Patent Application
20220120688
The present invention relates to an optical reader in accordance with claim 1. The invention also concerns a method of analyzing biological samples as defined in the other independent claim.
Enzyme-Linked Immunosorbent Spot Assay (ELISPOT) is a method that is typically used for monitoring cellular immune responses in humans and other animals or organisms. The ELISPOT method allows detecting cells that secrete various substances. The ELISPOT assay can be used, for example, for detecting antigen-specific antibody secreting cells or cytokine producing cells. The examined cells are placed into the wells of a microplate or into some other suitable small vessel and treated to trigger a reaction at least in part of the cells. The cells reacting to the treatment secrete biologically relevant molecules that can be detected as spots. In this assay, one spot represents one immunologically reactive cell. These spots can be detected and counted either manually using for example a microscope or automatically using a specific reader adapted to ELISPOT assays.
ELISPOT readers have been designed so that the samples are illuminated and a camera is used for taking an image of each sample. A problem with many known devices used for ELISPOT assays is the amount of stray light produced by the light illuminating the samples. The stray light decreases the contrast of the image and makes analyzing of the samples more difficult.
An object of the present invention is to provide an improved optical reader for analyzing biological samples arranged in the wells of a microplate. The characterizing features of the optical reader according to the invention are given in the claim 1. Another object of the invention is to provide an improved method of analyzing biological samples arranged in the wells of a microplate, which is arranged in a horizontal reading plane. The characterizing features of the method are given in the other independent claim.
The optical reader according to the invention comprises a horizontal reading plane for receiving a microplate comprising a plurality of wells, an illuminating arrangement comprising a light source and being arranged to illuminate the samples in the wells of the microplate, an imaging device arranged to receive light from the microplate, a beam splitter, which is arranged to direct part of the light from the illuminating arrangement towards the reading plane and to direct part of the light received from the microplate to the imaging device, and a lens system comprising at least one lens and being arranged between the beam splitter and the reading plane to focus the light received from the illuminating arrangement to a sample and to focus an image of the sample to the imaging device. The optical reader is configured to transmit from the illuminating arrangement to the lens system only light having a specific polarization, and the optical reader comprises a polarizer that is arranged between the lens system and the imaging device and configured to block polarized light reflected from the surfaces of the lens system.
The method according to the invention comprises the steps of producing polarized or unpolarized light by means of an illuminating arrangement, directing part of the produced light by means of a beam splitter towards a sample in a well of a microplate, in case of unpolarized light, polarizing the light received from the beam splitter, directing light with a specific polarization by means of a lens system to the sample, passing at least part of the light reflected from the microplate and the sample and/or emitted by the sample through the lens system, directing part of the light received from the microplate and the lens system by means of the beam splitter towards an imaging device, and forming an image of the sample by means of the imaging device. Polarized light reflected from the surfaces of the lens system are blocked by means of a polarizer arranged between the lens system and the imaging device.
By directing the light from the illuminating arrangement via the beam splitter and the lens system to the well of the microplate, it is possible to form a uniform and bright spot on the bottom of the well. Stray light around the well can be significantly reduced, which increases the contrast of the image. However, as the illumination beam goes through the lens system, stray light is formed on each lens surface. Stray light is also formed by scattering of the illumination beam inside the construction of the lens system and especially on metal surfaces. Both the reflections of the lens surfaces and scattering on the metallic surfaces of the lens system maintain the polarization of the light. Therefore, this stray light can be effectively removed by using polarized light for illuminating the samples and polarizing the light reflected by the sample before the light is captured by the imaging device. Unwanted stray light can thus be blocked by the polarizer. However, the light reflected from the microwell is not polarized, and can therefore partly pass through the polarizer between the beam splitter and the imaging device. As a result, images with high contrast can be obtained.
According to an embodiment of the invention, the illuminating arrangement is configured to illuminate the samples in the wells of the microplate with light having a first polarization direction, and a polarizer having a second polarization direction that is perpendicular to the first polarization direction is arranged between the beam splitter and the imaging device.
According to an embodiment of the invention, the light source is arranged to produce unpolarized light and the illuminating arrangement comprises a polarizer arranged between the light source and the beam splitter. For example a LED can thus be used as a light source.
According to an embodiment of the invention, a circular polarizer is arranged between the beam splitter and the lens system. With a circular polarizer, only one polarizer is needed.
According to an embodiment of the invention, the angle between the normal of the surface of the circular polarizer and the longitudinal axis of the lens system is in the range of 5-15 degrees. By inclining the circular polarizer, reflections from the surface of the polarizer to the imaging device are reduced and contrast of the image is further improved.
According to an embodiment of the invention, the imaging device comprises a camera sensor.
According to an embodiment of the invention, the illuminating arrangement comprises an integrating chamber, such as an integrating sphere. With an integrating chamber, uniform illumination with a desired beam diameter can be created.
According to an embodiment of the invention, the illuminating arrangement comprises at least one LED for producing the light used for illuminating the samples.
According to an embodiment of the invention, the imaging device is arranged above the beam splitter.
According to an embodiment of the invention, the optical reader comprises a reference detector arranged to measure the intensity of light produced by the illuminating arrangement and passed through the beam splitter. With the reference detector, the effects of varying intensity of the light produced by the light source on the image can be compensated.
According to an embodiment of the invention, the optical reader comprises a first filter that is arranged between the illuminating arrangement and the beam splitter and configured to pass through light only in one or more first predetermined wavelength ranges, and a second filter that is arranged between the beam splitter and the imaging device and configured to pass through light only in one or more second predetermined wavelength ranges. The first filter can be configured to pass through certain wavelengths that are needed for excitation of samples in fluorescence measurements. The second filter can be configured to pass through only those wavelengths that are emitted by the samples. The same reader can thus be used for both ELISPOT and FluoroSpot assays.
According to an embodiment of the invention, the lens system comprises an aperture, through which the light from the beam splitter is directed to a well of the microplate, and the diameter of the aperture is at most the same as the diameter of the well. This prevents vignette in the samples and the imaging device.
In a method according to an embodiment of the invention, light with a first polarization direction is produced by means of the illuminating arrangement and light received from the lens system and the beam splitter is polarized by means of a polarizer having a second polarization direction that is perpendicular to the first polarization direction.
In a method according to an embodiment of the invention, polarized light is produced in the illuminating arrangement by producing unpolarized light and passing it through a polarizer.
According to an embodiment of the invention, light received from the beam splitter is polarized by means of a circular polarizer that is arranged between the beam splitter and the lens system.
According to an embodiment of the invention, unpolarized light is produced by means of at least one LED.
According to an embodiment of the invention, unpolarized light is directed towards the beam splitter from an integrating chamber, such as an integrating sphere.
According to an embodiment of the invention, the light from the illuminating arrangement is reflected to the sample by the beam splitter.
According to an embodiment of the invention, the intensity of light produced by the illuminating arrangement and passing the beam splitter is measured.
According to an embodiment of the invention, the samples are arranged in a microplate, where the walls of the wells are black. The bottoms of the wells can be white. The black walls reduce straylight and improve the contrast of the image.
Embodiments of the invention are described below in more detail with reference to the accompanying drawings, in which
ELISPOT assays allow detecting cells that secrete various substances. ELISPOT assays can be used, for example, for detecting antigen-specific antibody secreting cells or cytokine producing cells. The examined cells are placed into the wells 2 of a microplate 1 and treated to cause a reaction at least in part of the cells. The cells reacting to the treatment secrete biologically relevant molecules that can be detected as spots. In this assay, one spot represents one immunologically reactive cell.
FluoroSpot assay can be considered as a modification of the ELISPOT assay. While the ELISPOT assay utilizes an enzyme-labeled antibody and a precipitating enzyme substrate for color development, the FluoroSpot method uses fluorophores. Different fluorophores can be used for detecting different analytes and cells secreting several analytes can be studied using multiplexed assays with multicolored spots.
A microplate (also called e.g. as a microtiter plate, microwell plate, multiwell plate or multiwell) is a flat plate comprising a plurality of wells, i.e. cavities that are arranged in rows and columns. In
The optical reader comprises an illuminating arrangement 4. The function of the illuminating arrangement 4 is to produce polarized light, which is used for illuminating the samples in the wells 2 of the microplate 1. In the optical reader of
From the first polarizer 9, the light is directed to a beam splitter 7. The beam splitter 7 is an optical device, which is configured to reflect part of the light and transmit the rest of the light through it. In practice, part of the light received by the beam splitter 7 is absorbed. The beam splitter 7 is arranged to direct the reflected light towards the reading plane 3. The beam splitter 7 can be made, for instance, of two triangular glass prisms that are glued together. Alternatively, the beam splitter 7 can be a coated glass plate. Beam splitters 7 are available with different properties. The optimal beam splitting ratio for the optical reader is 50-50%, i.e. the amount of light reflected by the beam splitter 7 equals the amount of light transmitted by the beam splitter 7. Half of the light that is not absorbed by the beam splitter 7 is thus reflected and half of the light is transmitted. However, the portion of the reflected light could be, for example, in the range of 40-60 percent.
Between the beam splitter 7 and the reading plane 3, there is arranged a lens system 8 comprising at least one lens 16. In
The bottom of the well 2 of the microplate 1 and the sample in the well 2 reflect part of the light back towards the lens system 8. In case of FluoroSpot assays, the samples can also emit light. The lens system 8 is configured to focus an image of the sample to an imaging device 6. The same lens system 8 is thus used for focusing the light used for illuminating the sample and for focusing the light received from the microplate 1. From the lens system 8, the light is directed to the beam splitter 7. Part of the light is reflected from the beam splitter 7 towards the illuminating arrangement 4, but part of the light can pass the beam splitter 7 and reach the imaging device 6. 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 6 can comprise a digital camera sensor 6a. The imaging device 6 is configured to take one or more images of each sample.
The aperture 17 between the lenses 16 of the lens system 8 and the reading plane 3 is dimensioned to have a diameter that is at most the same as the diameter of the wells 2 of the microplate 3. This eliminates vignette in both the samples and in the imaging device 6. The aperture 17 can be adjustable to allow the optical reader to be used for analyzing samples in different microplates 1. With the aperture, the size of the illuminated area at the bottom of the well can be adjusted. For instance, in a typical 96-well plate the diameter of the illuminated area could be approximately 6.6 mm and in a 384-well plate 2.5 mm.
The beam splitter 7 and the lens system 8 maintain the polarization of the light received from the illuminating arrangement 4. The sample and the bottom of the well 2 of the microplate 1 are thus illuminated with light consisting substantially of polarized light. The bottom of the well 2 of the microplate 1 is configured to depolarize the light. The bottom can for example have white matt finish. The light reflected from the bottom of the well 2 therefore consists of waves with different polarizations. The bottom can comprise for example a PVDF membrane. The walls of the well 2 can be black to reduce straylight, although that is not necessary. A suitable microplate can be manufactured for example by making a black microplate and then applying a white membrane on the bottom of each well of the microplate. Alternatively, a black plate with clear bottom could be provided with white membranes applied to the bottoms of the wells. Instead of membranes, some other kind of coating could be used in the wells.
A second polarizer 10 is arranged between the beam splitter 7 and the imaging device 6. Also the second polarizer 10 is a linear polarizer. The second polarizer 10 can be a polarizing filter. The second polarizer 10 has a second polarization direction, which is perpendicular to the first polarization direction. The second polarizer 10 thus blocks light having the first polarization direction. For instance, if the light from the illuminating arrangement 4 is s-polarized, i.e. the electric field of the light is normal to the plane of incidence, the second polarizer is p-polarizing, i.e. it transmits only light with its electric field along the plane of incidence. Reflections of light from the lens surfaces of the lens system 8 with the first polarization direction are thus blocked, whereas the light reflected from the microplate 1 and consisting of light with different polarization directions can partly pass the second polarizer 10. Stray light can thus be effectively reduced while still enabling image formation in the imaging device 6.
The polarization direction of the light received from the illuminating arrangement 4 is preferably chosen so that the reflection by the beam splitter 7 is maximized.
The light source 5 can be, for instance, a LED or a group of LEDs. The illuminated area on the bottom of the well 2 of the microplate 1 should cover the whole 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 affected by the lens system 8. However, it may be beneficial to increase the size of the illuminated area by arranging an integrating sphere, also known as an Ulbricht sphere, around the LED or other light source.
Instead of using a light source 5 producing unpolarized light and the first polarizer 9, the light source 5 could produce polarized light. The light source 5 could thus be a laser. A laser beam is typically narrow, and a beam expander could therefore be arranged after the light source to increase the diameter of the beam.
The optical reader further comprises a plate moving device (not shown), which is configured to move the microplate 1. The microplate 1 is moved in the reading plane 3 so that one well 2 at a time is below the lens system 8. An image or several images of the sample is taken and the microplate 1 is then moved so that a next well 2 is below the lens system 8.
The optical reader of
In the embodiment of
In the embodiment of
The light source 4 could comprise more than one LEDs. For instance, the light source 4 could comprise three LEDs of different colors. Three black-and-white images of each sample could be taken and the images could be used for constructing a color image.
In the embodiment of
The circular polarizer 19 comprises a linear polarizer 20 and a quarter-wave plate 21. Unpolarized light from the beam splitter 7 first reaches the linear polarizer 20. Only light with a specific polarization can pass through the linear polarizer 20. The linearly polarized light is passed through the quarter-wave plate 21, which can also be called as a quarter-wave retarder. The quarter-wave plate 21 converts the linearly polarized light into circularly polarized light. The light can be either in right circular polarization state or in left circular polarization state.
As the circularly polarized light reflects from the surfaces of the lens system 8, it remains circularly polarized. However, if the light passing through the quarterwave plate 21 is in right circular polarization state, the polarization is transformed in reflections into left circular polarization. In the same way, left circular polarization would be transformed into right circular polarization. As the circularly polarized light passes through the quarter-wave plate 21, it is converted into linearly polarized light. However, the polarization direction is perpendicular to the polarization direction of the light that was received by the quarter-wave plate 21 from the linear polarizer 20. As a result, the linearly polarized light is blocked by the linear polarizer 20.
As an example, the linear polarizer 20 is an s-polarizing filter. It receives unpolarized light from the beam splitter 7 and passes through s-polarized light. The quarter-wave plate 21 converts the s-polarized light into right circular polarized light. The right circular polarized light reflects from the surfaces of the lens system 8 and it is converted into left circular polarized light. The left circular polarized light is converted in the quarter-wave plate 21 into p-polarized light. The p-polarized light is blocked by the s-polarizing filter 20, and reflections from the surfaces of the lens system 8 can thus not pass to the beam splitter 7 and further to the imaging device 6.
The light reflected from the bottom of the microplate 1 loses its polarization. Part of the light reflected from the microplate 2 can thus pass through the circular polarizer 19 to the beam splitter 7 and further to the imaging device 6.
For further improving the image, the circular polarizer 19 may be inclined in respect of the axial direction of the lens system 8. The axial direction of the circular polarizer 19 thus differs from the axial direction of the lens system 8. The angle a between the normal 23 of the surface of the circular polarizer 19 and the longitudinal axis 22 of the lens system 8 can be for example in the range of 5-15 degrees. By inclining the circular polarizer 19, less light is reflected from the polarizer 19 to the imaging device 6 and the contrast of the image is improved.
An example of an ELISPOT assay, for which the optical reader could be used, is described below.
The bottom of the well 2 of the microplate 1 can be made of a PVDF membrane. The membrane is coated with appropriate antibody coating (capture antibodies). T-cells are dispensed into the wells 2. When the T-cells are treated with certain antigens, the cells will secrete corresponding cytokines. These cytokines are recognized by the capture antibodies resting on the PVDF membrane. The T-cells are washed away after the secretion phase.
Biotinylated (detection) antibodies are then dispensed into the wells 2, which also recognize the cytokine via a different epitope. The microplate 1 is then incubated with alkaline phosphatase-conjugated streptavidin or a similar detection system. Streptavidin binds to the biotinylated antibodies. Unbound molecules are washed away. Enzyme substrate molecules are added to the wells 2 and these will amplify label visibility on the bottom of the well 2. Spots are formed, and as the well 2 is illuminated with the light beam, the imaging device 6 can be used for taking an image of the bottom. The spots may be counted either manually or using image recognition.
In FluoroSpot assays fluorophores are used instead of an enzyme-labeled antibody and a precipitating enzyme substrate. In that case, the illuminating arrangement 4 is used for exciting the samples on the bottom of the well 2 with light having a certain wavelength, and the sample emits light with a longer wavelength according to the optical properties of the label molecule. Different fluorophores can be used simultaneously for detecting different analytes (assay multiplexing), as the cells secreting several analytes create multicolored spots.
It will be appreciated by a person skilled in the art that the invention is not limited to the embodiments described above, but may vary within the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20195073 | Feb 2019 | FI | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FI2020/050055 | 1/31/2020 | WO | 00 |
