METHOD FOR REDUCING LIQUID EVAPORATION FROM WELLS OF A MICROPLATE

Description

[0001] The invention relates to a method for reducing liquid evaporation from wells of a microplate and a microplate reader.

[0002] Microplate readers, which can be used to optically examine or analyze the contents of one or more wells of a microplate, have been known for a long time. In the context of the present invention, the term microplate refers to all multi-well plates which have a large number of wells or containers, which are arranged, for example, in an array. Particularly preferred microplates have at least approximately the mass and footprint of a microplate according to the SBS standard, as published by the American National Standards Institute (ANSI). For example, standard microplates are known whose wells are equipped with a round bottom, flat bottom, or V-bottom. What all of these standard microplates, with their very different well shapes, have in common is that the axial distance of the wells arranged in an array is also standardized (cf. ANSI_SBS 1-2-3-4-2004 Standard for Microplate Dimensions from 2006).This center distance is, for example, 18 mm for 24-well (4 × 6) plates, 9 mm for 96-well (8 × 12) plates, 4.5 mm for 384-well (16 × 24) plates, and 2.25 mm for 1536-well (32 × 48) plates. The height of a standard microplate can vary greatly depending on the type and is typically between 10.4 mm (e.g., 1536 V-bottom deep well plate) and 44 mm (e.g., 96-well Masterblock ®< from Greiner).

[0003] Known microplate readers are equipped with appropriate light sources and / or detectors to examine samples or samples containing test solution, also called samples, in the wells of microplates based on their absorption, fluorescence and / or luminescence, for example. The samples are located in a test solution that is exposed to environmental influences. Particularly in long-term series of experiments with cell cultures in the wells, which are typically carried out in stand-alone microplate readers for hours or even days and possibly at temperatures higher than room temperature, evaporation problems can arise for the samples or the test solution containing the samples. The evaporation of the test solution leads to thickening and thus to a change in the concentration of buffer substances and molecules to be examined (analytes).This can, for example, alter the growth conditions for cell-based experiments and / or the response of cells to experimental influences. It has also been observed that the test solution from wells arranged in the corners of a standard microplate is more exposed to such evaporation problems than from wells arranged in the center of a microplate. This, in turn, means that thickening does not occur evenly across all wells of a microplate, but rather leads to differences, and thus to incomparable results, within the same series of experiments.

[0004] Devices for preventing or reducing such evaporation problems are known from the prior art. For example, patent EP 2943797 B1 discloses an incubation cassette for reducing liquid evaporation from microplate wells. The incubation cassette comprises a frame for accommodating a microplate. Furthermore, the incubation cassette provides a liquid-fillable reservoir in the form of a liquid channel that surrounds the inserted microplate. This enriches the atmosphere in the immediate vicinity of the microplate wells, thus delaying the thickening of the sample liquid due to evaporation.

[0005] US 4,800,164 A describes a very compact, automated device for analyzing and cloning cell cultures, as well as for bacteriological analysis, which allows for the maintenance, experimental, and optical analysis phases. The device comprises a small chamber containing a plate with several wells made of a transparent material, an optical observation unit, a device for aspirating / injecting the cell medium or cells, and a motor unit for driving the plate. The small volume of this chamber ensures automatic and constant humidification through evaporation of the cell medium contained in the wells. The injection device works in conjunction with a device for controlling the level of the liquid medium injected into each well.

[0006] However, with the current technology, the liquid in the liquid channel can completely evaporate, which means the atmosphere in the immediate vicinity of the microplate wells is no longer enriched. Therefore, the liquid level or liquid content in the liquid channel must be frequently checked and manually refilled to maintain functionality. Checking and manually refilling the liquid channel is laborious and time-consuming. Another disadvantage is that the liquid channel cannot be refilled with liquid for some measurements, especially during long-term measurements, without introducing interference into the experiment.

[0007] It is therefore an object of the present invention to propose a method for reducing liquid evaporation from wells of a microplate and a microplate reader in which disadvantages known from the prior art are eliminated.

[0008] This object is achieved by a method for reducing liquid evaporation from wells of a microplate according to claim 1. Furthermore, the object is achieved by a microplate reader according to claim 7.

[0009] The inventive method for reducing liquid evaporation from wells of a microplate comprises: a) Providing the microplate having a plurality of wells b) Adding a sample to at least one of the wells of the microplate c) Inserting the microplate or an incubation cassette containing the microplate into a microplate reader d) Injecting a liquid into a liquid reservoir provided in the microplate and / or the incubation cassette e) Performing measurements on the samples in the respective wells f) Optically measuring a liquid level in the liquid reservoir of the microplate and / or the incubation cassette through a substantially transparent portion of the liquid reservoir of the microplate and / or the liquid reservoir of the incubation cassette g) Re-injecting the liquid into the liquid reservoir of the microplate and / or the liquid reservoir of the incubation cassette when the liquid level falls below a predetermined thresholdh) repeating steps e) to g) until a predetermined number of measurement cycles is reached, i) removing the microplate or the incubation cassette containing the microplate from the microplate reader, wherein the liquid reservoir is provided between the wells and is delimited by a wall of the microplate and is designed to hold a liquid, wherein the microplate is provided with a liquid reservoir and comprises at least one transparent section, and / or wherein the incubation cassette comprises a frame for holding a microplate with wells, wherein the frame comprises a central first opening surrounded by an inner wall, the dimensions of which are designed for inserting a microplate, and the frame has a substantially parallel to the inner

[0010] Wall extending outer wall, which connects to the inner wall via an intermediate floor, wherein the liquid reservoir is formed by the two walls and the intermediate floor as a liquid reservoir surrounding the first central opening for receiving a liquid, wherein at least one section of the incubation cassette forming the liquid reservoir is provided at least in sections with at least one transparent section.

[0011] The sequence of the steps of the method according to the invention can be changed. For example, the liquid can also be injected into the liquid reservoir of the microplate and / or the incubation cassette outside the microplate reader, i.e., before the microplate or the incubation cassette containing the microplate is inserted into the microplate reader.

[0012] An incubation cassette for reducing liquid evaporation from wells of a microplate comprises a frame for receiving a microplate with wells, wherein the frame comprises a central first opening surrounded by an inner wall, the dimensions of which are designed for inserting a microplate, and the frame comprises an outer wall running substantially parallel to the inner wall, which is connected to the inner wall via an intermediate floor, so that a liquid reservoir surrounding the first central opening for receiving a liquid is formed by the two walls and the intermediate floor, wherein at least one section of the incubation cassette forming the liquid reservoir is provided at least in sections with at least one transparent section.

[0013] A microplate reader for carrying out the method according to the invention comprises a housing and a transport support that can be extended from the housing. The transport support comprises a support surface for placing a microplate or an incubation cassette. Furthermore, the microplate reader comprises a liquid injector for the automated dispensing of liquid into the liquid reservoir of the microplate or incubation cassette.

[0014] Further preferred and inventive features emerge from the further claims.

[0015] Advantages of the invention include: The liquid level in the liquid reservoir of the microplate and / or the incubation cassette can be monitored automatically, regardless of the duration of the experiment, with intervalic measurement of samples in the microplate wells. These wells are optically accessible from below through the transparent section of the incubation cassette and / or the transparent section of the microplate inserted into the incubation cassette, allowing optical measurements to be used to determine the liquid level in the liquid reservoir. The light for the respective optical measurement passes through the respective transparent section. The liquid reservoir of the microplate and / or the incubation cassette can be filled or refilled with liquid manually or automatically by an injector as soon as it is detected that the liquid level in the liquid reservoir falls below a predefined liquid level.This ensures that the liquid reservoir remains reliably filled with liquid. The subsequent injection of liquid into the liquid reservoir of the microplate and / or incubation cassette can be carried out using an injector already included in the microplate reader. Thus, the samples, or the samples containing test solution, are advantageously exposed to reduced fluctuations in temperature and / or atmosphere. For automated monitoring of the liquid level, at least one optical measurement device can be used, in particular an absorption measurement device. The transparent section of the microplate or incubation cassette allows light to fall onto the liquid from below for optical measurement of the liquid level, for example, using absorption measurement.The corresponding device for optically measuring the liquid level in the liquid reservoir of the microplate and / or the incubation cassette can already be included in the microplate reader, such as a device for optically analyzing samples in the microplate wells, in particular for absorbance measurement. This saves costs and space, as a device already included in the microplate reader can be used. By determining the light absorption, the liquid level of the liquid in the liquid reservoir of the microplate or incubation cassette can be precisely determined. In general, the proportion of light absorbed by the liquid allows conclusions to be drawn about the concentration of the liquid or the liquid level.The incubation cassette is dimensioned and designed so that it can be easily inserted (manually or robotically) into the transport tray of a microplate reader and just as easily removed (manually or robotically) from this transport tray. To shield the enriched atmosphere from the environment, a lid can be placed on the incubation cassette. This lid can be lifted off and replaced within the microplate reader so that the microplate wells are freely accessible for all necessary operations. If a microplate is used without an incubation cassette, a lid can also be placed on the microplate. The gas exchange between the microplate wells and the environment required for cell cultures or cell-based experiments can be achieved by sporadically lifting the lid of the incubation cassette orThe microplate lid can be removed by the operating personnel, by a robot, or by a corresponding device in the microplate reader itself. Any microplates can be placed in the incubation frame or incubation cassette, although standard microplates according to ANSI_SBS 1-2-3-4-2004 are preferred. This eliminates the need for special microplates to conduct experiments with cell cultures or cell-based experiments. Advantageously, the liquid in the reservoir of the incubation cassette and / or microplate and the option of automated refilling of the reservoir means that long-term experiments can be conducted in a microplate reader, even at elevated temperatures (e.g., at 37°C).

[0016] The invention is illustrated by way of example in the accompanying drawings using schematic figures. The figures are intended to illustrate selected embodiments of the subject matter of the invention, but do not limit the scope of the present invention. They show: Fig. 1: a view of an incubation cassette with the lid removed on a transport support of a microplate reader, the incubation cassette being provided with transparent sections; Fig. 2A,B: detailed views of a microplate with differently filled liquid reservoirs; Fig. 3A,B: a view of a frame of an incubation cassette with an inserted microplate in a view from above, as well as a detailed view of a transparent section of the frame of the incubation cassette; Fig. 4: a perspective view of a frame of an incubation cassette with an inserted microplate and a partially lifted lid; Fig. 5A,B: vertical partial sections through a covered incubation cassette in different designs, each with a schematically illustrated transparent section; Fig.Fig. 6 shows a vertical section through a microplate reader in which a transport support with an incubation cassette and a microplate inserted therein is extended; Fig. 7 shows a vertical section through another microplate reader in which a transport support with a microplate inserted therein is extended; Fig. 8A,B shows curves of an absorption measurement of the liquid in the liquid reservoir at different wavelengths, each plotted as a function of time; Fig. 9A,B shows a flow diagram of a method for reducing liquid evaporation from wells of a microplate inserted in an incubation cassette; and Fig. 10A,B shows a flow diagram of a method for reducing liquid evaporation from wells of a microplate.

[0017] The Figure 1shows an embodiment of an incubation cassette 1, which is placed on a transport support 2 of a microplate reader 3 for transporting the incubation cassette 1. A microplate, e.g., a standard microplate, can be inserted into the incubation cassette 1 (not shown).

[0018] The incubation cassette 1 comprises a frame 4, onto which a lid can be placed (not shown). The frame 4 comprises a central first opening 5, the dimensions of which are designed for the complete insertion of the microplate. The central first opening 5 is surrounded by a preferably substantially vertical inner wall 6, wherein preferably at the lower end of which several substantially horizontal support surfaces 7 are arranged, at least in sections. These support surfaces 7 serve to support the inserted microplate (not shown). The frame 4 of the incubation cassette 1 also comprises an outer wall 8, which preferably runs substantially parallel to the inner wall 6 and is connected to the inner wall 6 via an intermediate floor, so that a channel surrounding the central first opening 5, also called a liquid reservoir 9, for receiving a liquid is formed by the two walls 6, 8 and the intermediate floor (see also Fig. 3A , 5A,B ).

[0019] The liquid reservoir 9 is provided in sections with at least one transparent section TA. In other words, at least one section of the incubation cassette 1 forming the liquid reservoir 9, preferably the intermediate floor, is provided at least in sections with at least one transparent section TA. As used here, sections of the liquid reservoir 9 comprise the inner wall 6, the outer wall 8 and / or the intermediate floor. The transparent section TA is arranged such that it is in contact with the liquid held in the liquid reservoir 9. In the example shown, and preferably, the transparent section TA is arranged in the intermediate floor. Although not shown, the transparent section TA can be arranged in at least one of the walls 6, 8. The transparent section TA contains an optically transparent material, which can be optically transparent to light, e.g.a measuring device for measuring the absorption of the liquid and thus for determining an optical path length or a level of the absorbed liquid. Thus, the transparent section TA enables the liquid content or liquid level in the liquid reservoir 9 to be monitored by optical measurement. The respective light for the optical measurement passes through the transparent section TA from below the incubation cassette 1. The optical measurement can be performed automatically, regardless of the duration of a respective measurement or analysis of samples in wells of a microplate.

[0020] The Figures 2A,Bshow detailed views of a microplate 10. The microplate 10 contains several wells, which protrude upwards from the bottom of the microplate 10. The microplate 10 is in turn delimited by a wall. The space thus formed forms a reservoir, also referred to as a liquid reservoir, for receiving liquid F. In the microplate 10 shown in the figures, the outer wells, which run parallel to the wall, are connected by webs 12. These webs 12 divide the liquid reservoir into two sub-reservoirs. As can be seen in the figures, a first sub-reservoir is located in the section of the microplate 10 between the webs 12 (and outer wells) and the wall and is in Fig. 2A shown filled with liquid F. Another partial reservoir is formed by the remaining (inner) section of the microplate 10 and is in Fig. 2Bshown filled with liquid F. Although not shown, both partial reservoirs can also be filled with liquid F. The microplate 10 can be made entirely or partially of transparent material.

[0021] Fig. 3A shows a view of a frame 4 of an incubation cassette 1 with inserted microplate 10 (e.g. a 384-well standard microplate) in a view from above, and Fig. 3B shows a detailed view of a transparent section TA of the frame 4 of the incubation cassette 1. The frame 4 of the incubation cassette 1 is in the area of ​​the liquid reservoir 9 (see also view in Fig. 1) with, for example, six transparent sections TA, through which (from below) optical measurements can be taken to determine the liquid level in the liquid reservoir 9 of the incubation cassette 1. The microplate 10 itself can be completely transparent, so that, additionally or alternatively, a liquid level of a liquid held in the liquid reservoir of the microplate 10 can be detected through it.

[0022] Fig. 4shows a perspective view of a frame 4 of an incubation cassette 1 with an inserted microplate 10, with a partially lifted lid 11. The lid 11 of the incubation cassette 1 serves to cover the frame 4 with the inserted microplate 10. The lid 11 can be placed on or removed manually or by a robot inside or outside a microplate reader (not shown). The lid 11 shown comprises a plate 12 with a magnetizable surface 13, which can cover only part of the plate 12. Alternatively, several such small magnetizable surfaces or a single large magnetizable surface could be provided, which covers at least approximately the entire plate 12.The magnetizable surface 13 can be selected from a group comprising a self-adhesive metal foil, an overmolded metal plate, and an adhesively bonded metal plate, and wherein the metal can comprise: iron, nickel, and their alloys. A microplate reader can comprise a magnetic device integrated in the housing for lifting and placing the lid 11 of the incubation cassette 1 placed on a transport support. In an alternative example, the lid 11 can be lifted and placed by means of a gripping cup or suction cup (not shown). The lid 11 is preferably made of a chemically inert plastic, e.g., by injection molding.

[0023] Fig. 5A,Beach show schematic, vertical partial sections through an incubation cassette 1 covered with a lid 11';11" with an inserted microplate 10. The inner wall 6 is connected to the outer wall 8 via an intermediate floor 14. The outer wall 8, the intermediate floor 14 and the inner wall 6 define the liquid reservoir 9 into which the liquid F can be filled. As shown schematically in the Figs. 5A,B As shown, the intermediate base 10 is provided with the transparent section TA, through which light can pass for optical measurement, e.g., a corresponding measuring device (not shown). The liquid reservoir 9 of the incubation cassette 1 is filled with liquid F in both embodiments shown. The liquid level of this liquid F can be measured or monitored through the transparent section TA.

[0024] In the incubation cassettes 1 shown here, the inner wall 6 can comprise recessed areas 15 so that, when the lid 11';11" is in place, each recessed area 15 connects the microplate 10 to the surrounding liquid reservoir 9. It can be provided that the inner wall 6 of the incubation cassette 1 extends continuously less high than the outer wall 8, so that, when the lid 11';11" is in place, a circumferential gap connects the microplate 10 to the surrounding liquid reservoir 9. This creates a coherent gas atmosphere above the liquid reservoir 9 and above the wells of the microplate 10.

[0025] The incubation cassette 1 is designed such that light for optical measurement from below can pass unhindered through the transparent section TA. In other words, there should be no sections that could block an optical axis passing through the respective transparent section TA. The incubation cassette 1 is preferably made of a chemically inert plastic and, for example, by injection molding. The transparent section(s) TA can be formed integrally with the incubation cassette 1. The Fig. 5A The lid 11' of the incubation cassette 1 shown comprises a substantially flat plate 12 and a downwardly projecting and circumferential edge 16', which is preferably integrally formed on this plate 12. This edge 16' allows the lid 11' to be securely and centrally placed and removed without being displaced, for example, when the incubation cassette 1 is moved sideways.

[0026] Fig. 5B shows an example of an arrangement comprising incubation cassette 1 and attached lid 11", wherein the lid 11" comprises a downwardly projecting and circumferential edge 16" which engages in a circumferential recess 17 formed on the incubation cassette 1. Furthermore, a downwardly projecting, circumferential web 18 can be formed on the lid 11", which can strike with a surface against the outer wall 8 of the incubation cassette 1. In this way, the lid 11" can be placed and removed further securely and in a centered manner without being displaced, for example, if the incubation cassette 1 is moved sideways. Reliable centering of the lid 11" on the incubation cassette 1 is achieved. At the same time, slipping of a placed lid 11" on the incubation cassette 1 is prevented.

[0027] The Fig. 6shows a vertical section through a microplate reader 3 for drawing an incubation cassette 1 into a measuring chamber 19, wherein a microplate 10, e.g., a 96-well standard microplate, is inserted into the incubation cassette 1. Although not shown, the incubation cassette 1 can be covered with a lid. The microplate 10 comprises, for example, wells containing biological structures. In the context of the present invention, the term "biological structures" includes: tissue parts, e.g., from humans, animals, or plants; cell cultures or parts thereof; single cells; cell organelles; macromolecules such as nucleic acids or proteins, as well as single molecules such as nucleotides, amino acids, hormones, and metabolites.

[0028] The microplate reader 3 comprises the transport support 2 for receiving the incubation cassette 1. The transport support 2 can preferably be extended far enough out of the measuring chamber 19 of the microplate reader 3 that the incubation cassette 1 can be placed onto or lifted off the transport support 2 by hand or by means of a microplate handling robot (not shown). The transport support 2 is shown here as already partially retracted because the microplate 10 and the incubation cassette 1 surrounding it are currently being inserted into the microplate reader 3. While the incubation cassette 1 is being inserted or extended, a flap is opened which, when closed, can seal the measuring chamber 19, preferably in a light-tight and / or gas-tight manner, so that no ambient light that could influence the examinations can enter the measuring chamber 19 and / or the gas concentration in the measuring chamber 19 can be reliably regulated independently of the ambient environment.

[0029] In addition to receiving the incubation cassette 1 loaded with the microplate 10, this transport support 2 also serves to position the microplate 10 with the wells containing biological structures (e.g., metabolites, macromolecules, cells, or cell cultures) relative to light sources 21, 22.1 and relative to measuring devices 22.3, 23 of the microplate reader 3 or relative to the optical axes 24', 24" of the measuring devices 22.3, 23. The light sources 21, 22.1 serve, for example, to induce an interaction between at least one of these light sources 21, 22.1 and biological structures in specific wells of the microplate 10, and to elicit or generate a measurable signal. Such signals include, for example, fluorescence emission, luminescence emission, reflected light, and / or transmitted light.

[0030] In the illustrated embodiment, the microplate reader 3 contains a fluorescence module 22 with a first light source 22.1 including a wavelength selection device, e.g., a monochromator or wavelength filter (not shown) for illuminating a sample (excitation light) along the first optical axis 24'. The fluorescence module 22 additionally contains a semi-transparent or dichroic mirror 22.2 for coupling the light reflected back by the sample (emission light) out of the path of the excitation light (=first optical axis 24'). The emission light is guided to a first measuring device 22.3 via the mirror 22.2. In fluorescence top-reading mode, a sample in a well is irradiated directly from above with the fluorescence module 22, and the emission light reflects back from the sample upwards.In bottom-reading mode, the excitation light is directed beneath the microplate via a light guide 25, irradiating the sample from below through the bottom of each well. The emission light reflects downward from the sample and is guided via the light guide 25 to the fluorescence module 22.

[0031] In the embodiment shown, the second light source 21, including a device for wavelength selection, e.g., a monochromator or wavelength filter (not shown), is used to irradiate a sample or biological structures in wells of this microplate 10, and a second measuring device 23 (here, e.g., in the form of a photodiode) is used to measure the absorbance of the sample with respect to the second optical axis 24". The absorbance is calculated by comparing the light intensity passing through the sample to the second measuring device 23 with the transmitted reference light intensity. If, on the other hand, the luminescence of samples is to be detected, it is even possible to dispense with a light source and measure the light signal using, e.g., photomultiplier tubes.

[0032] Such light sources are, for example, selected from a group that includes arc lamps, flash lamps, incandescent lamps (such as halogen lamps), lasers, laser diodes, and light-emitting diodes (LEDs). The corresponding wavelengths for exciting the fluorescence, as well as the corresponding fluorophores and their emission characteristics, are known to the person skilled in the art and are selected depending on the application. The non-invasive irradiation of cells or cell cultures to detect the absorption, as well as the light sources to be used for this purpose, are also familiar to the person skilled in the art. Measuring devices 22.3, 23 for detecting at least one integral signal that was induced or generated by the light source(s) 21, 22.1 in or on biological structures in the specific wells of the microplate 10, are preferably selected from a group that includes photomultipliers, photodiodes, photodiode arrays, and avalanche diodes. The measuring devices 22.3, 23 and light sources 21, 22.1, respectively.their optical input and / or output are preferably coupled via optical fibers 25, such as optical fibers or optical fiber bundles.

[0033] The second measuring device 23 of the microplate reader 3 can also be used to monitor or determine the liquid level in the liquid reservoir of the microplate 10 and / or incubation cassette 1. For this purpose, the incubation cassette 1 can be moved relative to the second optical axis 24" of the microplate reader 3 such that the second optical axis 24" passes through one of the transparent sections of the incubation cassette 1 and / or microplate 10. Thus, the measuring device 23 can also reliably measure the liquid level in the liquid channel of the incubation cassette 1 and / or the microplate 10. The arrangement comprising the light source 21 and the second measuring device 23 is preferably used to measure the absorption of the liquid in the liquid reservoir of the incubation cassette 1 and / or in the liquid reservoir of the microplate 10.

[0034] As previously described, the incubation cassette 1 is provided with the transparent section TA, through which the liquid level in the liquid reservoir of the incubation cassette 1 can be monitored. Alternatively or additionally, in the case of a transparent microplate 10, the liquid level in the liquid reservoir of the microplate 10 can be monitored through the transparent material of this microplate 10. As already mentioned, the arrangement comprising the second light source 21 and the second measuring device 23 is preferably used to measure the absorption of the liquid in the liquid reservoir of the incubation cassette 1 and / or microplate 10. Further details on the absorption measurement preferably used here will be described in connection with Figures 8A,B described.

[0035] The microplate reader 3 contains, for example, two injectors 26', 26", whereby a test solution TL is dispensed into the wells of the microplate via a first injector, also called test solution injector 26', and liquid F is filled or topped up into the liquid reservoir of the incubation cassette 1 and / or the microplate 10 via a second injector, also called liquid injector 26". The liquid reservoir is, for example, automatically topped up as soon as the measuring device 23 of the microplate reader 3 detects, for example by means of absorption measurement, that the level of liquid F in the liquid reservoir of the incubation cassette 1 and / or the microplate 10 has fallen below a predefined level. A controller 27, which is used, for example, to control the first light source 21, the first measuring device 23, the movement of the transport support 2 of the microplate reader 3, etc.can be designed, can further be designed to control the second injector 26" for the automated dispensing of the liquid F into the liquid reservoir of the incubation cassette 1 and / or the microplate 10. Thus, it can be ensured that the liquid reservoir of the incubation cassette 1 and / or the microplate 10 is always sufficiently filled with liquid F, even during, for example, long-lasting analyses.

[0036] The microplate reader 3 also includes an internal or integrated processor 28, or it is designed to be connectable to an external processor (not shown). Such a processor can thus be a microprocessor integrated into the electronic control system of the microplate reader 3 or an attached personal computer.

[0037] Fig. 7shows a vertical section through a microplate reader 3 for drawing a microplate 10 (without incubation cassette) into the measuring chamber 19. The microplate 10 can be completely transparent. In the embodiment shown, an arrangement consisting of an imaging module with an optics / lens system 29 and an illumination source 30 is used. This illumination source 30 illuminates the microplate 10 from above. The imaging module with an optics / lens system 29 records an image from below through the transparent bottom of a well of the microplate 10. The optics / lens system 29 can be designed to optically enlarge or reduce images. The microplate reader 3 can, in addition to the illumination source 30 and the imaging module with an optics / lens system 29, comprise measuring modules as in Figure 6 shown. For example, the liquid level can be determined by measuring the absorption by the liquid.

[0038] Figures 8A,Bshow curves of a measured absorption (at different wavelengths) and a specific path length or fill level of a liquid in the liquid reservoir, each plotted as a function of time. Fig. 8A the measured absorbance of the liquid as a function of time, plotted at a wavelength of 900 nm and 977 nm. As expected, the liquid level decreases with time due to evaporation.

[0039] For the absorption measurement preferred here, the Beer-Lambert law can be applied according to the equation: A = εcl. The absorption (A) of the sample can be determined by the product of the extinction coefficient (ε) and the concentration (c) of the sample and the path length (l) through which the sample is measured. In this method, the absorption peak of the liquid, e.g., water, at room temperature (977 nm) and a background measurement at 900 nm can be used (see measured values, as in Fig. 8A The difference in optical density (i.e., 977 nm - 900 nm) can be divided by 0.18, which corresponds to the optical density of water at 1 cm. The result of the above calculation gives the path length, or liquid level, of the liquid in the liquid reservoir.

[0040] Using the Lambert-Beer law, the path length (i.e., level height or liquid level) can be determined by measuring the liquid (aqueous sample) at 900 nm to obtain a baseline absorption line and at 977 nm to obtain the specific absorption of the liquid (aqueous sample), using the equation: where: A 977 -> Absorption of the aqueous sample at 977 nm A 900 -> Absorption of the aqueous sample at 900 nm A Water -> A 977 - A 900 Water in a 1 cm cuvette

[0041] Using the equation, the level of the liquid in the liquid reservoir is determined as shown in Fig. 8BIn the example shown, a drop in the fill level from an initial level of approximately 1.1 cm to 0 cm is measured in approximately 18 hours. This precise measurement of the fill level allows the reservoir to be refilled with fluid in a timely manner, e.g., at a measured fill level of 0.6 cm, 0.4 cm, 0.2 cm, etc.

[0042] Fig. 9A , B show a flow diagram of a method for reducing liquid evaporation from wells of a microplate inserted in an incubation cassette. Fig. 10A , B show a flow diagram of a method for reducing liquid evaporation from wells of a microplate.

[0043] Regarding the Fig. 9A , B The flow chart shown refers to the respective Fig. 6 and 7 shown microplate reader, into which an incubation cassette is inserted, in which a microplate is inserted ( Fig. 6 ) or only a microplate (without incubation cassette) is inserted into the microplate reader ( Fig. 7). The method begins with a step S9_1 in which the transport support of the microplate reader is extended. In a step S9_2, the incubation cassette is placed on the transport support. The incubation cassette can, however, also be permanently inserted. In a step S9_3, the microplate is placed in the incubation cassette. Then, in a step S9_4, a lid is placed on the incubation cassette. The transport support with the incubation cassette prepared in this way is moved into the microplate reader in a step S9_5. In the microplate reader, the lid is robotically lifted (step S9_6). Furthermore, the gas concentration is measured in the microplate reader (e.g. the concentration of O 2 , CO 2 , etc.), and in a step S9_7, the system waits until a predetermined gas concentration is reached.The temperature is also measured in the microplate reader, and in a step S9_8 the system waits until a predetermined target temperature is reached, e.g. 37°C. As soon as the predetermined gas concentration and / or target temperature is reached, in a step S9_9 the test solution is added by means of an injector into predetermined wells of the microplate provided with substances. However, the test solution can also be added at an earlier point in time, e.g. outside the microplate reader, i.e. before the microplate is inserted into the microplate reader. Furthermore, in this step the liquid can be added to the liquid reservoir of the incubation cassette and / or the microplate. However, the liquid can also be added beforehand, e.g. outside the microplate reader, i.e. before the incubation cassette with the microplate is inserted into the microplate reader.

[0044] In a step S9_10, the samples in the wells are measured using measurement methods such as absorption, fluorescence, imaging, etc. Subsequently, in a step S9_11, the lid is placed on the incubation cassette. This reduces liquid evaporation from the wells of the microplate. In a step S9_12, a predetermined time interval is waited, e.g., 1 h, 2 h, etc. After the predetermined time interval has elapsed, the lid is robotically lifted in a step S9_13. In a step S9_14, the samples in the respective wells are measured. Subsequently, in a step S9_15, the liquid level in the liquid reservoir of the incubation cassette and / or in the liquid reservoir of the microplate is measured. In a step S9_16, based on the result of the fill level measurement, it is determined whether the fill level has fallen below a predetermined threshold value or not.

[0045] If it is determined in step S9_16 that the fill level has fallen below the threshold value (yes), the method continues with step S9_17, in which the liquid reservoir of the incubation cassette and / or the liquid reservoir of the microplate is moved by means of a transport support to an outlet of an injector line of an injector for adding liquid. Once this position is reached, the corresponding liquid reservoir is filled, refilled, or re-injected by the injector (step S9_18). The method then continues with a step S9_19 described later.

[0046] If it is determined in step S9_16 that the fill level has not fallen below the threshold value (no), the method continues with step S9_19, in which the lid is robotically placed back on the incubation cassette. In a subsequent step S9_20, it is determined whether a predetermined maximum number of measuring cycles has been exceeded. If it is determined in step S9_20 that the maximum number of measuring cycles has not been exceeded (no), the method returns to step S9_12. If it is determined in step S9_20 that the maximum number of measuring cycles has been exceeded (yes), the method continues with step S9_21, in which the transport support with the incubation cassette and the microplate inserted therein is moved out of the microplate reader. The method can then be terminated.

[0047] It should be noted that the order of the steps in the process can be changed. Likewise, the method described in the process is not limited to the incubation cassette, the microplate inserted therein, and the lid placed on the incubation cassette, as previously described as an example. In addition to the described incubation cassette with the lid attached, an incubation cassette without a lid can also be used. In this case, for example, no robotics is required to lift the lid. In this case, no reservoir(s) can be present in the microplate. In this case, the measuring space of the microplate reader should be as small as possible. In addition to the automated refilling of the reservoir by the injector described here, refilling can also be done manually.

[0048] Regarding the Fig. 10A , B The flow chart shown refers to the respective Fig. 6and 7 shown microplate reader, into which an incubation cassette is inserted, in which a microplate is inserted ( Fig. 6 ) or only a microplate (without incubation cassette) is inserted into the microplate reader ( Fig. 7 ). The method begins with a step S10_1, in which the transport support of the microplate reader is extended. In a step S10_2, the microplate is placed on the transport support. In a step S10_3, the transport support with the microplate placed on it is moved into the microplate reader. The gas concentration is measured in the microplate reader (e.g., the concentration of O 2 , CO 2 , etc.), and in a step S10_4, the system waits until a predetermined gas concentration is reached.

[0049] The temperature is also measured in the microplate reader, and in step S10_5, the system waits until a predetermined target temperature is reached. As soon as the predetermined gas concentration and / or target temperature are reached, in step S10_6, the samples in the respective wells of the microplate are measured using measurement methods such as absorption, fluorescence, imaging, etc., to generate a 'blank' value for subsequent measurements. In step S10_7, test solution is added to predetermined wells of the microplate containing substances using an injector. However, the test solution can also be added at an earlier time, e.g., outside the microplate reader, i.e., before the microplate is inserted into the microplate reader. Furthermore, in this step, the liquid can be added to the liquid reservoir of the microplate. However, the liquid can also be added beforehand, e.g.,outside the microplate reader, i.e., before the microplate is inserted into the microplate reader. In a step S10_8, a predetermined time period t can be waited for. In a step S10_9, the samples containing the test solution can be measured in the respective wells. Subsequently, in a step S10_10, the fill level of the liquid in the liquid reservoir of the microplate is measured. In a step S10_11, based on the result of the aforementioned measurement, it is determined whether or not the fill level has fallen below a predetermined threshold.

[0050] If it is determined in step S10_11 that the fill level has fallen below the threshold value (yes), the method continues with step S10_12, in which an alarm can be issued to a user of the microplate reader. In step S10_13, the transport support is extended from the microplate reader. As soon as the transport support is extended from the microplate reader, the liquid reservoir of the microplate is refilled with liquid in step S10_14, e.g., manually using a hand pipette. The transport support is then retracted into the microplate reader (step S10_15). The method then continues with step S10_16, described later.

[0051] If it is determined in step S10_11 that the fill level has not fallen below the threshold value (no), the method continues with step S10_16, in which it is determined whether a predetermined maximum number of measuring cycles has been exceeded.

[0052] If it is determined in step S10_16 that the maximum number of measurement cycles has not been exceeded (no), the method returns to step S10_8. If it is determined in step S10_16 that the maximum number of measurement cycles has been exceeded (yes), the method continues with step S10_17, in which the transport support with the microplate placed on it is moved out of the microplate reader. After this, the method can be terminated.

[0053] It should be noted that the order of the steps in the described procedure can be changed. Likewise, the method described in the procedure should not be considered limited to the microplate without a lid in place, as previously described as an example. In addition to the described microplate without a lid in place, a microplate with a lid can also be used, for example. The microplate is provided with at least one reservoir. When using a microplate without a lid in place, no robotics is required in the microplate reader to lift the lid. Here, too, the measuring chamber of the microplate reader should be as small as possible. The reservoir can be refilled using the injector or manually.

[0054] All described processes are to be considered exemplary and serve to explain the invention. The method steps described herein are not limited to the described sequences. The respective sequences may be variable. Method steps may deviate from the described sequences within the scope of the claims.

[0055] The same reference symbols in the figures denote the same or at least similar features, even if these are not always described in detail. Reference symbol 1 Incubation cassette 21 second light source 2 Transport support 22 Fluorescent module 3 Microplate reader 22.1 first light source 4 framework, incubation framework 22.2 semi-transparent or dichroic mirror 5 central first opening 6 inner wall 22.3 first measuring device 7 Wing area of ​​4 23 second measuring device 8 outer wall 24' first optical axis 9 liquid reservoir 24" second optical axis 10 microplate 25 light guide 11 Lid 26' first injector 11',11" Lid 26" second injector 12 plate 27 steering 13 magnetizable surface 28 processor 14 intermediate floor 29 Imaging module with optics / lens system 15 lowered area 16' Edge of 11' 30 Lighting source 16" Edge of 11" 31 optical axis 17 recess F liquid 18 web TA transparent section 19 measuring room TL Test solution

Claims

1. Method for reducing liquid evaporation from wells of a microplate (10), comprising: a) providing the microplate (10) with a plurality of wells, b) adding a sample to at least one of the wells of the microplate (10), c) inserting the microplate (10) or an incubation cassette (1) equipped with the microplate (10) into a microplate reader (3), d) injecting a liquid (F) into a liquid reservoir (9) provided in the microplate (10) and / or the incubation cassette (1), e) performing measurements on the samples in the respective wells, f) optically measuring a liquid level in the liquid reservoir (9) of the microplate (10) and / or the incubation cassette (1) through an essentially transparent section (TA) of the liquid reservoir (9) of the microplate (10) and / or the liquid reservoir (9) of the incubation cassette (1), g) re-injecting the liquid (F) into the liquid reservoir (9) of the microplate (10) and / or into the liquid reservoir (9) of the incubation cassette (1) when the liquid level falls below a predetermined threshold, h) repeating steps e) to g) until a predetermined number of measurement cycles is reached, i) withdrawing the microplate (10) or the incubation cassette (1) equipped with the microplate (10) from the microplate reader (3), characterized in that the liquid reservoir (9) is provided between the wells and is bounded by a wall of the microplate (10) and is formed to accommodate a liquid (F), wherein the microplate (10) is provided with the liquid reservoir (9) and comprises the at least one transparent section (TA), and / or that the incubation cassette (1) comprises a frame (4) for receiving a microplate (10) with wells, wherein the frame (4) comprises a central first opening (5) surrounded by an inner wall (6), the dimensions of which are designed for inserting a microplate (10), and wherein the frame (4) comprises an outer wall (8) extending substantially parallel to the inner wall (6) and which adjoins the inner wall (6) via an intermediate bottom (14), wherein the liquid reservoir (9) is formed by the two walls (6, 8) and the intermediate bottom (14) as a liquid reservoir (9) surrounding the first central opening (5) for receiving a liquid (F), wherein at least one section (6,8,14) of the incubation cassette (1) forming the liquid reservoir (9) is provided at least sectionally with the at least one transparent section (TA).

2. Method according to claim 1, wherein the liquid is injected or re-injected into the liquid reservoir (9) of the microplate (10) and / or the incubation cassette (1) by a liquid injector (26') comprised in the microplate reader (3) or manually.

3. Method of claim 1 or 2, wherein step c) is followed by a step c1): injecting a test solution into the samples in the wells of the microplate (10).

4. Method according to any one of the preceding claims, wherein optically measuring the liquid level in step f) is performed by optically measuring the absorption of the liquid (F).

5. Method according to claim 3 or 4, wherein the samples or the samples mixed with the test solution are measured by measuring the absorption, luminescence or fluorescence, or by imaging the samples.

6. Method according to any one of claims 1 to 5, wherein the microplate (10) further comprises webs (18) which are provided between outer wells running parallel to the wall and are designed to subdivide the liquid reservoir (9) into at least two partial reservoirs.

7. Microplate reader (3) for carrying out the method according to any one of claims 1 to 6 with a housing and a transport support (2) extendable from the housing, wherein the transport support (2) comprises a support surface for placing a microplate (10) or an incubation cassette (1), further comprising a liquid injector (26") for automated delivery of liquid (F) into the liquid reservoir (9) of the microplate (10) or the incubation cassette (1).

8. Microplate reader (3) according to claim 7, further comprising a fluorescence module (22), comprising a first light source (22.1), a means for wavelength selection for illuminating irradiating samples or samples provided with a test solution along a first optical axis (24'), a semitransparent or dichroic mirror (22.2) for outcoupling of light reflected by the samples or by the samples provided with a test solution, and a first measuring device (22.3) to which the reflected light is conducted.

9. Microplate reader (3) according to claim 8, further comprising a second measuring device (23), designed for recording the liquid level in the liquid reservoir (9) of the microplate (10) and / or the incubation cassette (1), wherein the extendable transport support (2) is designed for positioning the microplate (10) and / or the incubation cassette (1) in relation to a second optical axis (24") of the second measuring device (23) such that the second optical axis (24") passes through a transparent section (TA) of the microplate (10) and / or the incubation cassette (1).

10. Microplate reader (3) according to claim 9, wherein the second measuring device (23) of the microplate reader (3) is designed for the absorption measurement of the liquid (F) in the liquid reservoir (9) of the microplate (10) and / or the incubation cassette (1).

11. Microplate reader (3) according to claim 9 or 10, further comprising a controller (50) designed for controlling the second measuring device (23), a second light source (21) interacting with the second measuring device (23), the liquid injector (26") for the automated dispensing of liquid and / or the movements of the transport support (2) of the microplate reader (3).

12. Microplate reader (3) according to any one of claims 7 to 11, further comprising a device for lifting off and placing a lid (11) on an incubation cassette (1) placed on the transport support (2).