through the analysis area before, during, or after analysis. In some embodiments, after analysis, the sample is extracted from the optical test tube 600 and then delivered to another station (in a system with multiple stations) for further processing and/or for feeding One-step processing and/or analysis. Some embodiments may use gates in the system to control the sample flow.

FIG. 6B shows that there is only a single ring structure 604; however, it should be understood that in another embodiment of an optical test tube 600 formed as illustrated in FIG. 6B, an optical test tube 600 may have a plurality of ring structures 604. For example, an optical test tube 600 having a plurality of ring structures 604 may have concentric ring structures 604 of different sizes, and an outer ring structure 604 surrounds one or more inner ring structures 604. Such a ring structure 604 may include an analysis area 608 inside each ring structure 604. FIG. 6B only shows a single opening 602; but it should be understood that in another embodiment of the optical test tube 600 illustrated in FIG. 6B, an optical test tube 600 may have a plurality of openings 602. For example, an optical test tube 600 having a plurality of ring structures 604 (for example, a plurality of concentric ring structures 604) may have a plurality of openings 602 (for example, each ring structure 604 may have at least one opening 602). It should be understood that some embodiments may have more or fewer openings 602 in an optical cuvette 600. Some embodiments may link certain openings 602 so that a selected pair of openings 602 or other sets can access the same channel or chamber. As a non-limiting example, there may be an opening 602 at each end of an analysis area. Optionally, more than one opening 602 can be at one end of an analysis zone.

Several embodiments of the optical cuvette as illustrated in FIGS. 6A and 6B can provide a structure 604 above a selected area of an optical cuvette 600. In one embodiment, the structure 604 is a reinforcing rib, and the optical cuvette area (for example, the area 613) that is selected to have a controlled thickness is provided with structural support for the optical cuvette area. For example, the thickness can be selected to provide desired optical properties, including a desired path for light to follow before and after reflection inside the light cuvette 600. This kind of reflection can Partial internal reflection (PIR) or total internal reflection (TIR). Whether or not such reflection occurs depends on many factors, including the wavelength of the light; the angle of incidence of the light reaching a surface; the composition of the material (zone 613 and the environment or material outside the boundary of the zone 613); and other factors. In the embodiment shown in FIG. 6A, the structure 604 is rectangular in shape and has a rectangular cross section. In the embodiment shown in FIG. 6B, the shape of the structure 604 is a ring shape with a rectangular cross-section, or a trapezoidal cross-section, or a cross-section of other shapes. These structures can have any convenient cross-sectional shape. As illustrated in FIG. 8B, such a structure 604 may have a triangular cross-section (for example, a saw-tooth-shaped cross-section is formed when there are multiple reinforcing ribs). It should be understood that these structures 604 may also have other shapes and cross-sections (such as semicircular, elliptical, irregular, or other shapes), and in an embodiment, there may be more than one shape in the same system (such as An optical test tube may include a rectangular, triangular, or other shape structure). The structure 604 can be used when the thickness of the control thickness area 613 is relatively thinner than the thickness of certain areas of the optical test tube, and thus the mechanical support effect provided by the structure 604 is obtained.

In addition to providing structural support, it is useful for the structure 604 to provide materials and paths for internal reflection of the light inside the light test tube 600. As shown in FIGS. 8A-8D, the light reflected inside a light test tube 600 may include a structure 604 (such as a reinforcing rib or a structure having a triangular cross-section, as shown in the figure, or any other shape, such as a circle or a semicircle). Cross-section or other cross-sectional shape) inside the reflected light path. Such a structure 604 can provide convex features extending outward from the surface 614 of the optical test tube 600; or can provide concave features extending inward from the surface 614 of the optical test tube 600; or can provide a concave surface on a surface 614 of the optical test tube 600 and Convex two features. In this way, the structure 604 can provide a mechanical The support can provide the optical cuvette 600 with desired optical properties and the light path, and as disclosed herein can provide other desired and useful features and capabilities to an optical cuvette 600.

In this way, the support structure 604 can be useful for providing structural support including, for example, stiffening to a light test tube 600. The optical properties of an optical test tube 600 are very important for the optical imaging and other optical measurements of samples in an analysis area 608 and the cells, particles, and other components of these samples. The maintenance of the proper flatness of the surface of the optical test tube 600 includes the maintenance of the flatness of the base portion 606 of a surface 614 or 618; the maintenance of the proper directionality and configuration of the optical test tube 600 (for example, no torsion, bending, or other distortion); The maintenance of the proper positioning of the optical cuvette 600 (for example, on the base support 620, or inside the optical assembly) may be very important for the integrity of the optical measurement values and images obtained using the optical cuvette 600. Thus, for example, the design and composition of the supporting structure 604 and the base portion 606 may be important factors for providing and maintaining the proper optical properties of the optical cuvette 600. Maintaining the proper dimensions of an analysis area 608, including maintaining proper distances and relative angles between the upper and lower surfaces (or side walls) of the analysis area 608, is very important for providing accurate and consistent illumination of samples inside an analysis area 608. Maintaining the proper dimensions of an analysis area 608 may also be important to ensure that the volume of an analysis area 608 and thus the sample volume inside the analysis area 608 is correct. As discussed herein, a force (eg, compression) can be applied to an optical test tube 600 to further ensure proper flatness, or to reduce twisting or distortion, or otherwise ensure the proper shape, size, and directionality of an optical test tube during use. It must be understood that compression may not be necessary to ensure such proper flatness and proper shape, size, and directionality of a light test tube during use. For example, in reality In an embodiment, the separate structure 604 may be sufficient to assist or ensure that an optical test tube 600 has proper flatness and proper shape, size, and directionality during use. In addition, it should be understood that in the embodiment, compression alone may be sufficient to ensure such proper flatness and proper shape, size, and directionality of an optical test tube 600 during use. It should be understood that in the embodiment, the combination of structure 604 and compression may assist and ensure such proper flatness and proper shape, size, and directionality of an optical test tube during use.

An optical test tube 600 including a supporting structure 606 and a cover 612 can be made of any material with suitable optical properties. In an embodiment, an optical test tube 600 includes a supporting structure 606 and a cover 612 can be made of glass (for example, quartz, or borosilicate glass, or aluminosilicate glass, or soda silicate glass, or other glass) become. In an embodiment, a cover 612 or a base bracket 620 can be made of acrylic or clarified polymers (such as cycloolefin, polycarbonate, polystyrene, polyethylene, polyurethane, polyvinyl chloride, or Other polymers or copolymers), or other transparent materials. In addition to the optical properties of these materials, physical properties (such as hardness, stiffness, melting point, cutting ability, and other properties), compatibility with other materials, cost, and other factors may affect the production of optical test tube 600 Choice of materials. As discussed above, the existence of the structure 604, the utilization rate of compression (for example, can be applied through the structure 610, or directly applied to at least a part of the support structure 606 and the cover 612), and other factors may permit less rigidity than quartz (for example) The use of materials can still provide the required optical and mechanical properties for the systems and methods disclosed herein. In addition, the existence of structure 604, the utilization of compression, and other factors may permit the use otherwise there is no such structure, compression and other factors. Under the circumstances, it is impossible to use the manufacturing technology and tolerances (for example, due to the possibility of distortion or other factors). In addition, the existence of the structure 604, the utilization rate of compression, and other factors may permit the use of materials that are less expensive than those used in the absence of such structure, compression, and other factors, including cheaper materials.

In this way, the proper design, composition and materials of the supporting structure 604 and the base portion 606 are very important for the optical test tube 600 and its use.

In some embodiments, these control thickness regions 613 (for example, refer to FIGS. 8A, 8B, and 8D) are selected to be placed above the analysis region 608. In some embodiments, these controlled thickness regions 613 can impart certain optical properties above or near the analysis region. Several embodiments can be configured with these structures 604 to also impart certain optical properties to the light passing through the light cuvette 600. Optionally, in some embodiments, the structure 604 can be configured to have no effect on the optical properties of the optical cuvette 600. In this embodiment, the structure 604 can be configured to have one or more light absorbing surfaces. By way of example but not limitation, certain surfaces may be black. Optionally, some embodiments may have structures 604 made from light-absorbing materials. Optionally, the structure 604 can be configured to provide mechanical support, but does not interact with the optical properties of the optical cuvette 600 near the analysis zone.

For example, a surface 614 of a certain surface including the thickness control region 613 and a surface 618 of the structure 604 may be coated with a black or other color coating. Such coatings may include one layer, and may include multiple layers. For example, a suitable coating for surface 614 or 618 may include 2, 3, 4, 5, 6, 7, or more layers. In an embodiment, for example, the surface of the structure 604 (such as the surface 618) or the surface 614 may be covered with 3 or 5 layers of coating. Such coatings may include dyes, inks, paints, surface treatments, color bands, or other coatings or surface treatments. Yu Example Medium, black or other color markers (such as Paper Mate® or Sharpie® or Magic Marker® or other markers) can be used to coat one surface 614 or One surface 618 of structure 604. For example, the super large black marker can be used to apply multiple layers of black ink coatings to the surface 614 or to a surface 618 of the structure 604 to provide a light absorbing surface, and thereby improve the optical quality of the light test tube 600. In an embodiment, the surface 614 or 618 may be coated or treated to affect or reduce the reflectance of the surface (regardless of PIR or TIR). The reduction in surface reflectance may affect (or reduce) the background illumination from a surface.

In an embodiment, a surface 614 of a certain surface including the thickness control region 613 and a surface 618 of the structure 604 may be coated or covered with a material to improve the surface reflectance. The reflectance of the surface can be increased by, for example, coating the surface or attaching materials to a surface; suitable materials to increase the reflectance include aluminum, silver, gold, and dielectric materials (such as magnesium fluoride, calcium fluoride, or other materials). Salt or metal oxide; or other reflective materials or other dielectric materials). Such a coating or cap layer may include one layer or multiple layers. For example, a suitable coating or capping layer for surface 614 or surface 618 may include 2, 3, 4, 5, 6, 7, or more layers. The increase in surface reflectance may affect (e.g. increase) the transmitted illumination from the surface. The increase in surface reflectance may assist or enhance the imaging of samples inside an analysis area 608, or may assist or enhance the optical analysis of samples inside an analysis area 608.

It should be understood that the optical cuvette 600 is typically made from an optically transparent or optically transmissive material. Optionally, only a selected part of the optical cuvette 600 (for example, the analysis area or the area connected to the analysis area) is optically transparent or optically transmissive. select Alternatively, the selected layers or regions in the optical test tube 600 can also be configured to be non-optically transmissive. A part or a region of the light test tube can be covered or coated, so that it is light-absorbing; for example, a surface (or part thereof) can be coated with a dark or light-absorbing dye or ink. In another embodiment, a surface (or part thereof) may be covered with a dark or light-absorbing coating, such as a dark or light-absorbing material, such as tape, or tape, or paper, or rubber, or plastic.

6A, 6B, and 8A-8D illustrate an embodiment in which the optical test tube 600 is parked on a base support 620, and some or all of the base support 620 is made of optically transparent or transmissive materials. In some embodiments, the optically transparent or transmissive part is assembled to align with the analysis area of the optical cuvette 600 to permit the optical query of the sample in the analysis area. In a non-limiting embodiment, the base support 620 can move in the X, Y, and/or Z axis to move the optical cuvette 600 to a desired imaging position. In some embodiments, the base support 620 includes a platform or a table that moves only on two of the axes. Optionally, some support structures can only move in a single axis. The optical test tube 600 may be configured to be operatively coupled to the support structure 600 through friction, mechanical coupling, or through fasteners installed in one or both of these components. In an embodiment, a compressive force or other force may be applied to an optical test tube 600 or a base support 620 or both to ensure proper contact and proper matching between an optical test tube 600 and a base support 620. In an embodiment, such compression can help ensure that the optically transmissive surface of a light test tube 600 or a base support 620, or both, is optically flat and substantially free of distortion. For example, in the embodiment, an optical test tube 600 can press a base support 620 to reduce or eliminate any possible optical distortion that may be caused by defects or abnormalities in the optical surface of the optical test tube 600. To implement In an example, such force (such as compression) can assist in providing the desired optical properties, effectively permitting distorted light to pass through the interface instead of generating distorted light. In an embodiment, such a force (such as compression) can be applied at least partially through a structure 610 or through multiple structures 610.

Figures 6A, 6B, 8A, 8B, 8C, and 8D further show embodiments in which an illumination source 650 (such as but not limited to the ring light shown in the figure) placed under the base bracket 620 can be used to position the light emitting device in the light Below the height of the test tube 600, the light for dark field and/or bright field observation is provided. In this configuration, the upper area of the light test tube 600 that can be used by the burette, the sample processing system, or other equipment has an opening or other features on the top surface of the light test tube 600 that is not covered. Optionally, some embodiments can position an illumination source 660 (shown in dashed lines in the figure) above the light test tube 600 to replace a single or multiple illumination source with the bottom side light (for example, the bottom side illumination source 650 as shown in the figure). combination. An objective lens 670 can be positioned as shown in the figure or in other configurations to observe the luminescent sample. It should be understood that the relative movement between the optical cuvette 600 and the optical parts 650 and 670 can be used to allow the system to visualize different analysis areas in the optical cuvette 600. Optionally, only one of these components is configured to be movable to query different analysis areas in the optical test tube 600.

Referring now to FIG. 7A, one embodiment of a convenient imaging system will be described with further cells. FIG. 7A shows a schematic cross-sectional view of various components located under the base bracket 620. The cross-section is along arrow 7 in Fig. 6A to indicate this area.

FIG. 7A shows an embodiment in which the optical test tube 600 includes a base portion 606 and an analysis area 608 defined by a cover portion 612. Optionally, the analysis area 608 may be defined inside a single piece. Optionally, the analysis area 608 can be defined by more than two pieces, such as, but not limited to, a separate area for the analysis area 608. Cover pieces. In one embodiment, the layer 606 includes an optically transparent plastic, such as, but not limited to, a cycloolefin polymer thermoplastic polymer that delivers excellent optical components and applications. Some embodiments may be made of one or more layers or components from glass, acrylic, transparent polymers, or other transparent materials. The optical test tube 600 illustrated in FIG. 7A includes five separate analysis areas 608; these areas are shown in the drawings in cross-sectional views; the analysis areas 608 with such cross-sectional views can be rectangular, square, or other shapes. For example, the analysis area 608 may include a long channel, providing a shallow chamber with a relatively large surface area, whereby the sample can be observed. It should be understood that an optical test tube 600 may include a single analysis area 608; or may include two analysis areas 608; or may include three analysis areas 608; or may include four analysis areas 608; or may include five (as shown in Figure 7A). Show) or more analysis areas 608.

In this non-limiting embodiment, the sample to be queried can be completely or partially enclosed in the analysis area 608. As a non-limiting example, the optical element under the base bracket 620 may include a ring light 650 including a toroidal reflector 652 and a light source 654. Other lighting components suitable for dark field lighting can be used; in this way, the optical element can include other lighting sources, alone or in combination with such ring lights. Several embodiments may use a mirror. Several embodiments may use a coated reflective surface. Some embodiments may use reflectors different from those shown in the figure (for example, toroidal reflection may not be used when lighting the sample). Several embodiments may use parabolic reflectors. Several embodiments may use a parabolic reflector in the shape of an elliptical paraboloid. Several embodiments may use a plurality of individual reflector blocks. Some embodiments may not use any reflectors. Some embodiments can obtain oblique illumination through the use of a curved light source that is positioned to guide the light with or without further assistance from one or more external reflectors.

The embodiment illustrated in FIG. 7A shows laser energy sources 680, 682, and 684, such as but not limited to laser diodes of specific wavelengths, installed to guide light into the sample in the analysis area 608. In a non-limiting embodiment, in order to help simplify packaging, the energy sources 680, 682, and 684 can guide light to the dichroic element 690 (such as a dichroic mirror or a beam splitter), and then guide the laser wavelength into the analysis area 608. The wavelength of the laser causes the wavelength of fluorescence to be emitted by the fluorescent group of the label, dye, and/or other material in the sample. The emitted fluorescence wavelength passes through the objective lens 670, passes through the dichroic element 690, passes through the selective filter wheel 692, and enters the detector 700, such as but not limited to a camera system. As a non-limiting example, the two-color element 690 is configured to reflect the laser wavelength, but instead passes the fluorescent wavelength and any desired wavelength for optical observation.

In one embodiment, all fluorescent laser wavelengths illuminate the sample in the analysis area 608 at the same time. For example, the detector 700 can be coupled to the programmable processor 710, which can capture signals and/or images and deconstruct the wavelengths associated with the fluorescent group that emits light. In some embodiments, the laser source may illuminate sequentially or a subset of the entire number of laser sources. Of course, it must be understood that the system is not limited to fluorescence-based lasers based on the fluorescent groups in the sample. Other detection technologies and laser technologies can be used to replace or combine alone or in multiple combinations with fluorescence. For example, several embodiments can also combine fluorescence detection simultaneously or sequentially to collect dark-field illumination scattering information.

Light scattered by an object inside the sample container (such as cells, beads, or crystals) will be scattered at multiple scattering angles, where a scattering angle can be measured from the light source to the object. Such plural scattering fish include a certain range of scattering angles. Such a sample container may have as disclosed here The characteristic parts shown, and can be assembled to provide a path for internal light reflection. An objective lens equipped with an imaging object will collect and focus the scattered light, where the light can pass to a detector. The light focused by an objective lens and focused on the detector can form a light spot on the detector. In an embodiment, the light passing from the objective lens to the detector can be focused by another lens; this focusing can reduce the size of the light spot formed on the detector. Whether the light focused on the detector passes through another lens or not, it will include objects from the inside of the sample container that scatter light at multiple scattering angles.

The applicant hereby discloses a comparison of other methods, systems, and devices (such as sample containers) that may permit the detection of a smaller range of scattering angles, thereby proposing higher resolution and better imaging of samples and objects inside the samples. The applicant here discloses a characteristic part of the design of the optical test tube, which can be used to control the angle and intensity of the light incident on the sample through PIR and TIR, and effectively control the amount of scattered light.

Due to the limitations imposed by the non-imaging optical elements of many systems (such as the amount of optical expansion, or the degree of light expansion through the system), the scattering angle of the light reaching the detector can be wider than desired. For example, in some ring light-light tube combinations that use LEDs as the light source, the light hitting the sample can be spread to at least 20 degrees around the main angle. In other words, if the chief ray hits the sample at 60 degrees, the other rays of the light beam can hit the sample with a scattering angle of about 50 degrees to about 70 degrees. It should be understood that the spread of the scattering pyramid of the light collected by the objective lens depends on the numerical aperture of the lens. In this case, the light collected by the objective lens (for example, having a numerical aperture of 70 degrees) will have a cone shape of about 30 to 70 degrees. As a result, light scattered through a wide range of scattering angles will reach the detector; for example, such a system Measure all the light scattered by the sample within a large cone angle of about 60°±40°. However, as disclosed herein, certain applications require the detection of scattered light in a narrow range, such as light in a very narrow angle range (for example, 60±5 degrees). The applicant hereby discloses that in order to provide a light measure from this narrow range, an aperture can be placed on the Fourier plane (or back focal plane) of the objective lens (or any position conjugated to this plane). On the Futura plane, the angle information is spatially coded. Therefore, depending on the shape and size of the aperture, light from a specific angle from the sample can be prevented from reaching the detector (for example, being blocked or filtered out). The ring-shaped pores will block or filter out internal angles (e.g. 60±30 degrees). Therefore, the obtained metric can be adjusted to the desired angle.

In an embodiment, an aperture may be provided through which the light from the objective lens passes before contacting the detector. In an embodiment, an aperture may be provided through which light from another lens (after passing through the objective lens) passes through the aperture before contacting the detector. When the pore system is configured to pass through the detector, compared to the light passing through the detector when there is no such pore, the light passing through the detector will be reduced to light from a smaller scattering angle, and reduced to light from a smaller scattering angle. Range of scattering angle of light. In an embodiment, such pores may include single pores, such as round pores. In an embodiment, such an aperture may include a single ring, such as a circular ring through which light can pass, and a central area (such as a circular area) through which light does not pass. In an embodiment, such pores may include 2 or 3 or more concentric rings through which light can pass, and include a central area (such as a circular area) through which light does not pass. In an embodiment, the aperture may include a circular or annular shape.

The aperture arranged between an objective lens and a detector, such as another lens and a detector (where the light passes through an objective lens before passing through the other lens) provides a clearer discrimination of light scattered from the sample The advantages, change Enter the resolution of light scattering images (such as dark field images) obtained from the sample. In the embodiment, the light intensity may be a factor, and the configuration of the pores as disclosed here is relatively lacking. The configuration of the pores as disclosed herein can increase the applied light (for example, from a light source or from multiple sources). Light source) intensity.

A system may include a sample container with one of the features as discussed and described herein, as well as a light source, dichroic mirror, and other components as shown in FIG. 7A. As shown in the example of FIG. 7B, a system with similar characteristics (such as shown in FIG. 7A and other figures herein) may include a sample container 600, a light source 650 (such as a light source 654, or a laser source 680, or both ), an objective lens 670, an aperture 694, another lens 696, and a Fourier lens 698. The aperture 694 may have a single channel to allow light to pass to a detector 700. The detector 700 is operatively linked to a processor 710 (for example, a programmable processor). In an embodiment, an aperture 694 includes two channels to allow light to pass through the detector 700. In an embodiment, an aperture 694 includes three channels to allow light to pass through the detector 700. In an embodiment, an aperture 694 includes four or more channels to allow light to pass through the detector 700. In an embodiment, a channel in an aperture 694 may include a circular hole to allow light to pass through the detector 700. In an embodiment, a channel in an aperture 694 may include 2, or 3, or 4 or more circular holes to allow light to pass through the detector 700. In an embodiment, a channel in an aperture 694 may include a ring configured to allow light to pass through the detector 700, and may include a middle part which does not allow light to pass through the detector 700. In an embodiment, a channel in an aperture 694 may include 2 or more rings (for example, concentric rings in the embodiment), each of which is configured to allow light to pass through the detector 700, and such aperture 694 may Including a middle part which does not allow light to pass through the detector 700. Such rings and these rings can be It has a round, oval, or other ring shape.

Accordingly, the applicant disclosed a system for imaging a sample, including: a sample container; a light source used to illuminate an object held in the sample container; an objective lens which is assembled to collect and focus from the sample container Light scattered by an object inside the sample container, wherein the scattered light includes light scattered at a plurality of scattering angles; an optical aperture for passing the light from the objective lens; and is configured to combine the light from the objective lens Another lens focused on the optical aperture, wherein the optical aperture is configured to allow only a part of the light focused by the objective lens to pass through the aperture, thereby allowing the part of the light passing through the aperture to be caused only by the plural The composition of light scattered at one of the scattering angles.

As used herein, "surface" and "surface illumination" refer to illuminating a sample by light traveling in a direction that is substantially away from an objective lens or other optical element used to observe or image the sample. In this way, in the absence of fluorescence, the illuminated sample image formed by surface illumination is formed by light reflected or scattered from the sample (the light travels from the light source to the sample, and the sample is reflected or scattered back to the optical element for observation , Imaging, or measurement). As used herein, "penetration" and "penetration illumination" refer to illuminating a sample by light traveling in a direction that is generally toward an objective lens or other optical element used to observe or image the sample (light from The light source travels to and through the sample, and continues to the optical element for observation, imaging, or measurement). In this way, in the absence of fluorescence, the illuminated sample image formed by the transparency is formed by the light passing through or scattered from the sample.

When a light source is set on the same side as the objective lens or other optical element used to observe or image the sample, the light from the light source Travel directly to the sample, so the sample is typically observed or imaged by surface illumination. But even when the only light source is set on the same side of the sample as the objective lens or optical element, in addition to surface illumination, the sample container as disclosed herein can provide penetrating illumination of the sample. In this way, two illumination directions are permitted and the light source is not required to be placed on both sides of the same book. This configuration is simple and resource-saving, and because the light source and other optical components are only arranged on one side of the sample container, the configuration permits access to the side of the sample container without interference from optical components. As such, this configuration provides the advantages of permitting loading, mixing, and removal of samples and reagents from the sample container without interfering with optical imaging or measurement, or with equipment and components used for optical imaging or measurement.

Examples of the images shown in Figures 4A and 4B illustrate that adding penetrating illumination to the dark field image greatly enhances the image and greatly enhances the information obtained from the image. The method and system disclosed herein provide such a greatly enhanced image by using illumination from a single direction, and in the embodiment, illumination from only a single light source, by combining both surface illumination and penetrating illumination.

As disclosed here, a container such as a light test tube 600 (for example, as illustrated in Figures 8A-8D) is configured to allow internal reflection of light from a light source (including PIR or TIR), so the analysis of the light test tube 600 A sample container in zone 608 is illuminated by direct light (surface illumination; for example, light traveling along path 830), and also illuminated by indirect reflected light (permeable lighting; for example, light traveling along path 820 or 825). As disclosed herein, light from a light source disposed on the same side of the optical elements 670, 690, 700 of an optical cuvette 600 can provide both surface illumination and penetrating illumination of the sample.

Referring now to FIGS. 8A-8D, yet another embodiment will now be described. 8A-8D show a schematic cross-sectional view of a part of a light test tube 600 and a dark-field scattering illumination source, such as but not limited to a part of the ring light 650 shown in FIGS. 6A and 6B. The base bracket 620 is also shown in Figures 8A-8D. FIGS. 8A-8D include brackets and arrows to indicate structures or partial structures; for example, the brackets labeled 600 indicate the entire optical test tube 600 shown in the figure; the brackets labeled 612 indicate the cover portion 612 of the optical test tube 600. Arrows 621 to 626 in FIG. 8A indicate the dimensions of the indicated portion of the cover 612. It should be understood that these dimensions may be different in different embodiments of an optical test tube 600, and these variations may depend on the size, application, material, light wavelength, sample, and other related to the composition and use of an optical test tube 600 Components and factors. For example, in an embodiment, the spacing 621 of the support structure 604 may be about 0.1 millimeter (mm) to about 1 centimeter (cm), and in an embodiment may be about 1 mm to about 100 mm, or about 1.5 mm to about 50 mm , About 2mm to about 20mm. In a further embodiment, the spacing 621 of the supporting structure 604 may be about 0.5 mm to about 10 mm, or about 1 mm to about 5 mm. In an embodiment, the height 622 of the supporting structure 604 may be about 0.1 mm to about 100 mm, or about 0.5 mm to about 50 mm, or about 1 mm to about 25 mm. In a further embodiment, the height 622 of the support structure 604 may be about 0.1 mm to about 10 mm, or about 1 mm to about 5 mm. Similarly, in an embodiment, the height 623 of the controlled thickness region 613 may be about 0.1 mm to about 100 mm, or about 0.5 mm to about 50 mm, or about 1 mm to about 25 mm. In a further embodiment, the height 623 of the controlled thickness region 613 may be about 0.1 mm to about 10 mm, or about 1 mm to about 5 mm. In an embodiment, the thickness 624 of the layer 800 may be about 0.01 mm to about 10 mm, or about 0.05 mm to about 1 mm, or about 0.1 mm to about 0.5 mm. In an embodiment, the width 625 of the analysis area 608 may be about 0.05 mm to about 100 mm, or about 0.5 mm to about 50 mm, or about 1 mm to about 25 mm. In a further embodiment, the width 625 of the analysis area 608 may be about 0.1 mm to about 10 mm, or about 1 mm to about 5 mm. In an embodiment, the width 626 of the support structure 604 may be about 0.1 mm to about 100 mm, or about 0.5 mm to about 50 mm, or about 1 mm to about 25 mm. In a further embodiment, the width 626 of the support structure 604 may be about 0.05 mm to about 10 mm, or about 0.5 mm to about 5 mm.

It should be understood that as illustrated in any of the figures here, the optical components and configurations used for lighting, lasers, observation light emission, etc. can prompt components and configurations that can be applied to embodiments of other figures, even if these specific components or configurations are not This is also true if it is not explicitly shown in the figures. For example, although the ring light 650 or other illuminating source 650 is not included in FIG. 8D, in any one of the embodiments shown and in other embodiments, the ring light 650 or other illuminating source 650 (for example, refer to FIG. 8A, 8B, and 8C) can be used to illuminate the analysis area 608 (the analysis area 608 is shown in FIGS. 8A and 8B). As an example of an optical assembly suitable for an optical test tube 600, the ring light assemblies 652 and 654 are shown in FIGS. 8A, 8B, and 8D; other or other numbers of lighting assemblies may be used in the example. For example, the light source 654 may be white light or a light source, such as, but not limited to, a light emitting diode (LED) or a laser diode with a specific wavelength or wavelength output. Optionally, the ring of the light source 654 may be a fiber optic cable assembled to provide a light ring (for example, there are many splices). Alternatively, the light source 654 may be an LED with a specific narrow divergence angle controlled by a reflector. It may be desirable to control the divergence angle from the ring light 650 through the selection of the light source and/or the design of the reflector.

As a non-limiting example, a light source 654 may use laser light In order to provide a narrow light pattern, it results in a lower penetrating illumination background in the current surface-style lighting configuration (where the light-emitting components are all on one side of the sample), because the light source: provides a narrow light spot (Guide to the inside of the sample analysis area 608); provide light with a narrow bandwidth (for example, light within a narrow range of a specific dominant wavelength); and be a cohesive light source. Optionally, using LEDs as the illumination source 654 can also provide a small light spot (for example, a small spot size inside the analysis area 608), and provide several advantageous properties achieved by a laser light source. For this reason and other reasons, laser light sources (or LEDs that provide small light spots) can effectively reduce the background signal level compared to other lighting configurations. Compared to the typical use of more diffuse light sources, laser illumination can reduce scattered light, thus reducing light scattering from an adjacent channel (for example, from the adjacent second analysis area 608) into a channel (for example, in the first analysis area) 608 inside), can reduce the background of the channel. In this way, laser lighting can result in less penetrating and illuminating the background compared to illuminating the prospective with a more diffuse light source. Of course it is expected that the reduction in penetrating illumination is less than the reduction in the background, where a more significant reduction in the background results in a more distinguishable signal. Optionally, LEDs are used as the illumination source 654 to provide a diffuse light pattern with increased background and increased penetrating illumination. Of course, it is expected that the increase in penetrating illumination is greater than the increase in background.

Several optical test tube embodiments may include a light test tube formed from a plurality of individual layers bonded together, a light test tube molded from one or more materials, and/or a reflective layer added to the light test tube on different surfaces to enhance Single or multiple internal reflections (e.g. enhanced TIR or PIR).

In the embodiment, the system, optical cuvette, and optical element disclosed herein can be combined with fluorescent operation, and it may be desirable to use these systems and optical cuvettes The dark field lighting system is not white light. However, for example, if fluorescence detection is not used in combination with dark field and bright field microscopy, some embodiments may use only white light.

FIGS. 8A and 8B show that in several embodiments, the device may have multiple layers, such as layer 800, that are typically opaque to light in an optical cuvette 600. As shown in FIGS. This may be useful in embodiments where the light source 654 is diffuse and the light is not directed to a specific location. The layer 800 can block light entering the light cuvette 600 at an unexpected angle and/or position. The layer 800 can be configured to prevent illumination, except through the area below the analysis area 608. In some cases, only a specific area may be sealed off at the point closest to the analysis area 608. Several embodiments may have blocking materials or non-transmissive materials in more than one layer. Some may have blocking materials or non-transmissive materials in different directions, such as but not limited to one direction being horizontal, and one direction being vertical or non-horizontal.

In the embodiment, it should be understood that the layer 800 may be light transmissive. For example, FIG. 8D shows an embodiment in which the layer 800 may be light transmissive. In some embodiments, the layer 800 may include a light-transmitting material having a refractive index different from the refractive index of the controlled thickness region 613 or the base support 620 or both. In some embodiments, the layer 800 may include a light-transmitting material having a refractive index that is the same as the refractive index of the controlled thickness region 613 or the base support 620 or both.

In FIGS. 8A, 8B, and 8C, a light source is shown as being located under a light cuvette 600 (close to the optical elements 652 and 654), and provides directing light from below the base 606. It is understood that such a light source can also be located in the embodiment illustrated in FIG. 8D. As shown in this diagram, a light source 650 may include a ring light 654 and a toroidal reflector 652. Other components include, but are not limited to, lenses, filters, gratings, mirrors, and other reflective surfaces, optical fibers, ridges, and other components. In an embodiment, a light source may include a laser, or an LED, or other light sources; and may include an optical fiber carrying light from the light source to another location, and/or guiding the light toward an optical element. A design standard of the optical system is the divergence, or divergence angle, of the light from the light source; a light beam with a high divergence width D, a light beam with a low divergence width D provides a beam at a given distance from the light source smaller. Generally speaking, it is better to provide a light source 650 with low divergence. These optical elements and configurations can be designed to provide substantially collimated light, for example, most or all of the light is directed along a substantially parallel path toward the sample (e.g., toward an analysis area 608). However, in an embodiment in which diffused light or scattered light is preferable, a light source 650 with high divergence can be used.

As shown in FIG. 8C, an embodiment of an optical system suitable for use as one of the device or system components disclosed herein may include optical elements (such as a light source 650, such as a ring light 654 shown in FIG. 8C, and an objective lens 670), and a light test tube 600 , And a base frame 620 are assembled to hold and position an optical test tube for imaging. In the embodiment shown in FIG. 8C, a base support 620 may include an optical feature 802 configured to refract (or diffract, or otherwise change the light path) light from the light source 650. As shown in the example of FIG. 8C, the optical characteristic element 802 may include an array of small lenses. It should be understood that the optical characteristic member 802 may include any suitable optical characteristic member. In an embodiment, the optical characteristic element 802 may include a small lens, or a diffraction grating, or a Fezny lens, or a convex mirror, or a concave mirror, or other shapes and characteristics that can refract, diffract, or otherwise change the light. , Or a combination thereof. In an embodiment, the optical characteristic member 802 may include a different material from the base bracket 620, and may have a different refractive index from the base bracket 620. For example, the light received by the image of the optical feature 802 can be directly or through Reflections (such as internal reflections) applicable to the methods disclosed herein are indirectly directed toward an analysis area 608, for example, thereby providing both surface illumination and penetrating illumination of the sample in an analysis area 608. As an example of the embodiment shown in FIG. 8C, these embodiments also include an optical path that detours the optical characteristic member 802. Compared with the optical path required to pass through the optical characteristic 802 for imaging, this optical path may be more suitable for imaging a sample inside an analysis area 608. In an embodiment, two types of optical paths (ie, bypassing the optical characteristic member 802 and passing through the optical characteristic member 802) can be provided at the same time, so that a light source located on the same side of a light source 650 from a light test tube 600 is provided. The surface illumination and penetrating illumination both illuminate the same suitable optical element for image analysis.

The optical test tube 600 includes a characteristic component, which affects the optical path for illuminating the optical test tube and the sample inside the optical test tube. Such penetrating illumination is affected by the light reflected (for example, by internal reflection) inside the light test tube 600, including or mainly by, for example, partial internal reflection (PIR) from a surface 612, a surface 604, or other surfaces or combinations of surfaces. Or total internal reflection (TIR). Other examples of light paths for TIR are shown in FIGS. 8A, 8B, and 8D, for example.

As illustrated in FIG. 8D, in an embodiment, an optical test tube 600 of an optical system as disclosed herein and suitable for an apparatus or system of the method disclosed herein may include a characteristic element that affects the illuminating light test tube The light path inside 600, such as the light path of the illumination analysis area 608, and the sample inside the analysis area 608 of the light test tube 600. As shown in FIG. 8D, a layer 800 may include a characteristic member that refracts, diffracts, or otherwise affects or changes the optical path into the analysis region 608. This change in the light path may affect and possibly improve the illumination of the sample inside the analysis area 608. In the embodiment shown in FIG. 8D, the light The layer 800 is entered from the lateral direction; the optical path is changed by the shape (and material properties) of the layer 800 and is directed into the analysis area 608 as desired. For example, the outer surface of the layer 800 may be flat (e.g., outer surface 674) or may be curved (e.g., outer surface 676). For example, an inner surface of the layer 800 may be flat (not shown in FIG. 8D; but refer to these surfaces in FIGS. 8A and 8B (although 800 in FIGS. 8A and 8B is not optically transmissive, but these surfaces are shown as flat) )) or may be curved (for example, the inner surface 678 shown in FIG. 8D). In the embodiment, the change of the light path effectively provides both surface illumination and penetrating illumination of the sample in the analysis area 608.

8A, 8B, 8C, and 8D provide TIR and PIR embodiments of a cover 612 and/or surface 618 of a support structure 604. The same container, such as the light test tube 600, may have a light-transmitting surface through which light can pass; in an embodiment, such a light-transmitting surface allows light to pass through without significantly distorting or reducing light intensity. The sample container, such as the light test tube 600, can be made of a light-transmitting material, and light can effectively pass through the inside of the sample container. In the embodiment, the container here is made at least partly of light-transmitting material, and light can pass through the light-transmitting surface of the sample container and can travel inside the sample container. In an embodiment, the light traveling inside the sample container may be reflected on one or more surfaces and travel along the reflection path inside the sample container. When the light from a light source arranged outside the sample container enters the sample container through the transparent surface of the sample container, and this light can travel away from the light source inside the sample container, and can be reflected on the surface of the sample container, so after reflection, The reflected light can travel in the direction of the light source. This reflection can be PIR or TIR.

In other words, the light passing through the inside of the light test tube 600 can be reflected off a surface (for example, the surface 614 or the surface 618). This internal reflection can effectively use indirect light Illuminate a sample inside an analysis area 608; combined with direct illumination (where light is not reflected before hitting a surface), the sample can receive surface illumination (illumination from the same side of the optical detection element) and penetrating illumination in this way (Illumination from the opposite side of the optical detection element).

It must be understood that the wavelength, material, surface and configuration of the light to enhance or enhance the PIR may not be suitable or effective to enhance or enhance the TIR. It must be understood that the wavelength, material, surface and configuration of the light to enhance or enhance the TIR may not be suitable or effective to enhance or enhance the PIR. In this way, some designs and structures can be promoted in one or the other of PIR and TIR without the presence of the other. In the embodiment, some designs and configurations can be improved at both PIR and TIR. In the embodiment, there are some designs and configurations where neither PIR nor TIR can be improved.

As illustrated in the example of FIG. 8A, the supporting structure 604 may have a rectangular or square cross-section. It should be understood that the supporting structure 604 may have a cross-sectional shape other than a square or a rectangle; for example, as shown in FIG. 8B, the supporting structure 604 may have a triangular cross-sectional shape; Or irregular shape) is also applicable to the system and light test tube as disclosed herein. PIR and TIR are adjustable characteristic parts, which can be adjusted according to the material used for the optical test tube 600, any coating, cladding, or cap layer applied, and the controlled thickness zone 613 of the optical test tube 600 Choose from geometry and/or thickness. In the embodiment, PIR is preferred, and light, material, and configuration can be selected to improve PIR.

In the embodiment, TIR is preferred, and the wavelength of the light from the light source 650 can be selected to increase the TIR. In the embodiment, the optical test tube 600 can be selected Material, thickness, surface configuration, and other characteristic parts to improve TIR. For example, the height of the controlled thickness region 613 (measured from the bottom of the cover portion 612 of the contact layer 800) will affect the angle and intensity of the TIR reflected light reaching the analysis region 608. The configuration of the light test tube 600, which is one of the oblique illumination (lighting from above the sample) of the TIR-permitted sample of the light inside the light test tube, is desirable, especially for dark field microscopy. In some embodiments, it is desirable to maximize the TIR obtained from the sample above. Optionally, in some embodiments, an optical cuvette 600 can be configured to provide TIR only from the upper surface of the analysis area 608. Optionally, several embodiments may be configured to provide TIR only from the upper surface of the controlled thickness region 613 (for example, in the embodiment shown in FIGS. 8A and 8B, substantially above the analysis region 608). Optionally, in some embodiments, an optical cuvette 600 can be configured with light from other surfaces in the optical cuvette 600 to provide TIR; for example, it can provide TIR from light from other surfaces in the optical cuvette 600 In order to scatter the light at an oblique angle, the light is effectively directed back to the analysis area 608.

The design and materials used to compose a light test tube 600 can be selected and assembled to provide light TIR. For example, in some embodiments, configurations that provide TIR or provide increased or increased TIR include, but are not limited to: configurations where the size of the controlled thickness region 613 is compatible with TIR or promotes TIR ; Wherein the angle between the surface 614 or the surface 618 (for example, relative to the incident light) is compatible with TIR or promotes the configuration of TIR; where the shape, texture, or coating layer of the surface 614 or surface 618 is compatible with TIR Or promote the configuration of TIR; wherein the difference between the refractive index of the material composing the controlled thickness region 613 and the refractive index of the material or space contacting a surface 614 forming the boundary of the controlled thickness region 613 can be compatible with or promoted by TIR TIR configuration; which consists of supporting structure 604 The difference between the refractive index of the material and the refractive index of the material or space in contact with a surface 618 forming the boundary of the support structure 604 is compatible with or facilitates the configuration of TIR; and other configurations and designs. In order to provide TIR, the first material through which light will be (internally) reflected will have a higher refractive index than the second material through which light will pass if it is not internally reflected; since the second material is usually air, it has a value close to 1. Refractive index, so it is usually not difficult to ensure this point. The incident angle must be greater than the critical angle to provide TIR. For example, referring to the embodiment shown in FIG. 8, the material composing the controlled thickness region 613 and the supporting structure 604 (for example, the outer regions of the surfaces 614 and 618) must have a refractive index greater than that of air. In the embodiment where total internal reflection of the layer 800 is desired, the material of the layer 800 must have a lower refractive index than the material of the controlled thickness region 613 to ensure that TIR appears on the walls, as shown in Figures 8A, 8B, and 8D The example description. In an alternative embodiment, the material of the layer 800 may have a refractive index higher than the refractive index of the controlled thickness region 613, and TIR will be formed at the boundary (between the layer 800 and the controlled thickness region 613). The effective angle and material can be The transmitted illumination light component of the sample in the analysis area 608 is adjusted so as to be optimized.

In an embodiment, a surface 614 or 618 may be coated or treated to affect or reduce the reflectance of the surface (regardless of PIR or TIR). In an embodiment, a surface 614 or 618 may be coated or treated to reduce light leakage from the surface. For example, even if a surface 614 or 618 is compatible with TIR or provides the amount of TIR, some light can be transmitted or refracted out of the surface 614 or 618. A light-absorbing coating or material may be placed or applied to a surface 614 or 618 or a part or parts thereof to reduce the amount of stray light leaking from an optical test tube 600. Such light-absorbing coatings can be, for example, dyes, inks, paints, Surface treatment, black band or color band, or other coating or surface treatment. In an embodiment, a black or other light-absorbing solid material may be placed back against or adjacent to a surface 614 or 618 to provide an optically absorbing surface.

Optionally, in some embodiments, an optical cuvette 600 may be configured to not provide total internal reflection of light (or only provide a meaningless amount of total internal reflection) from one or more parts of the optical cuvette, or to provide Does not provide partial internal reflection of light (or only provides a meaningless amount of partial internal reflection). In some embodiments, an optical cuvette 600 can be configured to provide no light TIR or PIR from the support structure 604 (or only provide a meaningless amount of TIR or PIR). Optionally, in some embodiments, an optical cuvette 600 may be configured to provide no light TIR or PIR from a surface 618 (or only provide a meaningless amount of TIR or PIR). Configurations that do not provide light TIR or PIR, or only provide meaningless amounts of TIR or PIR include, but are not limited to: the dimension of the controlled thickness zone 613 is incompatible with TIR or PIR or will not improve TIR or PIR The configuration of one surface 614 or one surface 618 (for example, relative to the incident light) is incompatible with TIR or PIR or does not improve the configuration of TIR or PIR; one surface 614 or one surface 618 shape , Texture, or coating layer is incompatible with TIR or PIR or will not improve the configuration of TIR or PIR; wherein the refractive index and contact of the material constituting the controlled thickness region 613 form a surface at the boundary of the controlled thickness region 613 The difference between the refractive index of the material or space of 614 is incompatible with TIR or PIR or will not improve the configuration of TIR or PIR; wherein the refractive index of the material constituting the support structure 604 and contact form a surface of the boundary of the support structure 604 The difference between the refractive index of the material or space of 618 is incompatible with TIR or PIR or will not improve the configuration of TIR or PIR; and other configurations and designs.

Optionally, in some embodiments, the reflective material may be placed on or attached to a surface 614 and/or a surface 618. Such a reflective material can be, for example, a metal such as silver, or gold, or aluminum; it can be a dielectric such as magnesium fluoride or calcium fluoride, or other salts or metal oxides; or other reflective materials. Typically, such a reflective coating layer can be very thin (for example, it can be less than about 0.1 microns, or up to about 100 microns in thickness). Optionally, a reflective material (such as a reflective coating) may only be placed on or attached to the surface 614. Optionally, the reflective material may only be placed on or attached to the surface 618. Optionally, the surface 618 can be treated to be black to be light absorbing. In other embodiments, the surface 614 may be processed to be black to be light-absorbing. In some embodiments, the width of the controlled thickness area 613 may be wider than the analysis area 608. For some embodiments using laser illumination, the layer 800 can be removed or light transmissive because the laser illumination is sufficiently focused so that it does not need to be blocked between the analysis regions 608.

By way of example but not limitation, the use permission of PIR, TIR, or both comes from the adjacent area and the light traveling along the path 820 is guided into the analysis area 608. As shown in FIGS. 8A, 8B, and 8D, the light traveling along the path 820 is reflected toward the analysis area 608, and the light traveling along the path 825 undergoes multiple reflections when traveling inside the optical cuvette 600 and finally reaches the analysis area 608. As shown in the figure, the light traveling along the path 820 in FIG. 8B undergoes multiple reflections as it travels inside the optical test tube 600 and finally reaches the analysis area 608. As illustrated in the example of Figure 8B, these reflections can be PIR or TIR. In accordance with traditional terminology, the illumination by light traveling along paths 820 and 825 shown in FIG. 8A and the illumination by light traveling along path 820 shown in FIG. 8B are penetrating lighting. The illumination by light traveling along the path 830 shown in FIGS. 8A and 8B shows that the light comes directly from the ring light, instead of passing through the total internal reflection. Shooting from: This is surface lighting. The combination of two types of light components from a light source located under the sample (or only one side of the sample) may have improved performance compared to a light source that only provides one of the light components. This is particularly useful for dark field microscopy.

A non-limiting example of the application of the embodiment shown in FIGS. 8A-8D is dark field illumination to measure the scattering properties of cells in a sample. Dark field microscopy is an established method mainly used as a contrast enhancement technique. In dark field microscopy, the image background is completely dark because only the scattered light or reflected light from the sample is imaged. Quantitative dark field microscopy has never been used to measure the scattering properties of cells in a way comparable to the traditional "side scatter" parameters used in flow cytometry.

From a hardware point of view, the illumination used for dark field microscopy is expected to be oblique, that is, no light from the light source can enter the objective lens without first contacting the sample. By way of example but not limitation, the wavelength of light emission must be such that it does not excite any other fluorescent groups already present in the sample. Optionally, this type of luminescence permits the use of high numerical aperture (NA) lenses for imaging. By way of example but not limitation, NA may be at least about 0.3 for a conventional lens size coupled with an optical microscope. Optionally, NA is at least 0.4. Optionally, NA is at least 0.5. Optionally, some embodiments may utilize oil immersion objectives to obtain the desired NA, especially when the lens size is not limited to below a certain level.

Traditional dark-field illumination methods have used penetrating illumination, where the sample is placed between the imaging lens and the dark-field light source. As such, in this traditional configuration, the detection component and the illumination component are not on the same side of the sample. Traditional surface photo The bright method (where the imaging lens/objective and the light source are on the same side of the sample) requires the use of specially manufactured objectives, and typically does not permit the use of high NA objectives, thus limiting the capabilities of the entire system.

In contrast, at least several embodiments of the dark field lighting system described herein have the following attributes. In terms of hardware, the solution of the embodiment of FIGS. 8A-8D is "table" because the ring light used for dark field illumination is on the same side of the sample as the objective lens. This is desirable from a system point of view, but alternative embodiments with a light source on the other side can be used alone or in combination with the embodiments described here. In a non-limiting embodiment, the ring light is designed such that the LEDs and/or lasers of the light source 654 are all on the same plane and have the same directivity (the light source is on the same horizontal plane and directs the light upward). Some embodiments may have lights on the sample plane, but guide the light in a non-parallel manner, such as but not limited to a cone-like manner. Some embodiments may have lights on different planes but with the same directivity to draw light. Some embodiments may have lights on different planes, but guide the light in a non-parallel manner, such as but not limited to a cone-like manner. In some embodiments, the light is reflected by the toroidal mirror 652 to achieve oblique illumination of the sample.

In addition to the optical properties of the ring light and the toroidal reflector, the optical properties of the light test tube 600 shown in the embodiments of FIGS. 8A-8D are also significant for dark field illumination. In this embodiment, the cytometry light test tube 600 is designed so that the light from the ring lamp 650 directly illuminates the sample; but in addition, the light is also "reflected" from the specific part of the light test tube onto the sample, thus simulating "Penetration" lighting. Such reflection can be achieved by TIR and/or true reflection.

Note that any kind of penetrating lighting scheme allows the measurement of forward scattered light from the sample, while the surface-scheme only allows measurement of the reverse light from the sample. Scattered light. The intensity of forward-scattered light is usually greater than that of back-scattered light by the power of two. In this way, the use of penetrating illumination permits the use of much lower illumination intensity and reduces harmful side effects on the sample.

As can be seen in the embodiment of FIG. 8A, the ring light 650 (or other lighting source) and the light test tube 600 provide a system that can be adjusted so that the penetration-and surface-illumination intensity can be adjusted to be superior to traditional surface lighting The effectiveness of. Similarly, in the embodiment of FIG. 8B, the ring light 650 (or other illuminating light source) and the light test tube 600 provide a system that can be adjusted so that the penetration-and surface-illumination intensity can be adjusted to be superior to traditional surface illumination The effectiveness of. This adjustment can be achieved by controlling the angle and degree of total internal reflection by the selected material (for example, for its optical properties) and the design of the optical cuvette geometry.

As shown in FIG. 8C, the characteristic element 802 can change the path of the incident light, and thus is used to enhance both the penetrating illumination and the surface illumination. As shown in FIG. 8D, the shapes and configurations of the surfaces 674, 676, and 678 can change the path of incident light (such as lateral illumination), and thus can be used to provide or enhance penetrating illumination, or surface illumination, or both.

FIG. 8E presents a schematic representation diagram of an optical test tube 600 being transferred from a sample preparation position to a sample observation position close to an optical detector D. As the figure indicates, the sample container 600 can be moved from one position to a position adjacent to or positioned on a detector D. A detector D may include a pedestal system configured to receive, hold, and position an optical test tube 600. The sample can be added to the sample container through the access port 602 (for example, six access ports 602 are shown in the embodiment shown in FIG. 8E), and then can be optically observed and optically measured in an analysis area 608 (Figure Is not shown in, the reason is that the light test tube shown in Figure 8E Inside the surface of 600 (for example inside the support structure 604)). The samples kept in the analysis area 608 can be illuminated and can be detected by a detector D. In an embodiment, a detector D can be configured for qualitative observation or imaging; in an embodiment, a detector D can be configured for quantitative observation or imaging.

A detector D as shown in FIG. 8E may include or be a part of a cytometry unit or a cytometry module. Such a cytometry unit or cytometry module may include an independent unit or module for sample analysis. In the embodiment, other analysis capabilities and devices can be included in a detector D, or can be covered with the same detector D, or can be combined with a detector D for use. In an embodiment, the system for sample analysis as disclosed herein may include such a cytometry unit or cytometry module, for example, including a detector D for analyzing a sample in an optical test tube 600. In an embodiment, the system for sample analysis as disclosed herein may include such a cytometry unit or cytometry module, and in addition to a detector D used to analyze the sample in the light test tube 600, other Analysis capabilities and other units or modules of the device. In these systems, these other units or modules can be covered with the same detector D, or can be combined with a detector D for use. These other analytical capabilities and devices can be applied to the sample; for example, these analytical capabilities and devices can be used to analyze samples or portions of samples that exist in an optical test tube 600. In an embodiment, these analysis capabilities and devices can be used to analyze different sample parts existing in an optical test tube 600 (for example, a sample can be divided into 2 or more liquid parts, where one liquid part is placed in one The optical test tube 600 receives cytometry analysis, and one or more other liquid fractions are covered, or approached, or combined with a cytometry unit or cytometry module operated by other devices for analysis). So, for example, and borrowed The analysis performed by this cytometry module is independent and irrelevant. The sample (or part thereof) can be used in a chemical analysis unit, or in a nucleic acid analysis unit, or in a protein analysis unit (for example, using antibodies or other specific binding molecules for analysis). Measurement and/or analysis within a unit of a sample), or other such units or combinations of units and capabilities. Such analysis may include the analysis of small molecules and elements present in the sample (for example, by general chemical units); the analysis of nucleic acid molecules present in the sample (for example, by nucleic acid units); and the analysis of proteins and elements in the sample. / Or analysis of antibody-reactive antigens (for example, by enzyme-linked immunosorbent assay (ELISA) unit); or a combination of these analyses. In addition, the system illustrated in FIG. 8E and as discussed herein may include a controller and control and schedule the operation of one or more of the units or modules.

FIG. 8F provides another detailed schematic representation of a system including a transport mechanism for transporting an optical test tube from a sample preparation position to a sample observation position close to the optical detector D. FIG. A system such as the system of the embodiment shown in FIG. 8F may include multiple sample analysis modules, which may be configured to work independently, or in some embodiments may be configured to work together. The system shown in FIG. 8F includes a single cytometry unit 707 with a detector D; in an embodiment, samples analyzed in any or all of the analysis modules 701, 702, 703, 704, 705, and 706 (Or a part thereof) can be transferred to the cytometry unit 707 for observation and measurement by the detector D. Independently of the analysis performed by the cytometry unit 707, the sample (or part thereof) can be measured and/or analyzed in a chemical analysis unit 715. Such analysis in the chemical analysis unit 715 may include analysis of small molecules and elements present in the sample (for example, by means of general chemical units); analysis of the nucleic acid present in the sample (for example, by means of Nucleic acid unit); analysis for proteins and/or antibody reactive antigens present in the sample (for example, by means of an enzyme-linked immunosorbent assay (ELISA) unit); or a combination of these analyses.

The system illustrated in FIG. 8F may include a controller to control and schedule the operations of one or more of the modules 701-707. The sample can be loaded into a sample container or other components for analysis in the system, as illustrated in the embodiment shown in FIG. 8E. These systems and system modules include, for example, sample handling system 708; burettes for obtaining, moving, and dropping samples, including extraction burette 711 and positive displacement burette 712; centrifuge 713; spectrophotometer 714 Chemical analysis unit 715; optical multiplier tube (PMT) 716; cassette 717 containing disposable supplies and tools, such as burette tips and other tips; and other components. The module and other components can be supported by a bracket 709 or other supporting structure. Samples, disposable supplies, tools, and other components can be transported within a module, and can be transported between modules (for example, between modules 701-706 and cytometry unit 707).

Figures 8E and 8F show that a sample container such as an optical test tube 600 can be transported from one location (such as a sample preparation location) and then to another location (such as to the detector D, as can be seen in Figures 8E and 8F). The optical test tube 600 does not release fluid into or on the detector D, but instead is a self-contained unit that holds all samples in it. There can be one or more, two or more, or three or more positions on the detector D or close to the detector D. The detector D has a transparent surface on which the light test tube 600 or other sample container can be joined to provide samples Transparent interface for signal detection. For further disclosure of the elements in FIG. 8F and related elements and their use, please refer to U.S. Patent Application No. 13/769,779, the full text of which is hereby incorporated. Enter the disclosure of this manual.

Dark field

At least some embodiments here include dark field illumination sources and light test tubes. The relevant characteristics of the optical cuvette 600 are related to the size of the optical cuvette and the design of the optical material and the geometrical shape of the optical cuvette. The light test tube transmits and reflects (for example, through TIR, or PIR, or both) to improve the degree of dark field illumination. In one embodiment, the system can use both the penetrating dark field and the surface dark field illumination of the sample at the same time.

In several embodiments disclosed herein, the light cuvette 600 combined with the light source 650 permits the use of a physical system to perform penetration- and surface-illumination in a surface configuration (that is, the light source and the objective lens are on the same side of the sample). The basic optical test tube is designed to contain biological samples and present for visualization. In an embodiment, the cover 612 may have a specific design. It is known that different materials can have different refractive indexes; materials with desired refractive indexes can be used to make a cover 612, or a base support 620, or other components and components of the optical test tube 600 and connected components and components. For example, in some embodiments, a cover 612 or a base bracket 620 may be made of glass. For example, in some embodiments, a cover 612 or a base bracket 620 may be made of quartz. For example, in some embodiments, a cover 612 or a base bracket 620 can be made of acrylic or clear polymers (such as cycloolefin, polycarbonate, polystyrene, polyethylene, polyurethane, Polyvinyl chloride, or other polymers or copolymers), or other transparent materials.

The material of the top cover 612 can be designed to assist in lighting and image collection. In an embodiment, in order to illuminate a sample, the light source 650 may be a ring light 650 (that is, a circular shape), and the light source 654 may be positioned in a discrete or continuous pattern. Formula, and a curved reflector 652 can be used to guide light to the sample.

In dark field microscopy, the sample is illuminated by oblique rays. In dark field microscopy, the light entering the optical elements of the microscope is scattered by the sample, allowing the measurement of the scattering properties of cells, particles, and other objects and structures in the sample. If no cells, particles, structures or other objects are present in the sample, the dark field image is black.

In this non-limiting embodiment, the reflector 652 and the LED 654 of the ring light 650 are designed to reflect light, so very few light components are guided back to the objective lens as a non-specific background. The system is designed to guide the light back to the analysis area 608 by means of TIR on the surface of the light test tube. The light reflected from a surface, whether reflected by TIR or other reflections, is thus directed to illuminate the specimen in the analysis area 608. The cells, particles, and structures in the sample in the analysis area 608 receive direct light from the ring lamp of the cell below (that is, through the surface illumination). In addition, as disclosed here, the light (reflection) from the top surface is also directed to the analysis area 608 (that is, through the penetrating illumination).

Thus, according to the system and method as disclosed herein, the ring light 650 is at the same position, and light from a single ring light source can be guided from two directions to illuminate the analysis area 608 (both surface illumination and penetrating illumination). In the embodiment, this lighting is oblique lighting. The design of the light test tube and the material used for the light test tube can control the relative intensity of the two light components.

This dark field lighting system is different from the conventional dark field. For example, in the embodiment disclosed herein, the dark field illumination is provided by TIR reflected light on the surface of the light test tube. As a non-limiting example, in the embodiment, the system as disclosed herein may use a reflective layer on the back side of certain surfaces of the cover 612 to reverse Shoot all the light. As a non-limiting example, in embodiments, the system as disclosed herein may use a reflective layer on the back side of certain surfaces of the light cuvette 600 to reflect all light. Several embodiments may use total reflection or selectively reflect the background.

For example, in the embodiment, it is desirable to guide the light at an oblique angle, and the light keeps illuminating the dark field. In some embodiments, the light source 654 can guide light at an angle, so the reflector 652 may be unnecessary or unnecessary. The reflector 652 can improve the manufacturability of the light source 654 because all the lights are on the same plane and directed in the same direction. Optionally, the angled light source 654 can also be used to replace or combine reflectors.

It should be understood that even though the light intensity of the penetrating illumination component of an illumination can be 10 times weaker than the corresponding surface illumination component, the intensity of scattered light from the cells or other objects in the sample due to the penetrating illumination can be 200 times stronger. In other words, when the scattering from the surface illumination is compared with the scattering from the equivalent penetrating illumination, the intensity of the scattered light caused by the penetrating illumination can be stronger than the scattered light illuminating the surface of the cells or other objects in the sample. 200 times. For example, a small amount of penetrating illumination can significantly enhance the scattered light from the cells.

When only the surface is illuminated, the light collected by the objective lens is only the reflected light from the sample. However, diffraction is a substantial component of scattering, and penetrating illumination is used to provide a certain amount of diffraction (such as diffracted light by the sample). However, the light collected from the surface illumination does not include the light diffracted by the sample (the light is not reflected back toward the light source after being diffracted). In this way, when using penetration-and surface-illumination, there are reflection, refraction, and diffraction components for the light collected by the objective lens. The traditional method uses full penetration dark field illumination. Since the optical components are located on both sides of the sample, the group Coupled with it consumes a significant amount of space. On the contrary, the system and method as disclosed herein use the combination of optical elements separately used for surface illumination to provide both surface-illumination and penetration-illumination. The embodiments disclosed herein can achieve the space saving of the surface lighting configuration, and provide both surface-illumination and penetration-illumination effects at the same time.

The sample container and light source are designed together, and the surface illumination configuration is allowed to increase the amount of penetrating illumination of the sample, and more specifically, it can provide consistent penetrating illumination. Several embodiments may use mirrored surfaces. Several embodiments use TIR, which can be adjusted to produce the desired penetrating illumination, including penetrating illumination that is uniform and oblique to the analysis area 608 for dark field illumination of the sample. A light test tube 600 can be configured to use reflection, such as using TIR, PIR, or both, to provide penetrating illumination of the analysis area 608 only from a light source in a surface illumination configuration. In a non-limiting embodiment, the thicker cover 612 allows TIR (or PIR, or both) to be reflected back to the analysis area 608. In addition, the systems and methods disclosed herein not only provide light that returns to the analysis area 608 due to TIR (or PIR, or both), but also provide light that returns to the analysis area 608 consistently. The embodiments of FIGS. 8A, 8B, and 8D have certain surfaces at certain angles, certain black surfaces, and certain reflective surfaces, so that light uniformly returns to the analysis area 608, effectively providing samples in an analysis area 608 The consistent penetrating lighting. Optionally, a total reflection surface (such as, but not limited to, a flat cover portion 612, as shown in FIGS. 7A and 7B) can be placed on the top, and optionally on the top of the area 613 in FIGS. 8A, 8B, and 8C. Area above). On the contrary, the light traveling inside the traditional hardware can undergo several reflections, including TIR (or PIR, or both), but the light may not return to the area 608.

For non-limiting examples, the examples disclosed herein are used to form Image-based platforms, instead of using highly complex and high-cost systems, such as 16 laser light sources, a more integrated detection system is used in this embodiment to allow imaging and identification of cell and type differences in samples.

In a non-limiting embodiment, the combination of all these different types of information is useful and effective to achieve the desired analysis goal. This may include quantitative measures and/or qualitative measures linked to quantitative measures, or images linked to quantitative measures. The method and system disclosed herein provide different fluorescent channels, where each channel has one or more targeted specific molecular markers (ie, quantitative information). The methods and systems disclosed herein can include and can be used for microscopy. The embodiments disclosed herein provide observation and measurement of staining (such as whether it is in the cytoplasm, whether it is concentrated on the surface, in the nucleus or elsewhere). Background, it can link the generated images and/or qualitative information to the generated quantitative information. In this way, if the quantitative measurement triggers an alarm or meets a critical value that requires further analysis, the link to the original image that produced the quantitative result can be used for further analysis. In this embodiment, the background image and information of staining of cells in a sample inside an analysis area 608 can be queried. These images and information allow to determine whether the staining is in the cell, such as in the cytoplasm, in the nucleus, in the cell membrane, or other organelles or cell locations.

In several embodiments of the methods and systems disclosed herein, the combination of quantitative scattering properties, cell shape, and/or cell size of cells can be observed and measured, and used to identify and/or characterize samples. In several embodiments of the method and system disclosed herein, the device simultaneously observes and measures the physical properties, optical properties, and biological/biochemical properties of a sample or part thereof in the same device. All these measurements and observations can be combined in a programmable processor or It processes the system to link various types of information to achieve the goal of verification analysis (for example, to achieve the clinical goal of verification analysis).

Although traditional devices may be suitable for one or other types of observation or measurement, they are not suitable for surface illumination and penetrating illumination from a single light source; there is no link between these different types of information. For example, in some of the embodiments disclosed herein, the image information that generates a quantitative measurement can be retrieved there, and these systems and methods can be used for tissue morphological measurement. Optionally, the system can be applied to Pap smears, which is more similar to traditional cytometry. Can be extended to any inspection using traditional microscopy. For urine, at least part of the embodiments of the present invention can observe and analyze crystals rather than cells. Observe the inorganic salt and chemical substance crystals obtained from urine samples, and some quantitative readings are produced in part of the online graph. In addition, it is possible to observe and analyze the cells and particles present in the blood, including the analysis of blood cells of different types and ethnic groups, such as but not limited to the different areas of the visible data in Figure 1A are circled. Image information of certain data areas can be obtained for further analysis of potential cell images, and the resulting measurement values can be plotted as line graphs or graphs.

Several embodiments here combine imaging features and pathological features. For example, the preparation of the tissue may occur inside a device or system configured to include the optical elements disclosed herein (a system may be or, for example, may include a module or module configured to perform optical and other analysis of a sample). Multi-module), and these prepared materials can be imaged on this platform. Then the image or analysis can be sent to the server for image analysis, diagnosis, or digital pathology analysis to effectively assist or enable the pathologist to analyze the sample.

The embodiments of the method, system, and device as disclosed herein include For example, the systems and devices illustrated in Figures 8C and 8D provide a wide range of cytometry capabilities that can be used together to analyze samples. These cytological capabilities include cytological imaging, such as typically limited to microscopy; microscopy imaging and image analysis of these biological samples are provided by the devices, systems, and methods as disclosed herein. In addition, the systems and devices as disclosed herein are configured to provide spectrophotometric analysis of biological samples. Such image analysis includes dark field, bright field, and other image analysis. A novel and improved method of applying both surface illumination and penetrating illumination from a single light source is disclosed, which allows more sensitive and accurate imaging and analysis of blood samples. In combination with the methods disclosed herein, separate metrics about RBC, WBC, and their subtypes can be obtained. The image analysis and spectrophotometric analysis as disclosed herein can be used to identify and quantify different groups of WBCs, and are useful for determining the characteristics of blood samples and the diagnosis of many clinical conditions. For example, the devices and systems disclosed here can be used to provide clinical reports, which include general chemical analysis information, nucleic acid-based analysis information, antibody (or protein or exon)-based analysis information, and spectrophotometric analysis information And in addition, provide images of the cells and samples being analyzed. The ability to generate such information and provide such reports, including images and other clinical information, is believed to provide novel and unexpected capabilities and results.

In addition, this information and these reports can be generated in a short period of time (for example, less than 1 hour, or less than 50 minutes, or less than 40 minutes, or less than 30 minutes, or other short periods of time). In addition, this information and these reports can be generated from small samples such as small blood or urine samples. The size of such a small sample is not greater than about 500 microliters, or less than about 250 microliters, or less than about 150 microliters, or less than about 100 microliters, or less than about 75 microliters, or less than about 50 microliters, or Less than about 40 microliters, or less than about 20 microliters, or less than about 10 microliters, or other small volumes. In the embodiment where the sample is a blood sample, such a small sample can be collected from a finger puncture. Typically, only a small amount of blood is collected from a finger puncture (for example, the amount of blood may be about 250 microliters or less, or about 200 microliters or less, or about 150 microliters or less, or about 100 microliters or less, or About 50 microliters or less, or about 25 microliters or less, or other small amounts).

Including cytometry information and images (including images, scatter mapping, and other optical and imaging information) as disclosed here, and also general chemical analysis information, nucleic acid-based analysis information, antibodies (or protein or external display The clinical report based on sub) analysis information and spectrophotometric analysis information is believed to provide broad and clinically rich information, which is useful for the diagnosis and feature determination of many clinical conditions and to provide advantages over the art world. These reports can be quickly prepared at the point of service (or care point), and can be quickly communicated (for example, electronically via wireless, landline, optical fiber, or other communication links) to pathologists or other clinical experts for Analysis and interpretation. Then, this expert analyzes and interprets the rapid communication back (for example, electronic communication via wireless, land communication line, optical fiber, or other communication links) to the individual clinical caregiver, or back to the service point (or care point) location , Or both for quick feedback. If necessary, such quick feedback permits can be provided at the point of service or the location of the care point, based on available, analyzable, or both samples, by providing information and analysis for immediate treatment or avoiding unnecessary treatment. Such rapid analysis, reporting, and feedback provide advantages over time-consuming methods, and by permitting immediate treatment and by avoiding unnecessary treatment, it can provide more effective, efficient, and cost-effective Low clinical service and treatment. Borrow here The disclosed device, system and method can avoid these more time-consuming methods, including but not limited to: the delay and inconvenience caused by the individual having to travel from the home to the remote laboratory or clinic and away from the clinician entrusted to care for the individual ; The delay caused by the transfer of the sample from the collection site to the analysis site and the possible degradation of the sample; the delay caused by the transmission of these analysis results to the pathologist or other experts; the transmission of an expert opinion to the individuals clinician Delay; the delay in transmitting the clinical diagnosis and treatment of the individual after transmitting an expert opinion to the clinician. These delays, inconveniences, and possible sample degradation can be reduced or eliminated by using the methods, systems, and devices disclosed herein.

Figures 6A, 6B, 7, 8A, 8B, 8C, and 8D and other diagrams and as disclosed herein illustrate examples of the system and device embodiments that provide compact cytometry capabilities, including compact formats for one or more other Sample analysis capabilities. The applicant hereby discloses novel devices and systems that include novel cytometry capabilities in devices and systems as disclosed herein, as well as other sample analysis capabilities. The applicant hereby discloses devices and systems that provide novel cytological capabilities as disclosed herein, combined with devices and systems that use general chemical units for sample analysis; combined with devices and systems that use nucleic acid analysis units for sample analysis; combined with antibody assays Analysis (such as ELISA) unit for sample analysis devices and systems; and combinations thereof. In this way, the sample processing device as disclosed herein can be configured to perform a plurality of verification analyses on a sample. Such samples may be small samples.

In the embodiment, all the sample verification and analysis actions or steps are performed on a single sample. In the embodiment, all sample verification and analysis actions or steps are performed by a single device or system and can be inside the housing of a single device implement. These systems and devices include cytometry, especially cytometry that provides image analysis and spectrophotometric analysis or other optical analysis in a single unit is believed to be novel and unexpected. The provision of systems and devices includes cytometry, especially cytometry that provides image analysis and spectrophotometric analysis or other optical analysis in a single unit is believed to provide advantages previously unattainable in the art world.

As shown in Figures 6A, 6B, 7, 8A, 8B, 8C, and 8D and other diagrams and examples as disclosed herein, the embodiments of the system and the device provide portable cytometry capabilities, where these devices and systems can be Covered in an enclosure small enough to be easily transported. For example, these devices and systems can be easily transported to a care location (such as a physician's office, clinic, hospital, clinical laboratory, or other location). For example, these devices and systems can be easily shipped for use in service locations (in addition to the care locations discussed above, such as pharmacies, supermarkets, or other retail locations or service locations). The service location point may include, for example, any location where an individual can receive services (such as testing, monitoring, treatment, diagnosis, guidance, sampling, ID authentication, medical services, non-medical services, etc.). Service locations include, but are not limited to, the individuals home, individuals business, the location of health care providers (such as doctors), hospitals, emergency rooms, operating rooms, clinics, health care providers offices, inspection rooms, and retailers[ For example, pharmacies (e.g. retail pharmacies, clinic pharmacies, hospital pharmacies), drugstores, supermarkets, grocery stores, etc.), transportation vehicles (e.g. cars, ships, trucks, buses, airplanes, motorcycles, ambulances) Vehicles, action units, fire trucks/fire trucks, emergency service vehicles, law enforcement vehicles, police cars, or other vehicles that are configured to transfer a body from one point to another), travel medicine Nursing units, action units, schools, daycare centers, security screening locations, war locations, health-assisted living houses, government offices, office buildings, tents, locations for obtaining body fluid samples (e.g. blood donation centers), locations where individuals may wish to approach entrances Location or nearby location, device location or nearby location that the individual may wish to approach (if the individual wants to approach the computer, the computer is located), the location where the sample processing device accepts the sample, or any other service point described elsewhere herein.

Esoteric cytometry and special cytometry markers

A variety of traditional advanced or esoteric cytometry assays require traditional systems to measure a large number of markers on cells; typically, these markers are measured simultaneously. The general method in this field is high-energy instruments, including, for example, 6 or more lasers and 18 different PMT tubes to measure all these marks at the same time. However, in many clinical equipment, there is no need to measure multiple markers at the same time. For example, in many clinical requirements, how many cells are positive for one marker, or how many cells are positive for two or more markers or other such combinations of several markers. Here, several embodiments propose multiple combinations of dyeing schemes. For example, there can be a set of 10 markers in this place, and a set of 3-4 or 5-6 markers can be combined there, which can be combined even if two markers are of the same color. In some embodiments of the system, images and information can be deconvolved to determine which signal comes from which tag. This allows several embodiments of the system to reduce hardware requirements in terms of the number of light sources, the number of channels used for sample analysis, and other simplifications and efficiency. In this way, it is useful to use a subset of multiple markers, or use a predetermined pairing in a non-simultaneous manner or measure markers for esoteric cytometry. For example, some markers can be regarded as "gated" markers; and some markers are measured first, and if the preliminary measurement result is negative (for example, the markers are not present or exist only in low amounts) For samples), there is no need to use other subsequent markers for measurement. In embodiments, these non-simultaneous methods and systems can reduce the sample volume required for analysis, and can reduce the amount of markers required for analysis (for example, if follow-up tracking markers are typically only used in a small number of sample components to be analyzed).

It is important to understand that cytometric analysis and imaging of samples such as blood or urine permits the actual cell number to be obtained and is therefore more accurate than traditional cytometry methods that do not contain such measurements. Imaging of the sample The imaging of the cells (and particles or structures) in the sample is actually more accurate than other methods such as traditional flow cytometry. For example, traditional flow cytometry gating does not allow for actual counting. The gate control in flow cytometry is subjective and therefore varies from system to system. In addition, traditional flow cytometry fails to provide images of the cells in the sample.

Several embodiments here are also gated, but the gated control is based on an algorithm based on various factors rather than limited to patient health. The classification method is based on the patient's ethnic group for training to know whether it is healthy or sick. Several embodiments herein can mark a patient as abnormal and mark the patient for overview. Self-learning gate control can be based on whether the information conveyed by the patient's health status requires different gate control. In this way, in the embodiments disclosed herein, the gating of the sample is completed by algorithms, possibly using a programmable processor, and changing the gating according to the health of the patient.

In the embodiments of the imaging method and system, it may be desirable to minimize the amount of hardware and complexity required, and if possible, it is desirable to reuse part or all of the samples to reduce the amount of samples required. In this way, the higher the ability to extract information from a single image, the better is the maximization of information obtained from the sample and when possible from a smaller sample. In this way, it can be distinguished from the fewest images available The more information about different cell types, the more the sample size needed can be reduced.

Optionally, in a non-limiting embodiment, the light test tube used in the microscopy stage can be configured as follows (refer to the embodiments and components shown in FIGS. 7, 8A, and 8B). A middle channel layer includes a thin plastic film 800 core layer with pressure sensitive adhesive (psa) on both sides. One side is adhered to the window layer 606 and the other side is adhered to the molded top cover 612. The core layer is a black extruded film, mainly due to optical reasons such as preventing light scattering and optical crosstalk between different liquid channels. The thickness of the core film is preferably uniform along its length and width, for example, it can be made from black polyethylene terephthalate (PET) or black HDPE (polyethylene) extruded film. The pressure-sensitive adhesive (psa) sublayers on both sides are preferably as thin as possible to maintain a close and consistent size of the total liquid channel (for example, the analysis area 608), but are preferably thick enough to provide surrounding liquid Good fluid tightness of the channel. In the embodiment, the psa adhesive useful for this kind of sample container is acrylic in nature and has high adhesive strength to low surface energy plastics. The liquid channels, ports and other alignment features on the middle layer can be made by laser cutting or die cutting.

This embodiment also shows that magnetic elements such as but not limited to a magnetic scaler or ingot, or a metal scaler or ingot that can be attracted by a magnet can be mixed into the optical test tube. For example, these magnetic elements may be included in or may include a molded top layer of a sample container or light test tube. The magnetic element can be used to simplify the hardware used to transport the light test tube. For example, the treatment system can engage the magnetic feature in the optical cuvette to transport the optical cuvette without the need for an additional sample handling device.

Although the present invention has been described and illustrated with reference to certain specific embodiments, those skilled in the art will understand that it is possible to make various adjustments, changes, modifications, substitutions, changes, modifications, substitutions, and modifications to the procedures and solutions without departing from the essence and scope of the present invention. Delete, or add. For example, different materials can be used to create different reflective surfaces in the light test tube or other surfaces along one of the optical paths in the optical system. Optionally, the reflective surface is selected so that the reflection is only diffuse. Optionally, the reflective surface is selected so that the reflection is only specular. In some embodiments, such as Coumans, FAW, van der Pol, E., &amp; Terstappen, LWMM (2012), a flat top illumination scheme using a double microlens array on a surface fluorescent microscope, Cytometry, 81A: 324-331 .doi: 10.1002/cyto.a.22029 stated flat top lighting scheme, the document is fully cited here and incorporated into the disclosure of this specification for all purposes.

In addition, concentration, content, and other numerical data can be presented here in a range format. It should be understood that this range format is only used for convenience and simplicity, and should be interpreted flexibly to include not only the values explicitly quoted as the limits of the range, but also all individual values or small ranges covered within the range. , As if individual values or small ranges are clearly quoted. For example, the size range of about 1 nanometer to about 200 nanometers must be interpreted as including not only the explicitly quoted limit of about 1 nanometer to about 200 nanometers, but also individual sizes such as 2 nanometers, 3 nanometers, 4 nanometers, and small areas such as 10 nanometers to 50 nanometers, 20 nanometers to 100 nanometers, and other ranges.

The published documents discussed or quoted here are only because their disclosure date is before the filing date of this case. The content here is by no means interpreted as acknowledging that the date of the present invention precedes such public documents of Jin Ming. Furthermore, the publication date proposed here may be different from the actual publication date and needs to be confirmed individually. All the publications mentioned here are the disclosures that are cited here and incorporated into this specification, and describe the structures and/or methods cited in these publications. The following application The case is also cited here and incorporated into the disclosure of this specification for all purposes: U.S. Patent No. 7,888,125; U.S. Patent No. 8,007,999; U.S. Patent No. 8,088,593; U.S. Patent No. 8,088,593; U.S. Patent No. No. 8,380,541; U.S. Patent Publication No. US20120309636; PCT Application No. PCT/US2012/057155; PCT Application No. PCT/US2011/53188; PCT Application No. PCT/US11/53189; U.S. Patent Application No. 13 /769,779; US Patent Application 13/244,946; US Patent Application 13/244,947; US Patent Application 13/244,949; US Patent Application 13/244,950; US Patent Application 13/244,951; US Patent Application 13/ 244,952; US Patent Application 13/244,953; US Patent Application 13/244,954; US Patent Application 13/244,956; US Patent Application 13/769,798; US Patent Application 13/769,818; US Patent Application 13/769,820; US Patent Application 61/766,113; US Patent Application 61/673,245; US Patent Application 61/786,351; US Patent Application 61/697,797; US Patent Application 61/766,076; and US Patent Application 61/733,886, which The full texts of such patent cases and patent application cases are all cited here and incorporated into the disclosure of this specification for all purposes.

This patent document contains materials protected by copyright. When this patent appears in the patent files or records of the United States Patent and Trademark Office, the copyright owner (applicant in this case) does not object to anyone copying the patent document or patent disclosure, otherwise he retains all copyrights. The following note applies: Copyright 2012-2013 Silano (Theranos, Inc.).

Although the foregoing is a complete description of the preferred embodiment of the present invention Mentioned, but various alternatives, modifications and equivalents may be used. Therefore, the scope of the present invention should not be determined with reference to the detailed description in the preceding section, but instead should be determined with reference to the attached scope of patent applications and the full scope of their equivalents. Any specific element, whether it is preferable or not, can be combined with any other specific element, whether it is preferable or not. Unless the terminology limitation of functional means is explicitly quoted in a given scope of patent application using the term "means used", the various terms of the scope of patent application attached should not be interpreted as including such limitation. It should be understood that if the detailed description here and the following applies in the full text of the scope of the patent application, unless the content clearly indicates otherwise, the meaning of "one" and "the" include plural forms. In addition, it must be understood that as used in the full text of the scope of patent application in the detailed description here and later, unless the content clearly indicates otherwise, the meaning of "in" includes "in it" and "in it". Finally, it must be understood that if the detailed description here and the subsequent patent applications are used in the full text, unless the content clearly indicates otherwise, the meanings of "and" and "or" include conjunctions and transitional conjunctions and can be used interchangeably. Thus, in the context of using words such as "and" or "Yu", the use of these conjunctions does not exclude the meaning of "and/or" unless the context clearly indicates otherwise.