Sample analysis method, analysis device, and computer program

Abstract

1. A method for analyzing samples (3) in wells (2) of a microplate (1), comprising the steps of: generating (101) electromagnetic radiation having a first predetermined wavelength or range of wavelengths; irradiating (102) the samples (3) with said radiation by transmitting said radiation from above the microplate (1) through the top of the wells and into the samples (3); transmitting (103) light emitted from the samples (3) and having a second predetermined wavelength or range of wavelengths to a detection means (13) through the top of the wells (2); determining (104) the intensity of light emitted from one or more predetermined measurement areas (23) of the samples (3); determining a result value representing the total amount of light emitted from the samples (3) based on the determined intensity; and counting (106) the number of spots emitting light having the second predetermined wavelength or range of wavelengths.

Description

[Technical field]

[0001] The present invention relates to a method for analyzing one or more samples placed in wells of a microplate as defined in claim 1. The invention also relates to an analytical device and to a computer program for operating the analytical device according to the other independent claims. [Background technology]

[0002] Fluorescence-based assays are commonly used in life sciences. Fluorescence is a form of luminescence. Fluorescence refers to the emission of light (photons) by a substance that has absorbed light or other electromagnetic radiation. Absorption of energy excites the orbital electrons of the molecule to a higher electronic state, and relaxation to the ground state results in the emission of photons.

[0003] Fluorescence assays take advantage of the properties of fluorophores, which absorb light energy at one wavelength and, in response, re-emit it at another, longer wavelength. Each fluorophore has a well-defined range of wavelengths at which it absorbs light, and another well-defined range of wavelengths at which it emits light. This property allows them to be used for the specific detection of biological products by analytical instruments and techniques.

[0004] In a fluorescent assay, samples are typically placed into the wells of a microplate. A microplate is a flat plate that contains a number of wells, i.e., holes arranged in rows and columns. The wells are configured to receive the samples and act as small test tubes. Typical microplates contain 6, 24, 96, 384, or 1536 wells, although larger microplates also exist.

[0005] In certain types of fluorescent assays, fluorescent spots are formed and the analysis of a sample is heavily based on counting the number of fluorescent spots. The problem associated with spot counting is that the counting process is highly dependent on a human user inspecting the spots with the help of an analytical device. This makes the analysis time-consuming and highly subjective. A further problem is that the count becomes unreliable when the number of fluorescent spots in a sample is large. For example, the diameter of a well in a 96-well microplate is about 6 mm. When the number of spots in one well is, say, 500 or more, reliable spot counting becomes difficult as the spots are very close to each other. Summary of the Invention

[0006] It is an object of the present invention to provide an improved method for analyzing one or more samples arranged in wells of a microplate. The features characterizing the method according to the invention are set out in claim 1. It is another object of the present invention to provide an improved analytical device. It is yet another object of the present invention to provide an improved computer program for operating the analytical device. The features characterizing the analytical device and the computer program are set out in the other independent claims.

[0007] The method according to the invention comprises the steps of generating electromagnetic radiation having a first predetermined wavelength or range of wavelengths, irradiating a sample with said electromagnetic radiation by transmitting said electromagnetic radiation from the top of the microplate through the top of the wells to the sample, transmitting light emitted from the sample and having a second predetermined wavelength or range of wavelengths from the top of the microplate through the top of the wells to a detector, determining the intensity of light emitted from one or more predetermined measurement areas of the sample, and determining a result value representative of the total amount of light emitted from the sample and having the second predetermined wavelength or range of wavelengths based on the determined intensity of light emitted from the one or more predetermined measurement areas.

[0008] The analytical device according to the invention comprises illumination means for illuminating the samples from above the microplate through the tops of the wells of the microplate using electromagnetic radiation having a first predetermined wavelength or range of wavelengths, detection means for determining the intensity of light emitted from one or more predetermined measurement areas of the sample through the tops of the wells having a second predetermined wavelength or range of wavelengths, and means configured to determine a result value representative of the total amount of light emitted from the sample having the second predetermined wavelength or range of wavelengths based on the determined intensity of light emitted from said one or more predetermined measurement areas.

[0009] The computer program according to the invention comprises instructions which, when said program is executed by a computer, cause an analysis device to carry out the method steps defined above.

[0010] Using the method, apparatus and computer program according to the present invention, sample analysis will be less dependent on judgment by the user. The result value represents an integral or estimate of the total amount of light emitted by the sample.

[0011] According to one embodiment of the invention, the sample comprises a number of predefined measurement areas and a result value is calculated based on the intensity of light emitted from said number of measurement areas.

[0012] According to one embodiment of the invention, the measurement area covers the entire bottom of the well.

[0013] According to one embodiment of the invention, to determine the intensity of light emitted from the one or more predefined measurement areas of the sample, the sample is illuminated using a light beam having a diameter that is up to 60 percent of the diameter of the well.

[0014] According to one embodiment of the invention, to determine the intensity of light emitted from said one or more predefined measurement areas of the sample, one measurement area at a time is illuminated.

[0015] According to one embodiment of the present invention, a photomultiplier tube is used as a detector to determine the intensity of light emitted from the one or more predetermined measurement areas of the sample.

[0016] According to one embodiment of the present invention, the method includes counting the number of spots emitting light having a second predetermined wavelength or range of wavelengths. This method allows for rapid screening of samples by providing high sensitivity. Furthermore, this method provides a larger dynamic signal range than simply counting fluorescent spots, since it allows measurements even when there are too many fluorescent spots present, making spot counting unreliable.

[0017] According to one embodiment of the present invention, to count the spots, the entire bottom of the well is illuminated and a camera sensor is used to form an image of the sample.

[0018] According to one embodiment of the invention, the illumination means comprises a bulb, an LED or a laser.

[0019] According to one embodiment of the invention, the analytical device includes means for selecting a wavelength for irradiating the sample.

[0020] According to one embodiment of the invention, the means for selecting a wavelength for illuminating the sample comprises a filter or a monochromator.

[0021] According to one embodiment of the invention, the detection means comprises a photomultiplier tube or a silicon photodiode.

[0022] According to one embodiment of the invention, the detection means comprises a camera sensor.

[0023] According to one embodiment of the invention, in order to determine the intensity of light emitted from a number of measurement areas of a sample, the apparatus is configured to sequentially illuminate said number of measurement areas, one measurement area at a time.

[0024] According to one embodiment of the invention, the apparatus is configured to form an image of the sample in order to count the number of spots emitting light having a second predetermined wavelength or range of wavelengths.

[0025] According to one embodiment of the invention, the device is configured to illuminate the entire bottom of the well to form an image of the sample to count the number of spots that emit light having a second predetermined wavelength or range of wavelengths.

[0026] According to one embodiment of the invention, the apparatus comprises means configured to count the number of spots emitting light having a second predetermined wavelength or range of wavelengths.

[0027] Embodiments of the invention are explained in more detail below with reference to the accompanying drawings. [Brief description of the drawings]

[0028] [Figure 1] 2 illustrates, as a flow chart, the steps of a method according to one embodiment of the present invention. [Diagram 2] 1 shows the main elements of an analytical device suitable for carrying out the method according to the invention. [Diagram 3] 1 shows an example of a control system for an analytical device. [Figure 4] 1 shows an example of a microplate suitable for use in an analytical device. [Diagram 5] 1 shows an example of measurement points within a well of a microplate. [Figure 6] 1 shows a schematic diagram of an example of a fluorescence measurement system of an analytical device. [Figure 7] 1 shows a schematic diagram of an example imaging system for an analytical instrument. [Figure 8] 1 shows an example of the measurement results of a fluorescence assay obtained by the method according to the present invention. [Figure 9] The results of spot counts based on the same assay as in FIG. 8 are shown. [Figure 10] 1 shows a schematic of the workflow of the ELISpot / FluoroSpot assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention relates to a method for analysing one or more samples disposed in the wells of a microplate, an analytical device and a computer program for operating the analytical device.

[0030] The method according to the invention measures the intensity of light emitted from a sample and can therefore be used in fluorescence-based assays commonly used in life sciences.

[0031] Fluorescence is a form of luminescence. The fluorescence procedure is based on the emission of light (photons) by a substance that has absorbed light or other electromagnetic radiation. The absorption of energy excites the orbital electrons of the molecule to a higher electronic state, and relaxation to the ground state releases a photon.

[0032] Fluorescence assays such as FluoroSpot exploit the properties of fluorophores: they absorb light energy at one wavelength and, in response, re-emit it at another, typically longer, wavelength. Each fluorophore has a well-defined range of wavelengths at which it absorbs light, and another well-defined range of wavelengths at which it emits light. This property allows them to be used for the specific detection of biological products by analytical instruments and techniques.

[0033] The FluoroSpot assay is a variant of the commonly used ELISpot assay. Figure 10 shows a schematic diagram of the FluoroSpot / ELISpot assay. The FluoroSpot assay can be used to study immune responses. The bottom of the wells 2 of the microplate is equipped with a membrane and coated with a suitable antibody 30 (capture antibody). Cells 31 are cultured and stimulated in the wells 2 to produce an analyte 32, which can be, for example, a cytokine, a chemokine, or an immunoglobulin. Such an analyte 32 secreted by the cells 31 is captured by the antibody 30 bound to the plate. The cells 31 are then removed from the wells 2. To detect the secreted analyte 32, a fluorescently labeled detection fluorophore antibody is added to the wells 2. For example, a biotinylated detection antibody 33 and a streptavidin-enzyme complex 34 can be used. In this way, fluorescent spots fixed on the membrane at the bottom of the well are formed, which can be detected using an analysis device. The well on the right shows a FluoroSpot assay, and the well on the left shows an ELISpot assay. One spot is assumed to correspond to one cell that secreted the analyte. By counting the number of fluorescent spots, it is possible to determine the frequency of antigen-specific T cells, a parameter that is highly relevant in immunological applications.

[0034] In a fluorescent assay, the samples are typically placed in the wells of a microplate. Figure 4 shows an example of a microplate 1. A microplate 1 (also called, for example, a microtiter plate, a microwell plate, a multiwell plate, or a multiwell) is a flat plate that contains a number of wells 2, i.e. holes arranged in rows and columns. The wells 2 of the microplate 1 are configured to receive samples and function as small test tubes. A typical microplate contains 6, 24, 96, 384, or 1536 wells, although larger microplates also exist. The wells 2 are arranged in a rectangular matrix with a side ratio typically of 2:3. Figure 4 shows a microplate 1 with 96 wells 2. However, other microplate sizes can also be used with the method and device according to the invention.

[0035] FIG. 1 shows the steps of a method according to an embodiment of the invention. In a first step 101 of the method, electromagnetic radiation having a first predefined wavelength or wavelength range is generated. In a second step 102 of the method, the sample 3 is irradiated with electromagnetic radiation by transmitting the electromagnetic radiation from above the microplate 1 to the sample 3. In a third step 103 of the method, light emitted from the sample 3 and having a second predefined wavelength or wavelength range is transmitted to a detector or detection means via the top edge of the well 2. The second wavelength or wavelength range is different from the first predefined wavelength or wavelength range. In a fourth step 104 of the method, the intensity of light emitted from one or more predefined measurement areas 23 of the sample 3 is determined. An example of a measurement area 23 is shown in FIG. 5. In the example of FIG. 5, there are multiple measurement areas 23 in the well 2. However, instead of multiple measurement areas 23, there can be a single measurement area. The single measurement area can cover the entire bottom of the well 2. In a fifth step 105 of the method, a result value representative of the total amount of light emitted by the sample 3 and having a second predetermined wavelength or wavelength range is determined based on the determined intensity of the light emitted from the measurement area 23. Thus, a single result value is determined for the analyzed sample. This value correlates with the number of fluorescent spots present in the sample. The fifth step 105 is computer-implemented. The method according to the invention can be performed by an analysis device.

[0036] The method may further include a sixth step 106. In the sixth step 106 of the method, the number of spots emitting light having a second predetermined wavelength or range of wavelengths is counted. The sixth step 106 is implemented in a computer. The determination of the result value and the counting of the spots do not have to be performed in the above order, but for example the spots can be counted before the result value is determined or at the same time as the fifth step 105 of the method. Either the same or different detection means can be used to determine the spot count and the intensity of the emitted light. The sample can be illuminated separately to determine the spot count and the intensity of the emitted light. For example, for spot counting it is possible to illuminate the entire bottom of the well at once, whereas for determining the intensity of the emitted light it is possible to divide the sample into multiple measurement areas and illuminate one measurement area at a time.

[0037] The advantage of the method according to the invention is that the analysis is less dependent on the user's judgement and, compared to simple counting of fluorescent spots, the method offers a wider dynamic signal range since it allows measurements even when the number of fluorescent spots is so large that the count becomes unreliable.

[0038] The method according to the invention can be carried out by an analytical device 10. Since a microplate 1 is typically used to hold the samples, such an analytical device is referred to herein as a microplate reader.

[0039] The main parts of a microplate reader 10 suitable for carrying out the method according to the invention are shown diagrammatically in FIG. 2. The microplate reader 10 may detect biological, chemical or physical events of a sample 3 in a microplate 1 (not shown in FIG. 2). The operation of the microplate reader may be based on different phenomena such as absorbance or luminescence. Since the method according to the invention can be used for fluorescence-based assays, the microplate reader 10 used to carry out the method is configured to at least measure fluorescence. However, the microplate reader 10 may also be a multi-mode reader that can be used for different assays.

[0040] The microplate reader 10 is configured to measure electromagnetic radiation emitted from samples 3 placed in the wells 2 of the microplate 1. The microplate reader 10 comprises an illumination device or illumination means 11 capable of generating electromagnetic radiation having a particular wavelength or range of wavelengths. The wavelength can preferably be selected by a user of the microplate reader 10. The electromagnetic radiation can be visible light (wavelength range about 380-750 nm), ultraviolet light (10-380 nm), or infrared light (750 nm-1 mm). The illumination means 11 is configured to illuminate the samples 3 placed in the wells 2 of the microplate 1. One sample 3 or a part of a sample can be illuminated at a time or the microplate reader 10 can be configured to illuminate multiple samples at a time.

[0041] The illumination means 11 comprises a light source 16, which may be a light bulb such as a xenon flash lamp or a halogen lamp. Alternatively, the light source 16 may be an LED or a laser. The illumination means 11 may comprise several light sources that can be used together or different light sources may be used for different measurement purposes. If the illumination means 11 comprises a laser, it can directly generate light having a specific wavelength. If the light source 16 generates light with a broader spectrum, the microplate reader 10 may comprise a means 17 for selecting the wavelength of light used to illuminate the sample 3, i.e. the excitation wavelength. The means 17 for selecting the excitation wavelength (excitation wavelength selector) may comprise one or more filters and / or a monochromator.

[0042] The microplate reader 10 further comprises a detection means 13. The detection means 13 is configured to measure the electromagnetic radiation emitted from the sample 3. The detection means 13 may comprise, for example, a photomultiplier tube, a camera sensor, or a silicon photodiode. The microplate reader 10 may comprise two or more different detection means 13 for different measurements. The detection means 13 is configured to measure the intensity of light emitted from one or more measurement areas 23 of the sample, as shown in the example of FIG. 5. The detection means 13 may also be used to form an image of the sample to enable counting of fluorescent spots.

[0043] The microplate reader 10 further comprises illumination optics and measurement optics 18. The illumination optics are configured to focus electromagnetic radiation from the light source 16 onto the sample 3. The measurement optics are configured to focus light emitted by the sample 3 onto the detection means 13. The illumination optics and the measurement optics may be at least partly the same.

[0044] The microplate reader 10 may further comprise a means 19 for selecting the emission wavelength to be measured. The means 19 (measurement wavelength selector) may for example be a filter placed in front of the detection means 13. The wavelength selection means 19 is used to pass only the wavelengths to be measured. Thus, the excitation light can be blocked from the detection means 13.

[0045] The microplate reader 10 may further comprise a positioning system or positioning means 29 for moving the samples 3 to a measurement position. The positioning means 29 may be configured to move the microplate. Alternatively, the positioning means 29 may be configured to move the detection means 13 and / or to control the illumination optics and measurement optics to measure the fluorescence of selected samples.

[0046] FIG. 3 shows an example of a control system of the microplate reader 10. The microplate reader 10 is controlled via the input means 14. The input means 14 may include, for example, operating buttons, a keyboard, and / or a touch display. Through the input means 14, a user of the microplate reader 10 can control the operation of the microplate reader 10, adjust parameters, and / or change the settings of the microplate reader 10. The results of the analysis can be displayed on the display 12. The display 12 can be an integral part of the microplate reader 10 or an external display connected to the microplate reader 10. The input means 14, the illumination means 11, the detection means 13, and the display 12 communicate with a central processing unit (CPU) 15. Also, the positioning means 29 is controlled by the CPU 15. The input means 14 and the display 12 do not have to be directly connected to the CPU 15. The microplate reader 10 can also be controlled via software installed on an external general-purpose computer 21, such as a PC. Thus, the input means 14 may include, for example, a keyboard connected to the external computer 21. The display 12 can also be connected to an external computer 21. All connections can be implemented by wires or any wireless means, and the external computer 21 can be a remote server or a cloud server.

[0047] When the microplate reader 10 is used for a fluorescence assay, the user first sets the desired wavelength for illuminating the sample. The user can select the desired excitation wavelength via the input means 14. The microplate reader 10 may also allow the selection of a specific excitation bandwidth, in which case the user can select a specific wavelength range for illuminating the sample. In practice, even if a specific excitation wavelength is selected, the microplate reader 10 can generate electromagnetic radiation with a specific bandwidth. Usually, a narrow bandwidth is preferred. The acceptable bandwidth depends on the application. In some cases, a bandwidth of 20 nm is sufficient. In some applications, the bandwidth should be a maximum of 10 nm. In some applications, the bandwidth should not exceed 2.5 nm.

[0048] Measurement data from the detection means 13 is collected by the CPU 15. This data is used to calculate a result value representative of the total amount of light, or to calculate an estimate of the total amount of light emitted from the sample 3 at a particular wavelength. Either the CPU 15 or an external computer 21 can be used to calculate the result value. The CPU 15 or the external computer 21 can be further configured to count the number of fluorescent spots.

[0049] The microplate reader 10 according to the invention can be configured both for measuring the electromagnetic radiation emitted by the sample and for forming an image of the sample. The microplate reader 10 can therefore include both a fluorescence measuring means and an imaging means. The means used for measuring the fluorescence and for imaging the sample may be at least partly the same, but in the following they will be described separately with reference to Figures 6 and 7.

[0050] FIG. 6 shows as a simplified diagram an example of a fluorescence measurement system of a microplate reader 10, which is configured to measure electromagnetic radiation emitted by a sample 3 when excited by an illumination means 11. In the example of FIG. 6, the illumination means 11 comprises a light source 16. The light source 16 can be a wide bandwidth lamp, such as a xenon flash lamp. The light source 16 can also be, for example, a quartz halogen lamp. The light source 16 generates electromagnetic radiation, such as visible light (wavelength range about 380-750 nm), ultraviolet light (10-380 nm) or infrared light (750 nm-1 mm), having a wide spectrum. To select a specific wavelength, the illumination means 11 further comprises a monochromator 17. The monochromator 17 generates a light beam with a narrow bandwidth. According to one example, the bandwidth of the light after the monochromator 17 is less than 2.5 nm. However, for some applications a wider bandwidth is sufficient. Instead of a monochromator, an interference filter can also be used as a means for wavelength selection. The light source may also be a narrow band light source such as an LED or laser, in which case monochromators, interference filters, or other external means for wavelength selection may not be required.

[0051] A light beam from light source 16 passes through the optics of microplate reader 10 and through monochromator 17. In the example of Figure 6, the optics between light source 16 and monochromator 17 include a mirror 23 and an entrance slit 24. However, the optics of microplate reader 10 can be constructed in many different ways.

[0052] In the example of Figure 6, light is transmitted from a monochromator 17 through an exit slit 25 and optical fiber 22 into a microplate. The light is focused by illumination optics 18 onto a sample placed in a well 2 of the microplate. The sample is illuminated from above. The intensity of the light emitted from the sample is measured by a detector 13, such as a silicon photodiode or a photomultiplier tube. The microplate reader 10 of Figure 6 is configured for top reading.

[0053] A wavelength filter can be placed between the microplate 1 and the detector 13 to separate emitted photons from excitation photons. The microplate reader 10 can further include an emission filter, such as a low-pass or band-pass emission filter, before the detector 13 to remove background fluorescence or to separate fluorescent signals originating from multiple fluorophores within a sample.

[0054] The fluorescence measurement system is preferably configured to measure the intensity of light emitted from multiple measurement areas 23 of the sample. However, a single measurement area may also be used. The measurement areas 23 or the single measurement area preferably cover the entire bottom of the well 2 of the microplate 1. The microplate reader 10 can be configured to illuminate one measurement area 23 of the sample 3 at a time. The measurement area 23 is illuminated and the intensity of light emitted from the area 23 is measured by the detection means 13. The diameter of the light beam used to illuminate the sample can also be, for example, 20 to 60 percent of the diameter of the well. By using a narrow beam it is possible to increase the illumination intensity and thus the sensitivity of the measurement. Based on the measured intensity a result value of the sample is calculated. In case of a single measurement area the result value is based on the measured intensity of the single measurement area.

[0055] 5, the distance between the center points of two adjacent measurement areas 23 is configured to correspond to the radius of the illumination beam. For example, if the beam has a diameter of 3 mm, this beam is moved 1.5 mm between two adjacent measurement areas 23.

[0056] The microplate reader 10 further comprises an imaging system for counting the number of fluorescent spots. Figure 7 shows an example of an imaging system of the microplate reader 10. The imaging system of Figure 7 is suitable for FluoroSpot and ELISpot assays, but can also be used for other assays. The microplate reader 10 comprises illumination means 11. The function of the illumination means 11 is to generate light that is used to illuminate the samples in the wells 2 of the microplate 1. In the microplate reader of Figure 7, one well 2 of the microplate 1 and one sample is illuminated at a time. The illumination means 11 comprises a light source 16. From the illumination means 11, the light is directed to a beam splitter 7. The beam splitter 7 is an optical device configured to reflect a part of the light and transmit the remaining light. In practice, a part of the light received by the beam splitter 7 is absorbed. The beam splitter 7 is configured to direct the reflected light to the reading surface 26 of the microplate reader 10. The beam splitter 7 can be made, for example, of two triangular glass prisms glued together. Alternatively, the beam splitter 7 may be a coated glass plate. The beam splitter 7 may have different properties. The optimal beam splitting ratio of an optical reader is 50-50%. That is, the amount of light reflected by the beam splitter 7 is equal to the amount of light transmitted by the beam splitter 7. Thus, half of the light is reflected and not absorbed by the beam splitter 7, and half of the light is transmitted. However, the portion of reflected light may be, for example, in the range of 40-60 percent.

[0057] Between the beam splitter 7 and the reading surface 26, a lens system 8 is arranged, which includes at least one lens 8a. Although only a single lens 8a is shown in FIG. 1, in practice the lens system 8 may include multiple lenses. The lens system 8 is arranged to focus the light received from the illumination device 11 and the beam splitter 7 onto the samples arranged at the bottom of the wells 2 of the microplate 1. The lens system 8 further includes an aperture 8b arranged between the lens 8a and the reading surface 26.

[0058] The bottom of the wells 2 of the microplate 1 and the samples in the wells 2 reflect part of the light back towards the lens system 8. In a fluorescence measurement, the samples emit light from the microplate 1 towards the lens system 8. The lens system 8 is configured to focus an image of the sample onto the imaging device 13, which acts as a detection means. The same lens system 8 is therefore used to focus the light used to illuminate the samples and to focus the light received from the microplate 1. The same components therefore form both the illumination optics and the measurement optics. From the lens system 8, the light is directed towards the beam splitter 7. Part of the light is reflected from the beam splitter 7 towards the illumination device 11, while part of the light can pass through the beam splitter 7 and reach the imaging device 13. If the beam splitting ratio of the beam splitter 7 is 50-50% and absorption by the beam splitter 7 is omitted, half of the light is reflected and half of the light is transmitted through the beam splitter 7. The imaging device 13 can include a digital camera sensor 13a. The imaging device 13 is configured to take one or more images of each sample.

[0059] The aperture 8b between the lens 8a of the lens system 8 and the reading surface 26 is dimensioned to have at most the same diameter as the diameter of the well 2 of the microplate 3. This eliminates vignetting in both the sample and the imaging device 13. The aperture 8b may be adjustable to allow the microplate reader to be used to analyze samples in different microplates 1. By using the aperture, the size of the illuminated area at the bottom of the well 2 can be adjusted. For example, in a typical 96-well plate the diameter of the illuminated area may be about 6.6 mm, and in a 384-well plate it may be 2.5 mm.

[0060] The light source 16 can be, for example, an LED or a group of LEDs. The illuminated area at the bottom of the wells 2 of the microplate 1 must cover the entire bottom. The diameter of a typical LED chip is much smaller than the diameter of the wells 2 of the microplate 1. The size of the illuminated area can be influenced by the lens system 8. However, it may be beneficial to increase the size of the illuminated area by placing an integrating sphere, also called an Ulbricht sphere, around the LED or other light source.

[0061] The microplate reader further comprises positioning means (not shown in FIG. 7 ) configured to move the microplate 1. The microplate 1 is moved within the reading plane 26 so that one well 2 at a time is under the lens system 8. Various images of the sample are taken and then the microplate 1 is moved so that the next well 2 is under the lens system 8.

[0062] The microplate reader of Figure 7 further comprises a reference detector 28 arranged to measure the intensity of the light generated by the illumination means 11 and transmitted through the beam splitter 7. If an LED is used as the light source 16, the intensity of this LED may change due to heating, which will affect the image taken by the imaging device 13. By measuring the light intensity, this effect can be taken into account when interpreting the measurements of the microplate reader.

[0063] The microplate reader further comprises a first filter 17 arranged between the illumination means 11 and the beam splitter 7. The first filter 17 is used in fluorescence assays. The first filter 17 is configured to pass only that part of the spectrum of the light source 16 that is required to excite the sample. The first filter 17 is therefore used as a means for selecting the excitation wavelength. A second filter 19 is arranged between the beam splitter 7 and the imaging device 13. Similar to the first filter 12, the second filter 19 is also used in fluorescence assays. The second filter 19 is configured to pass light consisting of wavelengths emitted by the sample.

[0064] In the embodiment of FIG. 7, the imaging device 13 is arranged directly above the lens system 8. The illumination means 11 are arranged in the same horizontal plane as the beam splitter 7. Thus, light from the illumination means is reflected to the microplate 1 and light from the microplate 1 is transmitted through the beam splitter 7. This arrangement allows the use of a reference detector 28 without disturbing the illumination of the sample. However, it would also be possible to switch the locations of the imaging device 13 and the illumination means 11. Thus, the light used to illuminate the sample can pass through the beam splitter 7 and an image of the sample can be reflected by the beam splitter 7 to the imaging device 13.

[0065] The images formed by this imaging system can be used to count the fluorescent spots in the sample. Thus, the central processing unit 15 of the microplate reader 10 or the external computer 21 can be configured to determine the number of fluorescent spots in the sample.

[0066] Thus, a microplate reader 10 according to the invention may include both a fluorescence measurement system, such as the system of FIG. 6, and an imaging system, such as the system of FIG. 7. The microplate reader 10 may thus be configured to measure the intensity of light emitted from the sample and form an image of the sample. This allows a more comprehensive analysis of the sample. Some of the components of the fluorescence measurement system and the imaging system may be the same. For example, the same light source 16 may be used in both systems. Also, the measurement optics and the illumination optics may be at least partially the same, both for measuring the intensity of the emitted light and for imaging the sample. The imaging system may also be used to measure fluorescence. The intensity of the emitted light may be determined by the camera sensor 13a, and thus a separate system for measuring the intensity of the emitted light would not be required.

[0067] Figures 8 and 9 show examples of results obtained by a method according to an embodiment of the invention. The method was applied to samples placed in wells of a 96-well microplate, the diameter of the well bottom was 6 mm. The diameter of the light beam used to illuminate the samples was 3 mm. The measurement areas were arranged as shown in Figure 5. They thus form a matrix of 5 rows and 5 columns. The light beam moved 1.5 mm at a time. Figure 8 shows the results of the step of determining the intensity of the emitted light, and Figure 9 shows the results obtained by the spot counting step. On the X-axis of both figures is shown the amount of antigen-presenting cells, in this case human peripheral blood mononuclear cells (PBMCs) (amount of PBMCs / well). Figure 8 shows on the Y-axis the fluorescence result value calculated based on the measured intensity of light emitted at a wavelength of 520 nm at several measurement points. The wavelength of the excitation light is 480 nm. Figure 9 shows on the Y-axis the number of spots counted from the sample. In both cases the number of IFN-γ producing cells is measured. The upper dots in Figures 8 and 9 represent stimulated samples and the lower dots represent blanks (unstimulated samples).

[0068] The readout of the multipoint fluorometry increases linearly over the entire range of cell concentrations (low and very high levels of cytokine production), while the spot number increases almost linearly until a plateau, which coincides with the maximum number of detectable spots per well. This is due to the inherent limitation of the image-based method to segment spots from the background when the spots become too crowded on the well. On the other hand, the detection limit of the spot counting method is better than that based on fluorescence intensity measurements. Thus, by combining both readouts, it is possible to keep the detection limit of the spot counting method low while expanding the dynamic range of the assay. For example, instead of spot counting, it is possible to use the measured fluorescence signal to make a prediction of the frequency of antigen-positive cells at high frequency values, which is less reliable in such cases.

[0069] The method according to the invention can be used, for example, for rapid screening of microplates. This method allows identifying samples to be taken for further analysis. This method is particularly useful when the number of fluorescent spots is too large to count a single spot. The combination of spot counting and calculation of the fluorescent signal provides a comprehensive solution for FluoroSpot data analysis and allows troubleshooting in case of spot count failure.

[0070] Those skilled in the art will understand that the invention is not limited to the embodiments described above, but that it can be varied within the scope of the appended claims.

Claims

1. A method for analyzing one or more samples (3) placed in wells (2) of a microplate (1), each well (2) containing one sample (3), comprising: - generating (101) electromagnetic radiation having a first predetermined wavelength or range of wavelengths; - irradiating (102) the samples (3) with electromagnetic radiation by transmitting said electromagnetic radiation from above the microplate (1) through the tops of the wells (2) to the samples (3) using a light beam with a diameter of up to 60 percent of the diameter of the wells (2); - transmitting (103) light emitted by said sample (3) and having a second predetermined wavelength or range of wavelengths through said top end of said well (2) to a detector (13); - determining (104) the intensity of light emitted from a number of predetermined measurement areas (23) of the sample (3), wherein in order to determine the intensity of light emitted from the number of predetermined measurement areas (23) of the sample (3), one measurement area (23) is illuminated at a time, and further comprising: - determining (105) a result value representative of the total amount of light emitted by the sample (3) and having the second predetermined wavelength or wavelength range, based on the determined intensities of light emitted from the at least two measurement areas (23); - counting (106) the number of spots of said sample (3) emitting light having said second predetermined wavelength or range of wavelengths.

2. The method of claim 1 , wherein the plurality of measurement areas (23) cover the entire bottom of the well (2).

3. 3. The method according to claim 1 or 2, wherein a photomultiplier tube is used as the detector (13) for determining the intensity of light emitted from the at least two predetermined measurement areas (23) of the sample (3).

4. The method according to any one of claims 1 to 3, wherein the entire bottom of the well (2) is illuminated and a camera sensor (13a) is used to form an image of the sample (3) for counting the spots.

5. 1. An analytical device (10) configured to receive a microplate (1) containing a plurality of wells (2), each of said wells (2) configured to receive a sample (3), said analytical device (10) comprising: - illumination means (11) for illuminating the samples (3) from above the microplate (1) through the upper edges of the wells (2) of the microplate (1) using a light beam having a diameter of up to 60 percent of the diameter of the wells (2) and using electromagnetic radiation having a first predetermined wavelength or range of wavelengths; - detection means (13) for determining the intensity of light emitted from a plurality of measurement areas (23) of the sample (3) through the top end of the well (2) and having a second predetermined wavelength or range of wavelengths; - means (15, 21) adapted to count the number of spots of said sample (3) emitting light having said second predetermined wavelength or wavelength range; - means (15, 21) configured to determine, based on the determined intensities of light emitted from the plurality of measurement areas (23), a result value representative of the total amount of light emitted by the sample (3) and having the second predetermined wavelength or wavelength range, In order to determine the intensity of light emitted from the plurality of predetermined measurement areas (23) of the sample (3), the apparatus (10) is configured to sequentially irradiate the measurement areas (23), one measurement area (23) at a time.

6. The analytical device (10) according to claim 5, wherein the illumination means (11) comprises a light bulb, an LED or a laser.

7. 7. Analytical device (10) according to claim 5 or 6, wherein the device (10) comprises means (17) for selecting a wavelength for irradiating the sample (3).

8. 8. The analytical device (10) of claim 7, wherein the means (17) for selecting a wavelength for illuminating the sample (3) comprises a filter or a monochromator.

9. The analytical device (10) according to any one of claims 5 to 7, wherein the detection means (13) comprises a photomultiplier tube or a silicon photodiode.

10. The analytical device (10) according to any one of claims 5 to 9, wherein the detection means (13) comprises a camera sensor (13a).

11. The analytical device (10) according to any one of claims 5 to 10, wherein the device (10) is configured to form an image of the sample (3) in order to count the number of spots emitting light having the second predetermined wavelength or range of wavelengths.

12. 12. The analytical device (10) of claim 11, wherein the device (10) is configured to illuminate the entire bottom of the well (2) to form an image of the sample (3) for counting the number of spots emitting light having the second predetermined wavelength or wavelength range.

13. A computer program for operating an analytical device (10), the program being executed by a computer to cause the analytical device (10) to A computer program comprising instructions for carrying out the method according to any one of claims 1 to 5.

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