Cell image analyzer, its method, and its software

The present invention relates to an image analyzer and method for microscopic images of cells obtained by an optical microscope and software for the same, and more specifically, membrane translocation observed in cells by analyzing a fluorescence microscopic image of cells. The present invention relates to an apparatus and method for determining whether or not a reaction has occurred, and software for that purpose.

In various cells, when a chemical stimulus such as a physiologically active substance or a drug or a physical stimulus such as light, electricity, or mechanical force is applied, certain intracellular proteins accumulate in the cell membrane. It is known that a phenomenon called "membrane translocation reaction" occurs. Membrane receptors, such as G protein-coupled proteins (GPCRs) on cell membranes When a receptor) or the like receives a specific substance (ligand) or photon from the outside, this becomes a trigger, that is, a stimulus, and is chained inside the cell, as is well known to those skilled in the art. Reactions of various substances proceed, and stimulus information is transmitted one after another inside the cell. A "membrane translocation reaction" is a protein that was released into the cytoplasm or was present in an organelle prior to stimulation as part of such intracellular stimulation or signal transduction processes. It is a phenomenon of migration to the cell membrane. Such a membrane translocation reaction can be directly observed in cells under an optical microscope by labeling a protein having membrane translocation activity (causing a membrane translocation reaction) or other biomolecule with a fluorescent dye or the like. Therefore, in the field of cell biology, medical or pharmaceutical research or drug discovery, it is determined whether or not any cell undergoes a membrane translocation reaction to any substance or stimulus under an optical microscope. By doing so, it is possible to evaluate whether or not the substance or stimulus has a bioactive effect on the cell (particularly as an intracellular signaling substance) or whether or not it is effective as a new drug or a therapeutic method (Drug Discovery). ) Is being tried.

When observing a membrane translocation reaction, conventionally, labeling a specific protein having a membrane translocation activity in a living cell means microscopically detecting a protein or a biomolecule fluorescently labeled on an individual cell. Skilled techniques such as injection were required, and the number of cells that could be observed at one time was small. However, a specific protein inside a large number of cells can be stained at once with a fluorescent dye that can be taken up into living cells and labeled with a specific substance, or a fluorescent protein such as GFP is fused by gene transfer technology. By culturing a large amount of cells expressing the protein at one time, it has become possible to observe the movement of the protein in a large number of cells using the fluorescent dye or the fluorescent protein as a label. .. For example, as described in Non-Patent Document 1, in adhesive cells HeLa (cervical cancer cells) and the like, a GFP fusion protein of protein kinase C (PKC), which is a membrane translocation active protein, is produced by gene transfer technology. A large number of expressed cells are prepared, and under a fluorescent microscope, a large number of GFP-fused PKC-expressing cells are treated with PMA, which is a membrane translocation signal, and other active agents, and whether or not a membrane translocation reaction occurs is observed. (PKC is released in the cytoplasm before stimulation, but it is known that when cells receive stimulation such as PMA, they bind to the inside of the cell membrane.) .. According to such an experiment, the responsiveness of cells to a certain stimulus can be observed and evaluated for a large number of cells at once, so that an intracellular signal transduction substance can be searched for and a substance having an arbitrary bioactive effect can be screened. It is very advantageous when doing.<patcit num="1"><text>JP 2004-54347</text></patcit><nplcit num="1"><text>The Journal of Biological Chemistry, Vol. 270, No. 50, 1995, pp. 30134-30140</text></nplcit>

<p num="0004"> When determining the presence or absence of a membrane translocation reaction using a fluorescence microscope, a fluorescence microscope image of cells (hereinafter referred to as "fluorescence image") obtained through an objective lens is visually or mounted on a microscope. It is observed by taking a fluorescence image. Then, at the discretion of the observer or the experimenter, it is determined that the membrane translocation reaction occurred in each cell when the fluorescence intensity on the outer periphery of the cell increased relatively after the stimulation. When the fluorescence image captured by the camera can be taken into an arbitrary image processing device (see, for example, Patent Document 1), the fluorescence intensity distribution inside and outside the cell can be graphed, so that the fluorescence image itself. Instead, by referring to the graph of the fluorescence intensity distribution for each cell, it is possible to determine whether or not the protein is accumulated around the cell membrane from the change in the fluorescence intensity on the outer periphery of the cell. Further, when screening for the bioactive action of an arbitrary substance, the number of cells that have undergone a membrane translocation reaction is counted, and the result of the count number is used for evaluating the action of the substance.</p><p num="0005"> However, in the above-mentioned determination, it is determined whether or not the fluorescence intensity on the outer periphery of the cell is recognized to be increased by the stimulus in a certain cell, that is, whether or not the membrane translocation reaction has occurred. Ultimately, it is left to the discretion of the observer, and therefore the judgment can vary. In fact, in living cells, the degree of change in fluorescence intensity due to the membrane translocation reaction varies depending on the individual cell, for example, the shape of the cell, the thickness of the cell (in the optical axis direction of the optical microscope), and the like. In some cases, when the presence or absence of the membrane translocation reaction is judged by the observer, the judgment criteria may differ from time to time or for each observer, and the judgment result may not be very objective or reproducible. Further, when determining the presence or absence of a membrane translocation reaction for one cell, it does not require much labor by using the graph of fluorescence intensity distribution, but when the number of cells to be determined increases, one It takes a lot of time and effort to make judgments one by one. Furthermore, since there are variations in the judgments of observers, if the total number of cells becomes large, an error will occur in the count result of the cells that have undergone the membrane translocation reaction, and the action in screening the bioactive action of any substance will occur. The reliability of the evaluation result will be reduced.</p><p num="0006"> Thus, if the determination of the presence or absence of a membrane translocation reaction can be performed without having to rely on the individual judgment of individual cells by the observer or experimenter, it will take time even if the number of cells to be determined increases. It is expected that reliable results will be obtained without any hassle.</p><p num="0007"> Therefore, one object of the present invention is a cell image analyzer or method for determining the presence or absence of a membrane translocation reaction to an arbitrary stimulus in an arbitrary cell using a fluorescence microscope, and is an individual cell. It is an apparatus or method for making a determination of a membrane translocation reaction in a cell so as not to be affected by a variation in judgment by an observer or an experimenter.</p><p num="0008"> Another object of the present invention is the device or method as described above, which can quickly determine the presence or absence of a membrane translocation reaction even if the number of cells to be determined increases. To provide. In such a device or method, it would be more preferable that after the cell sample is set in the optical microscope, it is possible to automatically obtain a fluorescence image of the cells and determine the presence or absence of a membrane translocation reaction. ..</p><p num="0009"> Furthermore, another object of the present invention is to provide software that realizes the above-mentioned cell image analysis apparatus or method.</p>

<p num="0010"> According to the present invention, a novel cell image analyzer for determining the presence or absence of a membrane translocation reaction that occurs inside a cell by giving an arbitrary stimulus to the cell regardless of the variation of the judgment of an observer or an experimenter. Computer programs and methods for cell image analysis are provided.</p><p num="0011"> The cell image analyzer according to the present invention is a means for acquiring a fluorescence microscopic image of a cell whose translocation molecule moving to the cell membrane during a membrane translocation reaction is stimulated by a fluorescently labeled cell, and a cell. A means for determining the region where the cell exists in the image data of the fluorescence microscope image of the cell, a means for determining the contour line of the cell having a width of a predetermined number of pixels from the outer edge of the region where the cell exists, and a means for determining the contour line of the cell. It is characterized by including a means for determining the presence or absence of a membrane translocation reaction based on a brightness value on a cell contour line and a brightness value inside the cell contour line in the image data of a fluorescent microscope image. ..</p><p num="0012"> According to the above-described configuration of the present invention, first, a fluorescence microscope image (fluorescence image) after stimulation of a cell in which a translocation molecule that moves to a cell membrane during a membrane translocation reaction is fluorescently labeled is usually obtained. Is acquired as image data in the above manner. Since the image data of the fluorescence image is a set of pixels having a brightness value and coordinates, if the coordinates are specified, the fluorescence intensity of the coordinates in the fluorescence image can be known. Therefore, in the apparatus of the present invention, the region where the cell exists in the image data of the fluorescence image is determined based on the fluorescence intensity corresponding to the coordinates in the acquired fluorescence image, and the cell image is obtained. A contour line having a width of a predetermined number of pixels is determined from the outer edge of the region where the cells exist. Then, the presence or absence of the membrane translocation reaction is determined based on the brightness value on the contour line of the cell and the brightness value inside the contour line of the cell in the image data of the fluorescence microscope image of the cell. In this case, preferably, the average of the brightness values of all the pixels located on the contour line of the cell and the average of the brightness values of the pixels located inside the contour line of the cell are calculated, and the average inside the contour line of the cell is calculated. The membrane translocation reaction may be determined when the ratio of the average on the contour line of the cell to the cell exceeds a predetermined value.</p><p num="0013"> According to such a configuration, the determination of the membrane translocation reaction is performed without being influenced by the subjective judgment of the observer or the experimenter, and therefore the reliability of the judgment result is increased. In addition, since the operation of the above device can be automated by using image processing technology, the observer or the experimenter does not need to judge the membrane translocation reaction in each measurement, and the membrane transformer does not need to be judged. The time and effort required for location reaction can be reduced.</p><p num="0014"> In the above configuration, the fluorescence microscope image may include fluorescence microscope images of a plurality of cells (that is, one image data may contain a plurality of cells), in which case. Means for determining the region in which a cell is present determines the region in which each of the plurality of cells is present, and means for determining the contour of the cell determines the contour of each of the plurality of cells, and the membrane trans The means for determining the presence or absence of a location reaction is to determine the presence or absence of a membrane translocation reaction based on the brightness value on each contour line of a plurality of cells and the brightness value inside the contour line. You can. Since such a configuration is also performed by automatic arithmetic processing using the pixel coordinates and the brightness value in the image data, even if the number of cells to be observed at one time increases, the observer or the experimenter can perform individual cells. It is possible to significantly reduce the labor and time required for determining the membrane translocation reaction without determining the membrane translocation reaction. Further, the criteria for determining the membrane translocation reaction is not based on the sense of the observer or the experimenter, but based on the brightness value on the contour line of each of the plurality of cells and the brightness value inside the contour line. Therefore, there is no variation in the judgment criteria for each cell, and the objectivity and reproducibility of the judgment results are improved.</p><p num="0015"> Determining the membrane translocation response is often used to assess the bioactive effects of any substance, as already mentioned. In that case, how many cells undergo a membrane translocation reaction in response to a certain stimulus is a measure of the bioactive effect. Therefore, in the apparatus of the present invention, the means for determining the region where the cells exist may have a means for counting the number of a plurality of cells in the fluorescence microscope image. In that case, the observer or the experimenter can save the trouble of manually counting the number of cells in the image data. Also, since the evaluation of bioactivity is often made by the ratio of the number of cells in which the membrane translocation reaction has occurred to the total number of cells stimulated, the cells in which such a membrane translocation reaction has occurred. The ratio of the number of the numbers may be calculated automatically.</p><p num="0016"> In addition, the above-mentioned device of the present invention may be used for determining the membrane translocation reaction of any cell. In that case, the amount of protein accumulated in the cell membrane or the state of accumulation of such protein is reflected in the fluorescence image depending on the cell type and experimental conditions. Therefore, the fluorescence in the vicinity of the cell membrane when the membrane translocation reaction occurs. The way the strength spreads will be different. In such a situation, if the width of the contour line is too narrow or too wide with respect to the width of the region where the protein accumulated near the cell membrane is distributed, in any case, when the membrane translocation reaction occurs. Since the difference in intensity when not occurring is reduced, it is not possible to satisfactorily capture the change in fluorescence intensity in the vicinity of the cell membrane due to the membrane translocation reaction. Therefore, in the above-mentioned apparatus of the present invention, a predetermined width of the contour line determined by the means for determining the contour line of the cell may be set by the user.</p><p num="0017"> Further, in the above-mentioned apparatus of the present invention, as understood from the above, the determination of the membrane translocation reaction depends on the width of the contour line of the cell and its brightness value, and the position of the focal point of the objective lens with respect to the cell. If it shifts, the spread and intensity of light in the region where cells exist in the fluorescent image and in the vicinity of the cell membrane will change, causing an error in the criteria for the membrane translocation reaction. It is desirable that the position is always constant. However, in the actual measurement, when the height or position of the objective lens of the fluorescence microscope shifts during the acquisition of the fluorescence image, or when the field of view of the objective lens is moved (actually, the position of the observation sample sample). The relative position of the bottom or top surface of the observation sample container with respect to the focal point of the objective lens with respect to the cells may shift. Therefore, in the apparatus of the present invention, when the cell is a cell that adheres to the bottom surface or the upper surface of the observation sample container, the cell is automatically focused on the bottom surface or the upper surface of the observation sample by focusing the objective lens of the fluorescence microscope. A means for controlling the position of the objective lens may be provided so as to focus the objective lens. As a result, if the fluorescence microscope used with the device of the present invention has a function of automatically adjusting the position of the objective lens, the observer or the experimenter does not need to confirm the position of the objective lens. The fluorescence image of the cell can be stably obtained, and therefore, the determination of the membrane translocation reaction based on the brightness value on the contour line of the cell can be stably executed (without variation).</p><p num="0018"> By the way, the above-mentioned function of the device of the present invention may be executed in any computer. Therefore, according to another aspect of the present invention, it is a computer program for cell image analysis that determines the presence or absence of a membrane translocation reaction generated inside a cell by giving an arbitrary stimulus to the cell, and is a membrane translocation reaction. The procedure for acquiring a fluorescent microscope image as image data after stimulation of a cell fluorescently labeled with a translocation molecule that moves to the cell membrane at the time of the above, and the presence of the cell in the image data of the fluorescent microscope image of the cell. The procedure for determining the region to be formed, the procedure for determining the contour line of the cell having a width of a predetermined number of pixels from the outer edge of the region where the cell exists, and the procedure on the contour line of the cell in the image data of the fluorescence microscope image of the cell. Provided is a computer program comprising causing a computer to execute a procedure for determining the presence or absence of a membrane translocation reaction based on a brightness value in the cell and a brightness value inside the contour line of a cell.</p><p num="0019"> In the above-mentioned computer program of the present invention, similarly to the apparatus of the present invention, in the procedure for determining the presence or absence of the membrane translocation reaction, the average of the brightness values of all the pixels located on the contour line of the cell and the cell The average of the brightness values of the pixels located inside the contour line of the cell is calculated, and when the ratio of the average on the contour line of the cell to the average inside the contour line of the cell exceeds a predetermined value, the membrane translocation reaction occurs. It may be determined that it has occurred. Further, when the fluorescence microscope image includes a fluorescence microscope image of a plurality of cells, the computer program of the present invention determines the region where each of the plurality of cells exists in the procedure for determining the region where the cells exist. On the contours of each of the plurality of cells in the procedure of determining and determining the contours of the cells, and in the procedure of determining the presence or absence of a membrane translocation reaction. The presence or absence of the membrane translocation reaction may be determined based on the brightness value in the area and the brightness value inside the contour line. In this case, the number of a plurality of cells in the fluorescence microscope image may be counted, and the ratio of the number of cells in which the membrane translocation reaction has occurred among the plurality of cells in the fluorescence microscope image may be calculated. ..</p><p num="0020"> Further, the feature in the apparatus of the present invention, that is, the region where the cell exists and the contour line are determined from the fluorescence image of the cell acquired as the image data, and then in the image data of the fluorescence microscope image of the cell. The configuration of determining the presence or absence of a membrane translocation reaction based on the brightness value on the contour line of the cell and the brightness value inside the contour line of the cell is generated inside the cell by giving an arbitrary stimulus to the cell. It can also be realized by a method for determining the presence or absence of a membrane translocation reaction. Therefore, according to still another aspect of the present invention, a fluorescence microscope image after stimulation of cells in which translocation molecules moving to the cell membrane during a membrane translocation reaction is stimulated is acquired as image data. The process of determining the region where the cell exists in the image data of the fluorescence microscope image of the cell, and the contour line of the cell having a width of a predetermined number of pixels from the outer edge of the region where the cell exists. The process includes a process of determining the presence or absence of a membrane translocation reaction based on the brightness value on the contour line of the cell and the brightness value inside the contour line of the cell in the image data of the fluorescence microscope image of the cell. A method for determining the presence or absence of a membrane translocation reaction is provided. According to such a method, it is possible to stably and quickly determine the presence or absence of a membrane translocation reaction without being influenced by the subjective or sensory criteria of the observer or the experimenter. ..</p><p num="0021"> In such a method, as in the case of the apparatus of the present invention described above, in the process of determining the presence or absence of the membrane translocation reaction, the average of the brightness values of all the pixels located on the contour line of the cell and the brightness value of the cell The average brightness value of the pixels located inside the contour line was calculated, and the membrane translocation reaction occurred when the ratio of the average on the cell contour line to the average inside the cell contour line exceeded a predetermined value. It may be judged. When the fluorescence microscope image includes a fluorescence microscope image of a plurality of cells, the region where each of the plurality of cells exists is determined in the process of determining the region where the cells exist, and the outline of the cells is determined. In the process of determining the contour line of each of a plurality of cells, and in the process of determining the presence or absence of a membrane translocation reaction, the brightness value and the contour on each contour line of the plurality of cells The presence or absence of a membrane translocation reaction may be determined based on the brightness value inside the line, and in the process of determining the region where the cells are present, the number of a plurality of cells in the fluorescence microscope image. , Or the ratio of the number of cells in which the membrane translocation reaction has occurred among the plurality of cells in the fluorescence microscope image may be calculated.</p><p num="0022"> In the above-mentioned invention, the membrane translocation reaction determined is, in part, a reaction in which a protein released in the cytoplasm of a cell is accumulated on the cell membrane of the cell by stimulating the cell. is there. Further, according to the present invention, it is possible to determine whether or not a reaction in which a protein existing in an organelle in the cell accumulates on the cell membrane of the cell by stimulating the cell also occurs. The cells are preferably adhesive cells that are easy to focus on the objective lens.</p><p num="0023"> Preparation of cells fluorescently labeled with translocation molecules that move to the cell membrane during the membrane translocation reaction is performed, for example, in a state in which the translocation molecules are fused with a fluorescent protein in advance before obtaining a fluorescence microscopic image of the cells. It may be done by introducing the gene for expression into the cell and expressing the translocation molecule fused with the fluorescent protein in the cell, or the membrane translocation reaction before obtaining the fluorescence microscopic image of the cell. At the time, a gene for introducing the tag molecule into the translocation molecule that moves to the cell membrane is introduced into the cell, the translocation molecule into which the tag molecule is introduced is expressed in the cell, and then the tag molecule is introduced into the cell. This may be done by introducing a fluorescent dye that specifically binds, thereby fluorescently labeling the translocation molecule. Of course, the translocation molecule may be fluorescently labeled by a method other than the above, and such a case also belongs to the scope of the present invention.</p>

<p num="0024"> As understood from the configuration of the present invention, according to the apparatus, program or method of the present invention, the observer or the experimenter can sensuously judge the judgment of the membrane translocation reaction from the fluorescent image of each cell. Therefore, there is no variation in the judgment of each observer or experimenter in the judgment result, and the objectivity and reproducibility are improved. Further, as described in relation to the configuration of the apparatus of the present invention, the determination of the membrane translocation reaction of the present invention is automatically performed by arithmetic processing using image data obtained by digitizing the fluorescence image of cells. Since it is feasible, the labor or time required for the determination is reduced, and even if the number of cells to be determined increases, the determination result can be obtained quickly.</p><p num="0025"> It should be understood that the apparatus, program or method of the present invention can also be used advantageously when assessing gene transfer efficiency. When evaluating gene transfer efficiency, for example, a gene transfer technique to be evaluated attempts to introduce a gene into a cell for fluorescent labeling of a protein involved in a membrane translocation reaction or a protein having translocation property. If gene transfer is successful, fluorescent labeling is performed, the protein is expressed, and a membrane translocation reaction is observed in response to a predetermined stimulus. The gene transfer efficiency is determined by the reaction rate of the membrane translocation reaction. In such a case, it is determined whether or not a membrane translocation reaction has occurred in a large number of cells, but according to the present invention, even in such a case, a rapid and reliable evaluation result can be obtained. Expected to be able to give.</p><p num="0026"> Other objects and advantages of the present invention will be apparent from the following description of preferred embodiments of the present invention.</p>

Some preferred embodiments of the present invention will be described in detail below with reference to the accompanying figures.

<u style="single">Configuration of fluorescence microscope observation system</u> FIG. 1 schematically shows a fluorescence microscope observation system incorporating a preferred embodiment of a cell image analyzer for determining a cell membrane translocation reaction according to the present invention. For the sake of simplification of the description, parts not particularly related to the configuration of the present invention are omitted.

With reference to the figure, the fluorescence microscope observation system includes an inverted fluorescence microscope 1 and an image analyzer 100. The fluorescence microscope 1 has a stage 2 that can be electrically moved in the XY direction (horizontal direction) on which a cell sample specimen P (for example, a 24-hole plastic microtiter plate) is placed, like a normal fluorescence microscope. , Illuminated light source 3 for observing transmitted light, objective lens 4, objective electric drive mechanism 5 for adjusting the position (height) of the objective lens, excitation light source 6 for fluorescence observation, dichroic mirror 7, and fluorescence of cells. It includes a camera 8 for taking an image and a multi-controller 10 that controls the operation of each of the above-mentioned parts of the fluorescence microscope 1 in cooperation with the image analyzer 100. When observing fluorescence with the fluorescence microscope 1, the excitation light EX from the excitation light source 6 is reflected by the dichroic mirror 7, illuminates the cell sample sample P through the objective lens 4, and is excited by the excitation light. The fluorescent FL from the intracellular fluorescent label passes through the objective lens 4 and the dichroic mirror 7, and is imaged on the light receiving surface (not shown) of the camera 16. As is well known to those skilled in the art, the dichroic mirror 7 has an excitation filter 7-1 and fluorescence so that the cell sample is excited by light of a specific wavelength and only the fluorescence from the sample is incident on the light receiving surface of the camera. A filter 7-2 for (or for light reception) is provided. Further, since the optical system of the illustrated optical microscope is a so-called "infinity system", an imaging lens is arranged in front of the camera, and an image of the focal plane of the objective lens 4 is formed on the light receiving surface. There is. The camera may be a video camera usually used in this field, such as a CCD or a cooled CCD. The excitation light source 6 may be any light source known in the art, such as a mercury lamp, a xenon lamp, or a laser. In the illustrated example, LEDs with emission wavelengths of R (620 nm), G (520 nm), B (460 nm), and UV (365 nm) are built-in, and the light of these LEDs is combined into one optical fiber 12. Introduced, the light emitted from the optical fiber is made parallel by the lens set 13

The objective lens 4 attached to the fluorescence microscope 1 may have an arbitrary magnification. In the present invention, since the whole cell or a plurality of cells having a size of about 10 μm to several tens of μm are observed at once and taken into the analyzer as image data, a 40x objective lens (empty) is typically used. Immersion) is used. Such an objective lens is usually provided with a correction ring 4a that adjusts the focal position of the objective lens according to the thickness of the cover glass P0 (or slide glass) on the surface of the cell sample sample P facing the tip of the objective lens. There is. Preferably, any mechanism may be provided in which the correction ring 4a is operated by the stepping motor 4b to automatically adjust the focal position of the objective lens.

Further, the illustrated fluorescence microscope 1 has an autofocus unit (automatic) having a function of driving and controlling the objective electric drive mechanism 5 to automatically adjust the focal position of the objective lens 4 to the surface on which the cells of the cell sample sample P are located. It is preferable that the focus adjustment mechanism) 20 is provided. As will be described later, in the analysis of the image of the cell of the present invention, the fluorescence intensity at the contour line of a predetermined width defined along the outer circumference of the cell is referred to, and the objective is during the measurement. If the focal position of the lens is deviated, the position and brightness of the contour line specified on the image data will change, and this change may affect the determination result of the membrane translocation reaction. Therefore, an autofocus unit is provided so that the focal position of the objective lens is maintained at a constant height in the cell sample P so that such a problem does not occur.

The autofocus unit 20 in the illustrated example is a so-called "pupil split type laser autofocus unit", and uses the reflected light of the laser beam applied to the bottom surface of the cell sample sample P through the objective lens 4 to use the objective lens 4 and the cells. The position of the sample sample P with respect to the bottom surface is automatically adjusted. To briefly explain the mechanism, first, the laser beam of infrared light having a wavelength of 785 nm (dotted line with an arrow in the figure) emitted from the laser diode 24 lit by the laser diode lighting circuit 22 is the aperture 26. , Passed through the beam splitter 28, reflected by the autofocus dichroic mirror 34 (reflecting light with a wavelength of 700 nm or more) provided in the optical path in the fluorescence microscope 1, incident on the objective lens 4, and cell sample. Reach the cover glass on the bottom of specimen P. At least part of the laser light that reaches the cover glass is reflected there, passes through the objective lens 4 again, is reflected by the dichroic mirror 34 for autofocus, and is then reflected by the beam splitter 28, and is reflected by the condenser lens 30. It passes through and is received by the detector 32. In the optical system having such a configuration, the incident position of the laser light on the objective lens 4 is eccentric with respect to the central axis thereof, so that the light is reflected by the bottom surface of the cell sample sample P and passes through the objective lens 4 again. The position of the spot of the laser light received on the detector 32 changes depending on the distance between the objective lens 4 and the bottom surface of the cell sample sample P. Therefore, the position of the spot of the laser beam on the detector 32 when the focal point of the objective lens 4 is aligned with the bottom surface of the cell sample sample P is specified in advance, and while the fluorescence image of the cell is taken, The objective electric drive mechanism 5 is controlled so that the position of the laser beam on the detector 32 is maintained at a predetermined position. Such control is automatically performed by the AF control circuit 36 based on the information on the position of the laser spot from the detector 32, and therefore, the distance between the objective lens 4 and the bottom surface of the cell sample specimen P is set in advance. Thereby, the observer or the experimenter then adjusts the focus of the objective lens. You don't have to do it. The AF control circuit 36 may also control the light emission output or ON / OFF of the laser diode 24 via the laser diode lighting circuit 22.

The autofocus unit 20 is not an essential configuration in the present invention, and if the microscope 1 is not provided, the observer or the experimenter manually adjusts the focal position of the objective lens 4. It may be adjusted, and such a case also belongs to the scope of the present invention. Further, an automatic focus adjustment mechanism other than the illustrated example may be adopted.

The multi-controller 10 controls the operation of the illumination light source 3 for observing transmitted light, the electric stage 2, the camera 16, and the autofocus unit 20 (objective electric drive mechanism 5) based on the command from the image analysis device 100, and also controls the operation of the camera 16. The fluorescent image of the cell sample sample taken in 1 is transmitted to the image analyzer 100. The configuration of the multi-controller 10 may be of any aspect known to those of skill in the art. Communication between the multi-controller 10 and the image analyzer 100 may be made in any form, for example CAN communication or USB connection.

The processing process in each means, each procedure and method in the cell image analysis apparatus of the present invention is realized by the configuration and operation of the image analysis apparatus 100. The image analysis apparatus 100 may be composed of a computer capable of performing any kind of image processing known to those skilled in the art. When the image analyzer 100 is activated, the image analyzer 100 executes a program for determining the membrane translocation reaction according to the present invention stored in the built-in storage medium in a manner described in detail below. The image analyzer 100 includes the operating status of each part of the keyboard 102 and the fluorescence microscope system into which the observer's or experimenter's instructions are input, and the cell sample taken by the camera 16 in the same manner as a normal personal computer. A monitor 104 on which a fluorescence image of the sample is projected is provided. In the illustrated example, the multi-controller 10 and the image analysis device 100 are configured as separate bodies, but they may be integrally configured with the image analysis device 100. Further, the image analysis device 100 may be a general-purpose personal computer, but may be configured as a unit or device specialized for cell image analysis according to the present invention.

<u style="single">Principle of determination of membrane translocation reaction</u> FIG. 2 schematically shows a fluorescence image before and after the membrane translocation reaction of a cell whose translocation molecule is fluorescently labeled (note that the cells are on the bottom surface of the well of the microtiter plate). Adhesive cells in a sticky state.). With reference to the figure, in the cells before the membrane translocation reaction (Fig. 2 (A)), the translocation molecules are free in the entire cytoplasm, so that the fluorescence intensity distribution of the cells is It has a shape as shown in Fig. 2 (B). When a specific stimulus is given to such cells and a membrane translocation reaction occurs, translocation molecules accumulate near the cell membrane and fluoresce as shown in FIGS. 2 (C) and 2 (D). The fluorescence intensity of the outer periphery of the cell increases relatively in the image (actually, the translocation molecule accumulates on the entire surface of the cell including the upper and lower surfaces of the cell, but the fluorescence intensity in the fluorescence image is shown in the figure. As shown in 2 (E), since the integration is performed in the focal depth direction of the objective lens, the fluorescence intensity of only the outer periphery of the cell is increased when viewed in a fluorescence image.)

When the distribution of fluorescence intensity changes due to such a membrane translocation reaction, the observer or the experimenter previously compares FIGS. 2 (A) and (C) or (B) and (D), so to speak. Since it was sensuously determined whether or not there was a change in the fluorescence intensity distribution due to the membrane translocation reaction, the determination criteria varied, and since the determination was made for each cell, it took time. Therefore, in the present invention, the region where the cells exist in the fluorescence image is specified based on the brightness (fluorescence intensity) in the fluorescence image, the contour line having a predetermined width is determined, and the contour line is defined. Based on the ratio of the fluorescence to the brightness inside the contour line, it is numerically determined whether or not the membrane translocation reaction has occurred (see Fig. 2 (D)). The determination of the film translocation reaction based on the ratio of the brightness in the contour line to the brightness inside the contour line is preferably determined in the average value of the brightness in the entire contour line and the entire area inside the contour line. It is determined by whether or not the ratio with the average value of the brightness to be squeezed exceeds a predetermined value. It should be understood that these processes are automatically performed in the image analysis apparatus 100 by performing arithmetic processing using the brightness value and the coordinate value of each pixel in the image data. The variability of the criteria will not affect the results, and the effort and time required of the observer or experimenter will be significantly reduced.

<u style="single">Preparation of cell samples</u> In the above-mentioned device or method for determining the membrane translocation reaction of the present invention, as already mentioned in the column of "disclosure of the invention", the cells are released into the cytoplasm by stimulating the cells. It is possible to determine the reaction in which the protein is accumulated in the cell membrane of the cell, or the reaction in which the protein existing in the cytoplasm in the cell is accumulated in the cell membrane of the cell by stimulating the cell. .. For example, in HeLa cells, which are a type of cervical cancer cell line, the reaction in which protein kines C (PKC) floating in the cytoplasm moves to the cell membrane by stimulation with PMA (phorpol myristyl acetate) or radiation. (Example of membrane translocation reaction from cytoplasm to cell membrane) In adipocyte line 3T3-L1, the molecule GLUT4 existing on the transgordi membrane in the absence of insulin is stimulated by insulin. Reactions that rapidly move to the surface of the cell membrane (an example of a membrane translocation reaction from the membrane of an organelle to the cell membrane. GLUT4 exerts an activity of taking up glucose into the cell by such a reaction). It is possible to determine the reaction.

In the cell in which the presence or absence of the membrane translocation reaction is determined in the present invention, the translocation molecule inside needs to be fluorescently labeled. The introduction of the fluorescent label into the cell is preferably carried out by expressing the translocation molecule in the cell in a fused state with the fluorescent protein or the halotag molecule by using a gene transfer technique. When a translocation molecule in which a halotag molecule is introduced is expressed in a cell, a fluorescent dye that specifically binds to the tag molecule is introduced into the cell after the expression, and the translocation molecule is fluorescently labeled. The introduction of the fluorescent label into such cells may be carried out by any method known to those skilled in the art.

For example, when determining the membrane translocation reaction of PKC stimulated by PMA in HeLa cells, which is a type of cervical cancer cell line, first, a PKC DNA vector in which a halotag molecule is introduced into the cells is used. Is introduced using any lipofection gene transfer technique. After that, the cells are cultured for 16 to 24 hours, washed appropriately, and the fluorescent dye TMR (tetramethylrhodamine) having a halotag ligand is added. The TMR with halotag ligand automatically invades the cell and specifically binds to the halotag of the PKC molecule expressed in the cell. Thus, after 15 minutes, the cells are appropriately washed to remove the fluorescent dye in the culture medium, further added with a normal medium, PMA is given to the medium, and the cells are allowed to stand for about 10 minutes. Here, if DNA transfer is successful, when HeLa cells respond to PMA stimulation, TMR-labeled PKC molecules migrate from the cytoplasm to the cell membrane, schematically shown in FIG. 2 (C). As shown above, the fluorescence intensity of the outer edge of the cell image is increased. Since the above-mentioned accumulation of PKC molecules near the cell membrane disappears relatively quickly, 80% ethanol is given to the medium at the time of observation to fix the cells in a state where a membrane translocation reaction is occurring. Is desirable.

As is well known to those skilled in the art, PKC used here phosphorylates and dephosphorylates in the process of intracellular signal transduction to reversibly control the functions of various proteins, and various cell functions. It regulates and is deeply involved in intracellular mechanisms involved in cell proliferation, differentiation, carcinogenesis and apoptosis. Therefore, the determination of the above-mentioned PKC membrane translocation reaction performed according to the present invention is used as a tool for searching for intracellular signal transduction substances, screening for substances having an arbitrary bioactive effect, and evaluating gene transfer efficiency. Expected to be used.

In addition, when determining the membrane translocation reaction from the transgordi membrane to the cell membrane surface by insulin stimulation of GLUT4 in the adipocyte strain 3T3-L1, the same gene transfer technique as in the case of Hela cells into the adipocyte strain Introduces a gene that expresses GLUT4 fused with a fluorescent protein such as GFP, and expresses the fluorescent protein fused GLUT4. When insulin stimulation is given to such cells by an arbitrary method for those skilled in the art, if gene transfer is successful, fluorescent protein-fused GLUT4 migrates to the vicinity of the cell membrane, as schematically shown in FIG. 2 (C). , The fluorescence intensity of the outer edge of the cell image will increase.

The sample sample placed on the stage of the fluorescence microscope is prepared as described above after the cell sample prepared as described above is dispensed into the well of the microplate P or the cells are dispensed into the well of the microplate P. It may be prepared by being fluorescently labeled by the procedure and given a stimulus. As already described, the brightness on the contour line of the cell is likely to change depending on the focal position of the objective lens, and therefore, it is advantageous to use a sample in which the objective lens is easily focused. Thus, the cells may preferably be any adhesive cells that adhere to the bottom surface of the wells of the microplate P. However, it should be understood that the cells may be spherical or non-sticking to the bottom of the well (in which case, preferably fixed in any way to stick to the bottom of the well of the microplate P). .).

<u style="single">Acquisition of fluorescence images of cells using a fluorescence microscope observation system</u> FIG. 3 (A) shows the processing process until the fluorescence image of the cell sample sample P prepared as described above in the above fluorescence microscope observation system is acquired as image data in the form of a flowchart. .. With reference to the figure, first, when the observer or the experimenter turns on the power of the image analysis device 100 (and the multi-controller) and operates the keyboard 102 in the image analysis device 100 to start the dedicated software, The settings of each part of the fluorescence microscope 1 are initialized, and a screen for instructing various settings and operations of the fluorescence microscope observation system is displayed on the monitor (step 10). At this stage, the observer or the experimenter can take a picture of the well to be photographed from the microtiter plate P into which the cell sample prepared as described above is dispensed from the image analyzer 100, the number of images taken for one well, Various settings such as the excitation light wavelength are input (step 20). Also, at this point, the objective should be maintained by the autofocus unit to determine the distance between the objective lens 4 and the bottom surface of the microtiter plate P so that the objective lens 4 is in focus on any cell sample in the well. The height of the lens may be determined. Further, the correction ring 6 of the objective lens 4 may also be automatically adjusted to match the thickness of the cover glass on the bottom surface of the microtiter plate P.

After that, when the observer or the experimenter gives a shooting start instruction from the image analyzer 100, the excitation light source 6 is turned on (or the shutter of the optical path of the excitation light source 6 (not shown) is opened), and the camera 16 is activated. The imaging of the fluorescent image is started (step 30). In photographing the fluorescent image, first, the electric stage 2 is moved in the XY direction under the control of the multi-controller 10 so that the well to be photographed input in step 20 enters the field of view of the objective lens 4. (Step 40). When the stage is moved, as described above, the autofocus unit keeps the relative position between the focal point of the objective lens 4 and the bottom surface of the well constant, so that the observer or the experimenter basically keeps the position constant. There is no need to adjust the height of the objective lens. Thus, when the movement of the well is completed, the fluorescence image formed on the light receiving surface of the camera is taken into the image analysis device 100 as image data (still image) via the multi-controller 10 (step 50). As will be understood by those skilled in the art, it should be understood that the capture of image data is performed in a manner corresponding to the format of the camera used. For example, in the case of a CCD camera or the like that outputs video at a video rate, images of an arbitrarily set number of frames may be integrated and captured as image data. Further, in the case of a cooled CCD, an image corresponding to the electric charge accumulated on the light receiving surface at an arbitrarily set exposure time is taken into the image analysis apparatus 100 as one image data. Thus, when the fluorescence image acquisition for the current well is completed, the stage is moved and the fluorescence image acquisition for the next well is performed (steps 40 and 50). Then, when the acquisition of the image data of the fluorescence image for all of the planned wells is completed (step 60), the analysis of the image data described later is executed.

<u style="single">Procedure for determining the membrane translocation reaction</u> FIG. 3 (B) shows the process of determining the membrane translocation reaction from the fluorescence image (FIG. 4 (A)) of the cell sample sample obtained in FIG. 3 (A) in the form of a flowchart. .. Although the image analysis described below is executed following the acquisition of the fluorescence image, the image data prepared and stored by another device or system may be imported into the image analysis device 100.

With reference to the figure, first, the region where the cell image exists is determined based on the fluorescence intensity in the fluorescence image containing the images of a plurality of cells (steps 100 and 110). As can be understood from FIG. 2, since the fluorescence intensity of the region where the cells are present is higher than that of the background, it is considered that the cells are present in the pixel region having the fluorescence intensity of a certain threshold value or higher. be able to. More specifically, since the fluorescence intensity distribution is slightly wider than the region where the cells really exist, a predetermined threshold value is arbitrarily set at the tail of the fluorescence intensity distribution, and a pixel region having a fluorescence intensity equal to or higher than the threshold value is set. It may be assumed that cells are present in. In the arithmetic processing, typically, the fluorescence image is binarized using a predetermined threshold value (1 for pixels having brightness above the threshold value and 0 for pixels having brightness below the threshold value). Give. Step 100, Fig. 4 (B)), after that, the cell exists in the area where the area given the brightness 1 in the XY direction of the image continuously expands over a predetermined area (number of pixels). Determined as an area (step 110). The coordinates of the determined region of the cell are recorded for each cell and used for later calculations.

A person skilled in the art may arbitrarily configure an algorithm for detecting a region in which a region given brightness 1 is continuously expanded by a predetermined area (number of pixels) or more. As shown in the cell images d and e in Fig. 4 (B), since the nucleus or other organelles are not fluorescently labeled, the brightness is inside the region where the pixels with brightness 1 are continuously spread over a predetermined area. When there is a region of 0, the pixel region having a brightness of 0 inside is also specified as an region in which cells exist.

Once the region in which the cells are present has been identified, the number of regions is used in later calculations as the number of cells in the fluorescence image (step 120).

As described above, after the individual regions of existence of the cells are defined and the number of cells is counted, further, in each of the pixel regions where the individual cell images are present, a predetermined width is set from the outer edge to the inside of the region. The contour line of is specified by the coordinates on the image data (step 130). The width of the contour line may be arbitrarily set by the observer or the experimenter. As already mentioned in the "Disclosure of Invention" column, the spread of fluorescence intensity near the outer edge of the cell in the membrane translocation reaction may differ depending on the cell type. As can be seen from Fig. 2 (C), if the width of the contour line is too wide than the extent of the increased fluorescence portion of the outer edge of the cell image, the contour line will contain a region where no increase in fluorescence can be seen. , The change in fluorescence intensity on the contour line will be reduced. On the contrary, if the width of the contour line is too narrower than the spread of the increased portion of fluorescence at the outer edge of the cell image, the number of pixels for picking up the luminance value is reduced, the reliability of the result is reduced, and the fluorescence is increased. There is a possibility that it will deviate from the increased portion, and in that case, the change in fluorescence intensity on the contour line will also be reduced. In order to set an appropriate contour line width, it is necessary to carry out a preliminary experiment and determine an appropriate width.

In the binarized image of FIG. 4 (B), a linear region having a width of several pixels from the outer edge of the cell image to the inside of the region, that is, a pixel on the contour line is given a brightness of 1, and the inside of the contour line is given brightness 1. When the brightness of 0 is given to the pixel, the image is formed only by the contour line as shown in FIG. 4 (C).

Thus, after the pixel region in which each cell exists and the contour line region are identified, the brightness value of the pixel in the contour line region is obtained from the image data of the fluorescence image for each cell, and the average value is calculated. Further, the brightness value of the pixel in the pixel region where the cells inside the contour line region are present is obtained from the image data of the fluorescence image, and the average value is calculated. Then, the ratio of the brightness average value of the contour line region to the brightness average value inside the contour line is calculated for each cell (step 140). As described above, since the coordinates of the pixel region where the cells exist and the contour line region are determined, such an operation is automatically executed by an arbitrary program in an image analysis device which is a computer. To. Thus, (Average brightness in area of contour line) / (Average brightness inside contour line)&gt; X When is established, translocation molecules are accumulated in the vicinity of the cell membrane, and it is determined that a membrane translocation reaction has occurred (step 150). The predetermined value X may be preset by the observer or the experimenter. Further, in the above-mentioned determination of the membrane translocation reaction, the magnitude of the ratio of the luminance average values is evaluated by a plurality of different values, and the degree of progress of the membrane translocation reaction may be classified into several stages. Since the predetermined value X is used fixedly for at least all of the cells currently being examined, variability in the criteria is avoided.

Further, after the presence or absence of the membrane translocation reaction of each cell is determined, the number of cells that have caused the membrane translocation reaction is counted, and the total number of cells counted earlier is used. (Number of cells generating membrane translocation reaction) / (Total number of cells) To calculate the reaction rate of the membrane translocation reaction (step 160).

Prior to the above-mentioned series of image processing, preferably, the image data of the fluorescence image may be shaded or background-corrected in a known manner. Further, the total number of cells may be determined by dyeing the cell nucleus with another dye, taking a fluorescence image of only the nucleus, binarizing the image, and counting the number of regions in which the nucleus exists. Since the shape and size of the nucleus are stable, the region where the nucleus of each cell exists can be easily and surely determined, and the number of cells can be accurately counted even when the cell images overlap. it can.

<figref num="1">FIG. 1 is a schematic diagram of a fluorescence microscope system incorporating a preferred embodiment of the cell image analyzer of the present invention.</figref><figref num="2">FIG. 2 (A) is a schematic diagram of a fluorescence image of cells that have not undergone a membrane translocation reaction, and FIG. 2 (B) shows the fluorescence intensity distribution on the horizon in (A). FIG. 2 (C) is a schematic diagram of a fluorescence image of cells undergoing a membrane translocation reaction, and FIG. 2 (D) shows the fluorescence intensity distribution on the horizon in (C). In (B), the fluorescence intensity of the central part of the cell is low because the fluorescently labeled protein is not distributed in the nucleus. In (C) and (D), the fluorescently labeled protein in the cytoplasm moves to the cell membrane, so that the contrast between the cytoplasm and the nucleus is almost lost. FIG. 2 (E) is a schematic cross-sectional view of a cell in which a fluorescently labeled protein (gray region) is accumulated near the cell membrane by causing a membrane translocation reaction, and when the fluorescent label is accumulated near the cell membrane. It is a figure explaining that the outer edge of a cell image looks bright.</figref><figref num="3">FIG. 3A is a diagram showing the process of acquiring a fluorescence image of a cell sample sample using the system of FIG. 1 in the form of a flowchart. FIG. 3 (B) is a diagram showing the process of determining the membrane translocation reaction from the fluorescence image obtained by the process of (A) in the form of a flowchart.</figref><figref num="4">FIG. 4 (A) is a schematic diagram of a fluorescence image of a cell sample sample after being stimulated. In the figure, the cells of ac, f, and g are in a state of causing a membrane translocation reaction, and the cells of d and e are in a state of not causing a membrane translocation reaction. FIG. 4 (B) is an image obtained by binarizing the image of (A) with a predetermined threshold value, and FIG. 4 (C) is an image showing the position of the contour line.</figref>

Code description

1 ... Fluorescence microscope 2 ... Electric stage 3 ... Light source for observing transmitted light 4 ... Objective lens 5 ... Objective electric drive mechanism 6 ... Excitation light source for fluorescence observation 7 ... Dichroic mirror 8 ... camera 10 ... multi controller 20 ... Autofocus unit 100 ... Computer (image analyzer)