Fluorescence measuring system

The invention relates to a. Fluorescence measuring system as it can be used, for example, in high-throughput screening in drug development.

One common method of non-contact detection or analysis of a substance is to label the substance with a selected fluorescent dye. The selection of the fluorescent dye generally depends on the substance property to be investigated. For such analyzes, the labeled substance is illuminated with light of a wavelength or a wavelength range which is tuned to the absorption behavior of the fluorescent dye used (generally the maximum of the absorption). The response of the fluorescent dye is the emission of the fluorescent dye characteristic emission light whose wavelength is generally longer wavelength than that of the exciting radiation. With a suitable choice of the labeling fluorescent dye, changes in the property of the substance, eg.

A subclass of substances are, for example, biomolecules (DNA, proteins, lipids) and cell fragments, biological cells or tissue or organic or inorganic carriers ('beats' or 'microspheres') on which biomolecules have been immobilized. It is state of the art that this biological material can be fluorescently labeled by appropriate techniques.

A common technique in drug discovery is to use fluorescence techniques for the evaluation of test substances by staining the test system to be tested (biomolecules and / or cells and / or biomolecule / cell immobilized carriers) with a suitable fluorescent dye, see above that the changes in parameters can be quantitatively determined by the interaction of test substances. Typical examples of this are the staining with ion-sensitive fluorescent dyes, such as those for the detection of K +, Ca ++ and their changes, or for the detection of changes in membrane potential or the intracellular pH are commercially available.

Frequently, appropriately prepared and fluorescently labeled cells are used in small reaction vessels for such tests. These are typically available on a surface of approximately 108 mm x 72 mm in a matrix arrangement as so-called standardized microtiter plates with a footprint of approximately 128 mm x 86 mm in the 96, 384 or 1536 test hole version. Often, biological cells grow in a nutrient solution on the bottom of the test holes and form a so-called cell layer, which can be excited to fluorescence in a variety of ways after appropriate staining. An automated standard method is to evaluate a microtiter plate using commercial microtiter plate readers with fixed fluorescence dye excitation and emission measurement geometry. For large-scale and complete evaluation, the microtiter plate is transported sequentially test hole for test hole electromechanically in the excitation / measurement position. There are measuring systems with different illumination geometries for the vertical excitation and measurement of fluorescence from above or through the transparent bottom of microtiter plates from below as it is eg in<patcit id="pcit0001" dnum="DE19720667A1"><text>DE 197 20 667 A1</text></patcit> (or parallel in <patcit id="pcit0002" dnum="US5933232A"><text>US 5,933,232</text></patcit>) is described.

Since the high-throughput screening of active ingredient research is intended to examine several million chemical substances in terms of their effects in the shortest possible time, one prerequisite for this high throughput is a high measuring speed. Here, in the conventional fluorescence readers, the mechanical movement of the plate is in the way. For a microtiter plate with 1536 test holes, which must be approached individually, the measurement times are in the range of minutes. Significantly faster work the technically more complex laser scanning systems (<nplcit id="ncit0001" npl-type="s"><text>K. Schroeder ea: FLIPR: A New Instrument for Accurate, High Throughput Optical Screening; J. of. Biomolecular Screening, Full, Number 2, 1996</text></nplcit>; <patcit id="pcit0003" dnum="US5355215A"><text>US 5,355,215</text></patcit>), in which the cells in all test holes of a microtiter plate are excited by a laser beam sequentially, but at the same time by a very fast Strahlführungnährungsweise to fluorescence. Scanning with the aid of rotating deflecting mirrors results in a quasi-planar excitation of the objects whose fluorescence is detected by an integrating image sensor. A glare system minimizes possible background fluorescence. Despite or because of the low residence time of the laser, the local irradiance is very high, so that there is a risk of fading of the generally not very light-stable fluorescent dyes and thus a misinterpretation of the measured values. In addition, the system is limited by the number of excitation wavelengths of the laser. Thus, excitation in the near UV at, for example, 360 nm with the common argon ion laser with its excitation maximum at 488 nm is hardly possible. A change of the laser is cumbersome; often necessary (water) cooling costly. The adjustment of the optics is difficult for a routine application and can only be carried out by specialist personnel; the mechanics (rotating deflecting mirrors) are potentially prone to failure.

The individual test wells of a microtiter plate contain in very many cases in the supernatant of the cells nor the fluorescent dye with which they have been stained. In some cases this is absolutely necessary (eg for distribution dyes for measuring the membrane potential); but also a washing process in which a washing out of the residual dye by dilution is problematic. During a washing process, it can not be ensured that the cell layer is not damaged or that the cells alone do not change their biological functionality due to the flow dynamics during aspiration and thus the measurement results are not falsified. An additional washing step also significantly reduces sample throughput.

In the conventional fluorescence measuring systems, their illumination geometry, in which the sample is excited vertically from below or from above, proves to be very disadvantageous. If fluorescent dye is still present in the cell supernatant as described above, then the background signal due to the excited liquid column in the supernatant can constitute a multiple of the cellular fluorescence change resulting from the interaction of a test substance with the cells. The reason for this is that the fluorescence-labeled cell monolayer generally only has a thickness of 10 μm, but the height of the supernatant is several millimeters. A falsification of the measurement results is thereby inevitable.

<patcit id="pcit0004" dnum="US5854684A"><text>US 5,854,684</text></patcit> discloses illumination of a planar substrate by two symmetrically arranged light sources at an angle &lt;90 ° from above and detection of the light reflected at the planar substrate by a camera located vertically above the planar substrate. This measurement arrangement is not suitable for microtiter plates, since shadowing effects can occur due to the depth of the test wells of the microtiter plate in this arrangement.

<patcit id="pcit0005" dnum="EP0751393A"><text>EP 0 751393 A</text></patcit> discloses the illumination of a microtiter plate through the transparent bottom. The illumination is provided by two arrays of light sources, each of which is provided with stray filters to achieve a homogeneous illumination of the bottom of the microtiter plate. In addition, the container in which the light sources and the detector system are located, provided with strongly scattering surfaces. This illumination arrangement causes significant portions of the excitation light at an opening angle greater than 30 ° reach the test holes, and thus a falsification of the measurement results by excitation of the supernatant in the test holes.

<patcit id="pcit0006" dnum="DE19914279C"><text>DE 199 14 279 C</text></patcit> discloses an arrangement for optically reading the information from a matrix substrate with a plurality of individual samples. However, the matrix-shaped substrate is not a microtiter plate. Illumination and detection through the bottom of the sample carrier is also not disclosed, nor are linear illumination sources arranged on opposite sides of a microtiter plate.

<patcit id="pcit0007" dnum="US6362006B1"><text>US 6,362,006 B1</text></patcit> discloses the illumination of a microtiter plate by two symmetrically arranged radiation sources, which are arranged at an angle &lt;90 ° to the microtiter plate, and the detection of the light reflected on the microtiter plate in a detector which is arranged vertically above the microtiter plate. This arrangement has the disadvantage that, because of the depth of the test holes in the microtiter plate, shading effects and hence uneven illumination of the samples at the bottom of the test holes may occur.

Quantitative fluorescence analysis thus revealed the need for a novel homogeneous area excitation method that meets the following requirements:<ul><li>Excitation of a fluorescent dye at least in the UV-VIS range (220 nm-800 nm) at freely selectable centroid wavelengths;</li><li>Minimization of disturbing background signals due to special illumination geometry;</li><li>Increase of the measuring speed by parallel, ie simultaneous excitation of all distributed over the measuring surface samples and parallel processing with imaging detection methods (in comparison to the sequential measurement);</li><li>Avoidance of fading effects due to minimal local radiant power;</li><li>Exclusion of mechanically moved components;</li><li>Avoidance of complex optical readjustments after setting up the system;</li><li>Detection sensitivity comparable to conventional fluorescence measurement systems.</li></ul>

The solution of the object according to the invention consists in a fluorescence measuring system with the features of claim 1.

The detector system is arranged at a substantially horizontal position of the microtiter plate below the microtiter plate.

The microtiter plate typically has formats with 96, 384 or 1536 test holes. The microtiter plate is transparent.

The illumination of the transparent bottom of the microtiter plate is preferably carried out with a homogeneity of ± 20%, preferably ± 10%. Such homogeneity can be achieved by arranging each of the linear illumination sources on each of two opposite sides of the microtiter plate. Each of the linear illumination sources illuminates the nearer area of ​​the bottom of the microtiter plate with greater intensity than the area further away, since the intensity I of the excitation light is I ~ 1 / R² decreases with increasing distance R from the illumination source. Due to the inventive geometric, that is symmetrical, arrangement of the illumination sources, the illumination intensity over the entire bottom of the microtiter plate is substantially constant. Preferably, the two linear illumination sources are arranged on each of the two longitudinal sides of a rectangular microtiter plate. However, it is also possible, for example, for four illumination sources to be present, which are arranged in pairs symmetrically on four different sides of the microtiter plate, so that the illumination intensities of the four illumination sources are superimposed and the illumination intensity over the entire bottom of the microtiter plate is substantially constant.

The linear illumination sources may be formed as linear halogen fuel rods, as fluorescent tubes or as LED rows. However, the linear illumination sources are preferably linearly arranged light-conducting fibers, which can be arranged in a special arrangement of a round fiber bundle to form a so-called fiber cross-section converter. Light-conducting fibers may be made of special polymer material such as PMMA (PolyMethylMeth acrylate) or PC (polycarbonate) or of standard glass or quartz glass and are suitable in the diameter range of about 5 microns to 2 mm. To compensate for inhomogeneities in the light irradiation on the input side of the light guide, the fibers of the cross-section transducer should be randomly mixed.

The linear illumination sources emit a wavelength corresponding to the excitation wavelength wavelength for fluorescence excitation of the samples in the test wells of the microtiter plate. The illumination sources can emit colored light or correspondingly filtered white light, for example, in that the light of corresponding lamps is fed to the input side of the fiber cross-section converter and is monochromatized via filters, preferably interference filters. Accordingly, the illumination sources can also emit the light of flash lamps for pulsed excitation or of UV lamps for fluorescence excitation in the UV range (220-400 nm). Likewise, a continuously radiating laser with a corresponding expansion optics can be coupled into the cross-sectional transducer adapted to the numerical aperture of the fiber optic. With a pulsed laser in the same arrangement, there is the possibility of imaging time-delayed fluorescence measurement (TRF). Due to the generally short fluorescence lifetime of the interfering background, the signal-to-noise ratio can be drastically improved given a corresponding fluorescence lifetime of the useful signal.

The detector system may include an imaging detector, such as a video measuring system, which preferably includes a residual light amplifier device. The residual light amplifier device can have a gating device, which can serve to make the camera sensitive to the useful signal only after a short laser excitation pulse (order of magnitude pico seconds) in the range of nano seconds.

Between the microtiter plate and the detector system, one or more filters may be arranged which select the fluorescent light prior to detection. Several filters that are to be used alternatively can be arranged in a filter change device.

The fluorescence measuring system can also have one or more dichroic mirrors between the microtiter plate and the detector system, by which the fluorescent light from the microtiter plate is split into two or more wavelengths and selectively decoupled and detected.

The advantage of the fluorescence measuring system according to the invention is that a microtiter plate is illuminated altogether at the same time, and not just the core region of each test hole.

The fluorescence measuring system according to the invention can be tuned by a suitable selection of excitation light sources and filters from the UV to the near infrared region (220-800 nm).

Because of the simultaneous and parallel measurement of all test holes of a microtiter plate, the fluorescence measuring system according to the invention is particularly suitable for kinetic measurements which can be carried out simultaneously with the fluorescence measuring system according to the invention on all test holes and thus saves a great deal of time over conventional kinetic measurements Mean systems.

<u>Figures and examples</u>

The figures show<dl id="dl0001" compact="compact"><dt>Fig. 1</dt><dd>Principle of homogeneous excitation</dd><dt>Fig. 2</dt><dd>Cross section of a glass fiber cross-section converter</dd><dt>Fig. 3</dt><dd>Construction of a fluorescence measuring system</dd><dt>Fig. 4</dt><dd>Illumination system with two cross-section transducers</dd><dt>Fig. 5</dt><dd>Measurement results on a 1536 test hole microtiter plate</dd><dt>Fig. 6</dt><dd>Spatially resolved fluorescence emission measurement from the test wells of a 384 test well microtiter plate with the fluorescence sensing system of the present invention</dd></dl>

<figref idref="f0001">Fig. 1</figref> shows how a homogeneous illumination of a microtiter plate 1 is achieved by two illumination sources 3. The microtiter plate 1 with the test holes 2 is illuminated from below by the two symmetrically arranged illumination sources 3. The volume 4 illuminated in the test holes 2 by the illumination sources 3 is colored dark and substantially constant for all test holes 2.

<figref idref="f0002">Fig. 2</figref> shows a cross section of a fiber cross-section converter 21. The excitation light is supplied through the Lichtleitbündel 22. In the cross-sectional transducer 21, the optical fibers of the bundle 22 are rearranged into a linearly extending row of fibers 23. The width of the line results from the diameter of the input beam and is about 1 mm with a length of 150 mm and an input cross section of 15 mm. The emitted light 24 typically has, for example in the case of a glass fiber cross-sectional converter, an aperture angle α of approximately 30 °, which corresponds to an aperture of 0.5 = sin α / 2.

<figref idref="f0002">Fig. 3</figref> shows the structure of a fluorescence measuring system. The fluorescence measuring system consists of a housing 330 with a lid 340. In the lid 340 there is a recess on which a microtiter plate 31 is positioned so that it can be illuminated from the interior of the housing 330. Inside the housing 330 are the other components of the fluorescence measuring system. The microtiter plate 31 with the base of about 128 mm x 86 mm and transparent bottom plate 360 ​​is illuminated by the bottom plate 360 ​​of two glass fiber cross-sectional transducers 32 at an angle of about 25 ° with the excitation light 33. The two glass fiber cross-sectional transducers 32 are arranged along the 128 mm long sides of the microtiter plate 31. The fluorescent light 34 emitted through the bottom plate 360 ​​of the microtiter plate 31 is directed via the dichroic mirror 35 via a lens 39 to an integrating CCD camera 350. Between the dichroic mirror 35 and the lens 39 is an interference filter 36 disposed in a filter wheel 37. The filter wheel 37 can be rotated by means of a motor 38.

<figref idref="f0003">Fig. 4</figref> shows an illumination system with two fiber cross-section transducers 41. The input light for the fibers is generated with a lamp unit 46. By way of a filter slide 45, light of different wavelengths can be filtered out of the excitation spectrum of the lamp unit 46. On the fiber mixing section 44, the optical fibers are randomly mixed and split at the distributor 43 into two strands 47. At the end of the strands 47 are the fiber cross-section transducers 41 with the linearly arranged fiber exit surfaces 42.

<figref idref="f0004">Fig. 5</figref> shows an example of the measurement of the fluorescence emission from the test holes of a 1536 test hole microtiter plate with the fluorescence measuring system according to the invention. All test holes contain the same fluorescence solution. The homogenous illumination results in an image of the microtiter plate on which the emission of all test holes within the given tolerances is uniformly bright.

<figref idref="f0005">Fig. 6</figref> Figure 14 shows an example of a spatially resolved measurement of fluorescence emission from the test wells of a 384 test well microtiter plate (24 x 16 holes) with the fluorescence sensing system of the present invention. In each test hole are different cell clones, which express a fluorescence protein, the so-called GFP (green fluorescent protein) differently well. We are looking for the clone with the best expression. It is obvious that the mean value over a test hole (principle: fluorescence reader) provides incorrect data / statements, since only the local resolution within a test hole identifies those cells which fluoresce particularly brightly, that is, a good expression