A ring-shaped spectroscopic detection module, a flow cytometer and a debugging tool

Abstract

The present application discloses an annular spectroscopic detection module, a flow cytometer and a debugging tool. The annular spectroscopic detection module is composed of a concave reflector, a circular array filter group, a circular array focusing lens group and a circular array light detector group which are coaxially arranged in sequence to form a fluorescence detection submodule. The fluorescence detection submodule uses the principle of repeated focusing of light and adopts a concave reflector and a group of circular array filters to form an annular reflection light path. During multiple reflections of fluorescence, adjacent channels are only reflected once by the concave reflector, so the light energy loss is small. In addition, the collimated light path is focused once each time it is reflected by the concave reflector, so that the imaging size on the channel light detector is almost not affected by the fluorescence collimation and the number of detection channels. Therefore, the annular spectroscopic detection module has a flexible channel configuration scheme, a simple light path structure, a small light energy loss and simple light path debugging using the debugging tool, and can meet the laser channel expansion requirements of the current flow cytometer.

Description

Technical Field

[0001] The present application relates to the technical field of medical analysis instruments, in particular to a flow cytometer, and specifically to an annular spectroscopic detection module for a flow cytometer and a tool for debugging the annular spectroscopic detection module. Background Art

[0002] Flow cytometers, as instruments for counting and classifying different cells, have been widely used in the medical and biological fields. The optical system of a flow cytometer is usually composed of a laser excitation system and a fluorescence collection and detection module, and the fluorescence collection and detection module system is composed of a fluorescence collection mirror and a detection module.

[0003] Since the fluorescence spectra generated by the excitation of the same specific fluorescent dye by multiple lasers of different wavelengths in the flow optical system will overlap, the fluorescence collection module is often used in current flow cytometers to separate the fluorescence excited by different lasers in space first, and then couple it into the optical fiber corresponding to different laser channels at the end of the multimode optical fiber. The optical fiber is connected to the optical fiber interface reserved on the detection module, and the fluorescence is finally divided into fluorescence signals of different wavelengths by the optical fiber collimator, the spectroscope, and the bandpass filter. The fluorescence is received by the detector, and the fluorescence signals of different wavelengths are analyzed one by one by the computer software to count the types and quantities of particles in the sample.

[0004] There are three main optical path structures of existing fluorescence detection modules. The first one adopts the classic "tree-branch type" splitting optical path. This fluorescence detection module mainly realizes the splitting function by cutting off with a long-pass or short-pass filter and filtering with a bandpass filter. However, due to the large steepness of the long-pass filter, for detection modules with a large number of channels and a channel bandwidth less than 20nm, it will inevitably cause a significant attenuation of channel fluorescence. In addition, the filter is expensive and bulky. The second one adopts a "star optical path system" to collect and detect fluorescence signals. In this fluorescence detection module, a long focal length objective lens is used to collect fluorescence and limit divergence, but the aberration and divergence angle of fluorescence during transmission limit the number of fluorescence wavelengths that can be detected; at the same time, the fluorescence spot incident on the photosensitive surface of the detector is greater than 3mm, so this fluorescence detection module can only use a large surface area photomultiplier tube (PMT) as a detector, and the system is large in size and high in cost (PMT is expensive). The third method uses "zigzag" multiple reflection spectrometry to collect and detect fluorescent signals. Multiple micromirrors are connected on the same side, and multiple filters arranged in a row are connected on the opposite side. The incident light zigzags back and forth between multiple continuous filters and continuous micromirrors, and the light is imaged to the side where the filters are located, and then enters the light detector through the focusing lenses of the channels where each filter is located.

[0005] With the expansion of existing flow cytometer laser channels, the number of channels of the fluorescence detection module also needs to be increased accordingly. However, the above-mentioned optical path structures have problems in practical applications, such as high light energy loss in multiple reflection channels, large volume, high cost, relatively complex optical path debugging, and poor channel scalability of the fluorescence detection module. Therefore, it is necessary to propose a new technical solution to solve the problems existing in the existing technology. Summary of the invention

[0006] The present application provides an annular spectroscopic detection module, a flow cytometer and a debugging tooling, which are used to solve the problems of high light energy loss and poor channel scalability of the fluorescence detection module channel in the existing flow cytometer.

[0007] In order to achieve the above objectives, this application provides the following technical solutions:

[0008] In a first aspect, the present application provides an annular spectroscopic detection module, comprising a collimated light path submodule, a spectroscopic submodule, a side scatter detection submodule and a fluorescence detection submodule;

[0009] The collimating optical path submodule includes a light collimating mirror for collimating the input fluorescence into an output;

[0010] The photon splitting module is used to receive the collimated emitted fluorescence and separate it into side scattered light and fluorescence;

[0011] The side scattered light detection submodule comprises a filter and a focusing lens for allowing the side scattered light to pass through in sequence, and a light detector for receiving the light spot passing through the focusing lens;

[0012] The fluorescence detection submodule comprises a concave reflector, a circular array filter group, a circular array focusing lens group and a circular array light detector group which are coaxially arranged in sequence; the circular array filter group comprises a plurality of filters arranged in a circular array, the reflection surfaces of the plurality of filters are oriented in the same direction and are coplanar, and each filter can transmit light of different wavelength ranges; the circular array focusing lens group comprises a plurality of focusing lenses arranged in a circular array, the circular array light detector group comprises a plurality of light detectors arranged in a circular array, and the filters, focusing lenses and light detectors correspond to each other to form a light detection channel; the fluorescence passes through the concave reflector and is incident on a filter, the filter reflects the reflected light back to the concave reflector and is reflected to the next filter, so that the incident light forms an annular reflection light path between the continuous filters and the concave reflector, and the light passing through each filter is incident on the corresponding focusing lens and is received by the target surface of the light detector.

[0013] Furthermore, in the above technical solution, each filter in the circular array filter group is equipped with a filter mounting seat, and each filter can be independently disassembled and assembled.

[0014] Furthermore, the filters in the circular array filter group are bandpass filters, each of which has a different wavelength and bandwidth, and each of which is coated with a film layer according to the wavelength of the fluorescence to be measured, so that the fluorescence of the target wavelength passes through, while the fluorescence of the non-target wavelength is reflected.

[0015] Furthermore, the circular array filter group is arranged at the exit end of the concave reflector, and the reflective surfaces of the filters in the circular array filter group face the concave reflector.

[0016] Furthermore, each focusing lens in the circular array focusing lens group is a plano-convex lens or a spherical lens with an anti-reflection film coated on the surface, and each of the plano-convex lens or spherical lens has the same physical parameters.

[0017] Furthermore, each photodetector in the circular array photodetector group is arranged corresponding to the fluorescence wavelength divided by each filter in the circular array filter group, and the photodetectors in the circular array photodetector group are avalanche diodes.

[0018] Furthermore, a stable adjustment relationship is formed between the concave reflector, the circular array filter group and the incident angle of the collimated light path, and the expression formula of the stable adjustment relationship is: Cos(2θ)=1-2d / R, wherein: 2θ is the angle between any two adjacent filters in the circular array filter group, d is the straight-line distance between the center point of the concave reflector and the filters in the circular array filter group, and R is the curvature of the concave reflector.

[0019] Furthermore, the fluorescence input into the collimating optical path module is introduced through a quartz optical fiber, the fluorescence introduced by the quartz optical fiber is collected by a micro-light lens, and the fluorescence collected by the micro-light lens is collimated by the optical collimator.

[0020] Furthermore, the light collimating lens is a double cemented lens.

[0021] Furthermore, the quartz optical fiber is a through-path optical fiber, the core diameter of the through-path optical fiber is 400um, and the NA at both ends is 0.22 respectively.

[0022] Furthermore, the NA value of the low-light lens is greater than 1.2, the half field of view is greater than 0.328 mm, and the magnification is less than 6.1.

[0023] Furthermore, the spectroscopic submodule includes a first spectroscopic filter, and the first spectroscopic filter is located on the outgoing light path of the light collimator.

[0024] Furthermore, the first spectroscopic filter is used to separate the incident light into side scattered light perpendicular to the direction of the incident light and fluorescence in the same direction as the incident light, the side scattered light detection submodule is used to receive the side scattered light, and the fluorescence detection submodule is used to receive the fluorescence.

[0025] Furthermore, the first spectroscopic filter is a long-pass filter capable of deeply cutting off the wavelength of the excitation light, and the OD of the first spectroscopic filter is greater than 4.

[0026] Furthermore, the size of the spot image on each filter in the circular array filter group is different, and the size of the spot image on each filter is sinusoidally distributed from small to large, the diameter of the maximum spot image is less than 5 mm, and the diameter of the spot image focused on the target surface of each of the light detectors is less than 0.8 mm.

[0027] In a second aspect, the present application provides another annular spectroscopic detection module, in which the circular array filter group in the above technical solution can be replaced by a concave circular array filter group, wherein the concave circular array filter group is composed of a plurality of concave filters arranged in a circular array, each of the concave filters is mounted on a concave base, the curvature of the concave base is the same as the curvature of the concave filter, and the mounting surface of each concave filter is coplanar;

[0028] The concave filters, focusing lenses and photodetectors correspond to each other to form a light detection channel; the fluorescence passes through the concave reflector and is incident on a concave filter, the concave filter reflects the reflected light back to the concave reflector and is reflected to the next concave filter, so that the incident light forms an annular reflection light path between the continuous concave filters and the concave reflectors, and the light passing through each concave filter is incident on the corresponding focusing lens and is received by the target surface of the photodetector.

[0029] Furthermore, a stable adjustment relationship is formed between the concave reflector, the concave circular array filter group and the incident angle of the collimated light path, and the expression formula of the stable adjustment relationship is: Cos(2θ)=1-2d / R, wherein: 2θ is the angle formed by the first reflection point and the next reflection point along the light propagation path on the concave circular array filter group relative to the center of the concave circular array filter group, d is the straight-line distance between the center point of the concave reflector and the center point of the concave filter in the concave circular array filter group, and R is the curvature of the concave reflector.

[0030] In a third aspect, the present application provides a flow cytometer equipped with an annular spectroscopic detection module, wherein the annular spectroscopic detection module is the annular spectroscopic detection module in the first scheme mentioned above, or the annular spectroscopic detection module is the annular spectroscopic detection module in the second scheme mentioned above.

[0031] In a fourth aspect, the present application provides a debugging tool for the above-mentioned flow cytometer, comprising a pinhole diaphragm and a side dispersion diaphragm;

[0032] The pinhole aperture comprises an annular plate, a plurality of observation pinhole structures are evenly arranged on the plate surface of the annular plate, the number of the observation pinhole structures is the same as the number of the light detectors in the circular array light detector group, the observation pinhole structure comprises a boss protruding outward from the plate surface of the annular plate, the boss is provided with a through observation pinhole along its axial direction, one observation pinhole corresponds to a target surface of the light detector, the inner diameter of the observation pinhole is 0.8 mm, the pinhole aperture is mounted on the target surface of the light detector of the circular array light detector group, after the optical path is debugged, the visible fluorescence of each light detection channel can be observed by human eyes through the pinhole aperture, and the infrared light of each light detection channel can be observed by an infrared observation instrument;

[0033] The side dispersion aperture stop is a transparent or translucent cylindrical structure, and a through observation hole is provided along the axial direction of the side dispersion aperture stop, and the inner diameter of the observation hole is 0.8 mm. The side dispersion aperture stop is mounted on the target surface of the light detector of the side dispersion detection submodule, and the observation hole is connected with the target surface optical path of the light detector. After the optical path is debugged, the light energy of the side dispersion channel of the side dispersion detection submodule can be observed by the human eye through the side dispersion aperture stop.

[0034] Compared with the prior art, this application has at least the following beneficial effects:

[0035] 1. Based on further analysis and research on the existing technical problems, the present application recognizes that the optical path structure of the fluorescence detection module of the traditional flow cytometer has the problems of high light energy loss in multiple reflection channels, large size, high cost, relatively complex optical path debugging, and poor channel scalability of the fluorescence detection module, which makes it difficult to meet the expansion requirements of the laser channel of the flow cytometer. To this end, the present application provides an annular spectroscopic detection module with a new optical path design. The fluorescence detection submodule in the annular spectroscopic detection module utilizes the "light repeated focusing principle" and adopts a concave mirror and a circular array filter group to form an annular reflection light path, which can divide the white fluorescence into several fluorescence channels. During the multiple reflection process of the fluorescence, adjacent channels are only separated by the concave reflection mirror. One reflection, so the light energy loss is small, and the collimated light path in the fluorescence detection submodule of the present application is focused once each time it is reflected by the concave reflector. Even if the input light spot of the collimated light path diverges, the reflected light spot can always be controlled within a certain range, so that the imaging size on the light detector of its light detection channel is almost unaffected by the fluorescence collimation and the number of detection channels; therefore, under the same number of channels, the annular spectroscopic detection module designed based on the "light repeated focusing principle" of the present application has a more flexible channel configuration scheme, a simpler structure, a smaller volume and a smaller light energy loss compared with the fluorescence detection module of the existing flow cytometer, and can meet the laser channel expansion requirements of the current flow cytometer.

[0036] 2. The annular spectroscopic detection module provided in the present application is universal and can be applied not only to full-spectrum flow cytometers, but also to ordinary flow cytometers on the market.

[0037] 3. In the scheme provided in the present application, the number of bandpass filters in the circular array filter group can be arbitrary, that is, the number of bandpass filters can be adjusted according to application requirements; in addition, the bandwidth parameters of the bandpass filters of each channel in the circular array filter group can be adjusted arbitrarily according to requirements; furthermore, the wavelengths and bandwidths of the bandpass filters in the circular array filter group are different, that is, the circular array filter group can realize selective filtering of light in multiple different wavelength ranges in the same array, and realize precise wavelength selection; in addition, each channel bandpass filter in the circular array filter group in the present application can be flexibly disassembled and installed separately, the arrangement order of each bandpass filter can be arbitrarily replaced, and the bandwidth parameters of each bandpass filter can be arbitrarily adjusted according to requirements; therefore, the annular spectroscopic detection module provided in the present application has a more flexible channel configuration scheme, which can meet the laser channel expansion requirements of current flow cytometers.

[0038] 4. The present application also provides a debugging tool for a flow cytometer equipped with the annular spectroscopic detection module, including a pinhole diaphragm and a side dispersion diaphragm. The pinhole diaphragm can debug each light detection channel in the fluorescence detection submodule to detect its spectroscopic effect, and the side dispersion diaphragm can debug the side dispersion channel in the side dispersion detection submodule to detect its spectroscopic effect. After the annular spectroscopic detection module is debugged using the pinhole diaphragm and the side dispersion diaphragm, the visible fluorescence of each light detection channel in the fluorescence detection submodule can be observed by the human eye through the pinhole diaphragm, and the infrared light can be observed by an infrared observation instrument. The purple light of the side dispersion channel can also be observed by the human eye through the side dispersion diaphragm, so that it can be verified whether the spectroscopic effect is consistent with the design result. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. It should be understood that the specific shapes and structures shown in the drawings should not usually be regarded as limiting conditions for implementing the present application; for example, based on the technical concepts and exemplary drawings disclosed in the present application, those skilled in the art are able to easily make routine adjustments or further optimizations to the increase / decrease / attribution division, specific shapes, positional relationships, connection methods, and dimensional ratios of certain units (components).

[0040] Figure 1 A schematic diagram of the optical design of the annular spectroscopic detection module provided by the present application in one embodiment;

[0041] Figure 2 Schematic diagram of the light path of the annular light path stability condition of the annular spectroscopic detection module provided in the present application, wherein (a) is a schematic diagram of the reflection light path of the concave reflector and the filter, and (b) is a schematic diagram of the reflection points in the circular array filter group;

[0042] Figure 3 This is a schematic diagram of a 16-channel light path simulation of a fluorescence detection submodule in an embodiment;

[0043] Figure 4 An instrument detection diagram of the maximum light spot on the target surface of the light detector of the fluorescence detection submodule in one embodiment;

[0044] Figure 5 is a schematic structural diagram of a flow cytometer equipped with an annular spectroscopic detection module in an embodiment;

[0045] Figure 6 A schematic diagram of the structure of a pinhole stop in an embodiment;

[0046] Figure 7 A schematic diagram of the structure of a side diffusion aperture in an embodiment;

[0047] Figure 8 An image observed by an infrared observer on a light detector target surface in a fluorescence detection submodule in an embodiment;

[0048] Fig. 9 Schematic diagram of channels 1 to 16 in the fluorescence detection submodule in Example 3, where numbers 1 to 16 in the figure are numbers of the channels respectively;

[0049] Fig.10 The target surface spot observation images of the light detectors of the 15th and 16th channels observed by the infrared display in the fluorescence detection submodule in the third embodiment;

[0050] Fig.11 Schematic diagrams of the structure of a concave circular array filter group, wherein (a) is a schematic diagram of the front view structure of the concave circular array filter group, (b) is a schematic diagram of the cross-sectional structure of the AA section in (a); (c) is a schematic diagram of the three-dimensional structure of the concave circular array filter group;

[0051] Fig.12 Schematic diagrams of the structure of a concave filter, wherein (a) is a schematic diagram of the front view structure of the concave filter, and (b) is a schematic diagram of the three-dimensional structure of the concave filter;

[0052] Fig.13 A schematic diagram of the optical path of the annular optical path stability condition of the annular spectroscopic detection module provided in Example 4, wherein (a) is a schematic diagram of the reflection optical path of the concave reflector and the concave filter, and (b) is a schematic diagram of the reflection points in the concave circular array filter group;

[0053] Fig.14 This is a schematic diagram of the simulation of the 16-channel optical path of the fluorescence detection submodule and the optical path of the concave reflector in Example 5;

[0054] Fig.15 This is a schematic diagram of the 16-channel light path simulation of the fluorescence detection submodule using the concave circular array filter group in Example 5.

[0055] Description of reference numerals:

[0056] 100. collimating optical path submodule; 110. optical collimating mirror;

[0057] 200, photon splitter module; 210, first photon splitter filter;

[0058] 300, side scatter detection submodule; 310, filter; 320, focusing lens; 330, light detector;

[0059] 400, fluorescence detection submodule; 410, concave reflector; 420, circular array filter group; 421, filter; 430, circular array focusing lens group; 431, focusing lens; 440, circular array photodetector group; 441, photodetector;

[0060] 500, pinhole aperture; 510, observation pinhole;

[0061] 600, side diffuser diaphragm;

[0062] 700, concave circular array filter group; 710, concave filter;

[0063] R, incident light; S, reflection point; DETAILED DESCRIPTION

[0064] The present application is further described below in detail through specific embodiments in conjunction with the accompanying drawings.

[0065] In the description of this application: unless otherwise specified, "plurality" means two or more. The terms "first", "second", etc. in this application are intended to distinguish the objects referred to, and do not have special meanings in terms of technical connotations (for example, they should not be understood as emphasizing the importance or order, etc.). Expressions such as "including", "comprising", "having", etc. also mean "not limited to" (certain units, components, materials, steps, etc.).

[0066] The present application proposes an annular spectroscopic detection module with a completely new optical path design, or an annular spectroscopic spectrum detection module, the optical path design of which mainly utilizes the "light repeated focusing principle", using a concave reflector and a group of circular array filters to form an annular reflective optical path, which can divide fluorescence (such as white fluorescence or other color fluorescence) into several fluorescence channels. Under the same number of channels, the annular spectroscopic detection module designed by the present application based on the "light repeated focusing principle" has a more flexible channel configuration scheme, a simpler structural form, a smaller volume, and a smaller light energy loss compared to the fluorescence detection module of the existing flow cytometer. The structural composition and layout of the annular spectroscopic detection module are described in detail below.

[0067] Embodiment 1

[0068] The present embodiment provides an annular spectroscopic detection module, which mainly includes a collimated light path submodule 100, a spectroscopic submodule 200, a side scatter detection submodule 300 and a fluorescence detection submodule 400. The structural components of the four submodules are described in detail below.

[0069] 1. Collimated optical path module

[0070] See also Figure 1The collimating optical path module 100 in the present application mainly includes a light collimating lens 110. The fluorescence input into the collimating optical path module is introduced through a quartz optical fiber, and the fluorescence introduced into the collimating optical path module is collimated by the light collimating lens.

[0071] In a specific embodiment, the light collimator 110 may be a double-cemented lens. A double-cemented lens is an optical lens formed by gluing two lenses of different materials together. The double-cemented lens mainly combines two glass lenses of different refractive indices together to reduce the refraction difference of light of different wavelengths in the lens, thereby effectively correcting chromatic aberration.

[0072] In other embodiments, the light collimator may also be an aspheric lens, a free-form lens, an optical waveguide collimator, a liquid crystal lens, a gradient refractive index lens, etc. That is, in addition to using a double-cemented lens, the light collimator in the present application may also use other types of lenses with collimation functions according to the actual application requirements of the optical system, the characteristics of the light beam (such as wavelength, divergence angle, power, etc.) and the accuracy requirements of the application.

[0073] In the present application, the optical fiber that introduces fluorescence into the collimated optical path submodule is a through-path optical fiber. In a specific embodiment, if a through-path optical fiber is used, a through-path optical fiber with a core diameter of 400um and a NA (numerical aperture) of 0.22 at both ends can be selected. In other embodiments, the optical fiber does not necessarily use quartz optical fiber, and plastic optical fiber (such as optical fiber made of polystyrene or polymethyl methacrylate, etc.), fluoride optical fiber (such as optical fiber made of calcium fluoride, magnesium fluoride, etc.), sulfide optical fiber (optical fiber made of compound materials containing elements such as sulfur, selenium or tellurium), photonic crystal optical fiber, etc. can also be used. Although quartz optical fiber dominates the communication field due to its low optical loss, wide transmission bandwidth and stability, different materials can be selected to make optical fiber according to specific application requirements, and the selection of which optical fiber material usually depends on factors such as the required transmission wavelength, optical loss, cost, flexibility and application environment.

[0074] In the present application, the fluorescence introduced by the optical fiber is collected by a micro-light lens, which is then collimated by a light collimator. In a specific embodiment, the NA value of the micro-light lens is greater than 1.2, the half field of view is greater than 0.328 mm, and the magnification is less than 6.1.

[0075] 2. Photon Module

[0076] See also Figure 1The spectroscopic submodule 200 in the present application mainly includes a first spectroscopic filter 210, which is located in the light emitting direction of the light collimator 110. The light collimator 110 collimates the incident light R and then incidents on the first spectroscopic filter 210. The first spectroscopic filter 210 is used to separate the incident light into side scattered light (light perpendicular to the direction of the incident light) and fluorescence (light in the same direction as the incident light), wherein the side scattered light enters the side scattered detection submodule, and the fluorescence enters the fluorescence detection submodule.

[0077] In a specific embodiment, the first spectroscopic filter 210 adopts a long-pass filter to deeply cut off the wavelength of the excitation light, and the OD (cut-off depth) of the first spectroscopic filter is > 4. That is, the long-pass filter in this embodiment can effectively cut off the excitation light (i.e., light with a shorter wavelength) and has a very high cut-off depth (OD>4), that is, it has a very strong attenuation ability for the excitation light, ensuring that the excitation light will hardly pass through the filter, and only allows light with a longer wavelength to pass, which can minimize the interference of the excitation light and improve the accuracy of the signal and the image quality. In addition to the long-pass filter, the first spectroscopic filter can also adopt a short-wavelength filter, a bandpass filter, etc. In specific applications, a suitable filter can be selected according to the spectral components to be separated.

[0078] In other embodiments, the spectroscopic submodule in the present application may also use a spectroscope, which can split the incident light into multiple light beams by reflection and transmission. Therefore, according to application requirements, a spectroscope can be used to split the incident light into side scattered light (light perpendicular to the direction of the incident light) and fluorescence (light in the same direction as the incident light), wherein the side scattered light enters the side scattered detection submodule, and the fluorescence enters the fluorescence detection submodule.

[0079] 3. Side Scattering Detection Submodule

[0080] See also Figure 1 In the present application, the side scattered light detection submodule 300 includes: a filter 310, a focusing lens 320 and a photodetector 330. The side scattered light separated by the first spectroscopic filter 210 passes through the filter 310 and the focusing lens 320 in sequence and is finally received by the photodetector 330 and converted into a photoelectric signal.

[0081] In a specific embodiment, the focusing lens 320 is a common plano-convex lens.

[0082] 4. Fluorescence Detection Submodule

[0083] See also Figure 1 , 3The fluorescence detection submodule 400 in the present application mainly includes: a concave reflector 410, a circular array filter group 420, a circular array focusing lens group 430 and a circular array light detector group 440.

[0084] In a specific embodiment, the substrate of the above-mentioned concave reflector 410 is K9 (an optical glass material, mainly composed of sodium silicate and a small amount of other chemical components, K9 glass is often used as the substrate of high-precision optical lenses), and the surface is coated with a metal dielectric film, and the reflectivity is >97% (365nm-850nm). That is, the concave reflector in this embodiment uses K9 optical glass as a substrate, and its surface is coated with a metal dielectric film to improve the reflection efficiency; within the spectral range of 365 nanometers to 850 nanometers, the reflectivity of the concave reflector exceeds 97%, that is, most of the incident light will be reflected back, and only a small amount of light will be absorbed or transmitted. Therefore, the concave reflector can meet the application scenarios that require efficient reflection.

[0085] In the present application, the above-mentioned circular array filter group 420 is composed of multiple filters arranged in a circular array, each filter includes a filter 421 (such as a bandpass filter) and a filter mounting seat. In the circular array filter group, the reflective surface of each filter 421 is oriented in the same direction and needs to be evenly distributed in a plane as a whole.

[0086] In addition, the number of bandpass filters in the circular array filter group 420 can be arbitrary, that is, the number of bandpass filters can be adjusted according to application requirements. Furthermore, the bandwidth parameters of each channel bandpass filter in the circular array filter group can be adjusted arbitrarily according to requirements.

[0087] In a specific embodiment, the wavelengths and bandwidths of the bandpass filters (the bandpass filters may be planar bandpass filters) in the circular array filter group 420 are different. That is, the circular array filter group 420 can selectively filter light of multiple different wavelength ranges in the same array, thereby achieving precise wavelength selection.

[0088] Traditional channel filters are glued to a glass block, and the filters cannot be disassembled or replaced individually. However, each channel bandpass filter in the circular array filter group in this application can be flexibly disassembled and installed individually, the arrangement order of each bandpass filter can be arbitrarily replaced, and the bandwidth parameters of each bandpass filter can be adjusted arbitrarily according to needs.

[0089] In the present application, the circular array focusing lens group 430 is composed of a plurality of focusing lenses 431 arranged in a circular array. In a specific embodiment, the circular array focusing lens group 430 is composed of a certain number of plano-convex lenses with the same physical parameters, and the surface of the plano-convex lenses is coated with an anti-reflection film.

[0090] In the present application, the circular array photodetector group 440 is composed of a plurality of photodetectors 441 arranged in a circular array. In a specific embodiment, the circular array photodetector group is a certain number of avalanche diodes. In other embodiments, the circular array photodetector group may also be a certain number of quantum dot photodetectors, single photon counters, or superconducting nanowire single photon detectors (SNSPDs).

[0091] In the present application, the above-mentioned concave reflector 410 is used to receive the fluorescence separated by the first spectroscopic filter 210; the above-mentioned circular array filter group 420 is coaxially arranged with the concave reflector 410, and the circular array filter group 420 is arranged at the light reflecting end of the concave reflector 410, and the light emitted from the circular array filter group 420 is incident on the circular array focusing lens group 430, and the light emitted from the circular array focusing lens group 430 is incident on the circular array light detector group 440.

[0092] Each filter in the circular array filter group can transmit light of different wavelength ranges, and reflect the reflected light to the concave reflector on the other side. The reflected light is focused by the concave reflector and then reflected to the next filter, so that the incident light forms a circular reflection light path between the continuous filters and the concave reflector. The light passing through each channel filter is incident on the focusing lens and finally enters the light detector. During the multiple reflections of fluorescence, the adjacent channels are reflected only once by the concave reflector. Figure 1 Schematic diagram of the optical design shown.

[0093] It should be noted that the curvature of the concave reflector and the distance between the circular array filter group and the incident angle of the collimated light path in this application need to meet certain stable adjustments to form a ring-shaped reflective light path. The schematic diagram of the light path under the stable condition of the ring light path is as follows: Figure 2 (a) to (b) in Figure 2 (a) shows that the incident light R reaches the filter of the circular array filter group after passing through the concave reflector 410, and is reflected back to the concave reflector 410 at number 2 at number 1, and so on, forming a circular reflection light path between the concave reflector and the circular array filter.

[0094] The above stability condition can be expressed by the formula: Cos(2θ)=1-2d / R, where 2θ is the angle between any two adjacent filters in the circular array filter group (that is, in the circular array filter group, each filter has a reflection point, and the angle formed by any two adjacent reflection points and the center of the circular array filter group is, such as Figure 2 As shown in (b), A in the figure represents the straight-line distance between the center of the circular array filter group and the reflection point on the filter), d is the straight-line distance between the center point of the concave reflector and the filter in the circular array filter group, and R is the curvature of the concave reflector.

[0095] In the present application, the image size on each filter in the circular array filter group is different, and is sinusoidally distributed from small to large, but the diameter of the largest spot is less than 5mm, and the diameter of the spot focused on the target surface of the light detector is less than 0.8mm.

[0096] Compared with the optical path structure of the existing fluorescence detection module, in the process of multiple reflections of the fluorescence in the present application, adjacent channels are reflected only once by the concave reflector, that is, the light energy loss is only one reflection loss (in the glass block scheme of the optical path structure of the existing fluorescence detection module, there will be reflection loss at the bonding interface between the filter and the glass block). At the same time, the light spots of adjacent channels will be repeatedly focused each time they are reflected by the concave reflector. Even if the input light spot of the collimated light path diverges, the diameter of the reflected light spot can always be controlled within a certain range (within 5 mm), and the signal received by each channel can be completely received by the light detector.

[0097] In other embodiments, based on the technical concept provided by the present application, the circular array filter group 420, the circular array focusing lens group 430 and the circular array photodetector group 440 in the present application are not limited to a perfect circle, but may also be an ellipse or a closed annular array arrangement consisting of a curved array, etc. In specific application practice, if an elliptical arrangement or a closed annular array arrangement is adopted, it is necessary to conduct multiple tests on the stable conditions for forming an annular reflection light path according to application requirements, so as to obtain the stable conditions for forming an annular reflection light path through the test.

[0098] Embodiment 2

[0099] The present embodiment provides a flow cytometer equipped with the annular spectroscopic detection module provided in the first embodiment, and the flow cytometer is provided with installation positions for installing and fixing the collimated light path submodule, the spectroscopic submodule, the side scatter detection submodule and the fluorescence detection submodule. The annular spectroscopic detection module is installed on the flow cytometer to realize the installation and debugging of the collimated light path submodule, the side scatter detection submodule and the fluorescence detection submodule.

[0100] A person skilled in the art can install the annular spectroscopic detection module designed in this application on a flow cytometer according to its structure and arrangement. The specific installation and fixing method of the various components of the annular spectroscopic detection module and the flow cytometer can be implemented by a conventional structural fixing method in the art. The flow cytometer can be a full-spectrum flow cytometer or a common flow cytometer. Figure 5 .

[0101] The annular spectroscopic detection module configured in the flow cytometer provided in this embodiment is specifically described below by taking a 16-channel fluorescence detection submodule as an example.

[0102] The annular spectroscopic detection module in the full-spectrum flow cytometer mainly includes a collimated light path submodule, a first spectroscopic filter, a side scatter detection submodule and a fluorescence detection submodule. The collimated light path submodule collimates the incident light and then incidents it on the first spectroscopic filter, so that the incident light enters the side scatter detection submodule and the fluorescence detection submodule respectively. Among them, the fluorescence detection submodule has 16 channels.

[0103] Specifically, the fluorescence detection submodule includes a concave reflector, a circular array filter group, a circular array focusing lens group and a circular array light detector group, wherein the circular array filter group includes 16 bandpass filters arranged in a circular array, each filter is coated according to the wavelength of the fluorescence to be measured, so that the fluorescence of a specific wavelength passes through, while the fluorescence of other wavelengths is all reflected, and the reflection surfaces of the 16 bandpass filters need to be in one plane; the circular array focusing lens group includes 16 spherical lenses arranged in an array, and both surfaces of each spherical lens are coated with anti-reflection dielectric films, and the spherical lens images the fluorescence passing through the first spectroscopic filter on the target surface of the light detector; the circular array light detector group includes 16 light detectors arranged in a circular array, and the light detectors are set corresponding to the fluorescence wavelengths divided by the bandpass filters, and the light detectors are used to receive fluorescence signals and convert the fluorescence signals into electrical signals. The circular array filter group and the concave reflector are arranged coaxially. Each filter in the circular array filter group can transmit light of different wavelength ranges and reflect the reflected light to the concave reflector on the other side. The reflected light is focused by the concave reflector and then reflected to the next filter. Finally, the incident light forms 16 evenly distributed light spot reflection rings between the continuous filters and the concave reflector. The light passing through each filter is incident on the focusing lens and finally enters the light detector. The schematic diagram of the 16-channel optical path simulation of the fluorescence detection submodule is shown in the figure. Figure 3 .

[0104] In this embodiment, the size of the fluorescent spot input by the collimated light path module is 4 mm, the diameter of the concave reflector is 25.8 mm, the curvature R = 400, and the straight-line distance d between the center point of the concave reflector and the flat bandpass filter in the circular array filter group is 15.224 mm. The instrument detection diagram of the maximum spot on the target surface of the light detector is as follows: Figure 4 It can be seen that the light spots on the target surfaces of the above 16 photodetectors are all smaller than 0.8 mm, and photodetectors with a target surface larger than 0.8 mm can be selected.

[0105] In a specific embodiment, a white light source is connected to the collimated optical path submodule of the flow cytometer equipped with the above-mentioned 16-channel fluorescence detection submodule. After the white light source is split by the first spectroscopic filter, a part of the white light source enters the side scatter channel of the side scatter detection submodule, and the other part of the white light source is divided into 16 parts by the fluorescence detection submodule. The fluorescence wavelength range received by the light detector of each channel is shown in Tables 1 and 2 below, wherein channels 1 to 14 are visible fluorescence, and channels 15 to 16 are invisible infrared.

[0106] Table 1 Fluorescence bandwidth of visible fluorescence of channels 1 to 14 in the fluorescence detection submodule

[0107]

[0108] Table 2 The fluorescence bandwidth of invisible infrared light in the 15th to 16th channels in the fluorescence detection submodule

[0109] aisle 15 16 Wavelength / nm 780 / 30 812 / 34

[0110] The light entering the side scatter channel of the side scatter detection submodule is purple light, and its fluorescence bandwidth is shown in Table 3 below.

[0111] Table 3 Fluorescence bandwidth of violet light in the side scattered channel

[0112] aisle Side channel Wavelength / nm 405 / 10

[0113] Embodiment 3

[0114] This embodiment provides a tool that can debug an annular spectroscopic detection module installed on a flow cytometer. The tool includes a pinhole diaphragm and a side dispersion diaphragm.

[0115] See also Figure 6 The pinhole aperture 500 in the present application includes an annular plate, and a plurality of observation pinhole structures are evenly arranged on the plate surface of the annular plate. The number of the observation pinhole structures is the same as the number of the light detectors 441 in the circular array light detector group 440. The observation pinhole structure includes a boss protruding outward from the plate surface of the annular plate, and the boss is provided with a through observation pinhole 510 along its axial direction. The observation pinhole 510 corresponds one-to-one to the target surface of the light detector 441.

[0116] The pinhole diaphragm 500 is installed on the target surface of the light detector 441 of the circular array light detector group 440. The observation pinhole corresponds to the target surface of the light detector one by one. The inner diameter of the observation pinhole is 0.8 mm. After the optical path is debugged, the visible fluorescence of each channel can be observed by the human eye through the pinhole diaphragm, and the infrared light can be observed by the infrared observation instrument.

[0117] See also Figure 7The side scattering aperture 600 in the present application is a transparent or translucent cylindrical structure, and a through observation hole is opened along the axial direction of the side scattering aperture 600, and the inner diameter of the observation hole is 0.8 mm. The side scattering aperture is installed on the target surface of the light detector of the side scattering detection submodule, and the observation hole corresponds to the target surface of the light detector. After the optical path is debugged, the purple light of the side scattering channel can be observed by the human eye through the side scattering aperture.

[0118] See also Figure 8 , is the image observed by the infrared observer on the light detector target surface in the fluorescence detection submodule; see Fig. 9 , is a schematic diagram of channels 1 to 16 in the fluorescence detection submodule, where numbers 1 to 16 are the numbers of the channels respectively; see Fig.10 , is the target surface spot observation image of the light detector of the 15th to 16th channels observed by the infrared display in the fluorescence detection submodule.

[0119] By placing a pinhole diaphragm at each photodetector target surface, after the optical path is debugged, the visible fluorescence of each channel can be observed by the human eye through the observation pinhole on the pinhole diaphragm, and the infrared light can also be observed by the infrared observer. The spectral effect detected by debugging the tooling is consistent with the design result.

[0120] Embodiment 4

[0121] This embodiment provides another annular spectroscopic detection module. The difference from the first embodiment is that the circular array filter group 420 in the first embodiment is replaced by a concave circular array filter group 700. Fig.15 .

[0122] In this embodiment, the concave circular array filter group 700 is composed of a plurality of concave filters 710 arranged in a circular array, each concave filter 710 is mounted on a concave base, the curvature of the concave base is the same as the curvature of the concave filter, and they need to be coplanar after being combined. Fig.11 , 12 , Fig.11 Schematic diagrams of the structure of a concave circular array filter group, wherein (a) is a schematic diagram of the front view structure of the concave circular array filter group, (b) is a schematic diagram of the cross-sectional structure of the AA section in (a); and (c) is a schematic diagram of the three-dimensional structure of the concave circular array filter group. Fig.12 Schematic diagrams of the structure of a concave filter, wherein (a) is a schematic diagram of the front view structure of the concave filter, and (b) is a schematic diagram of the three-dimensional structure of the concave filter.

[0123] Each concave filter is mounted on a concave base and the curvature of the base is the same as the curvature of the filter, which means that the shape of the base and the shape of the filter are consistent, and the two together form a curved surface, usually a concave surface.

[0124] In order to ensure that light can pass through each concave filter in the array according to the predetermined path, the installation positions of all concave filters must be ensured to be in the same optical plane, so as to ensure that the transmission and focusing of light are uniform. If these concave filters are not coplanar, it may cause the light to fail to accurately pass through each concave filter, thereby affecting the optical performance of the entire system. Therefore, the aforementioned "coplanar" means that after assembly, the installation surfaces of all concave filters should be on the same plane. Although each concave filter is concave, their installation positions (i.e., the surfaces contacted by the base) are coplanar. Therefore, there will be no height difference or misalignment between the multiple concave filters after installation.

[0125] In this embodiment, the curvature of the concave reflector 410, the distance between the concave circular array filter group 700 and the incident angle of the collimated light path need to meet certain stable adjustments to form a ring-shaped reflective light path. The stable condition optical path diagram of the ring-shaped light path realized by the concave circular array filter group is shown in FIG. Fig.13 (a) to (b) in Fig.13 (a) shows that the incident light reaches the concave filter of the concave circular array filter group after passing through the concave reflector 410, and is reflected back to the concave reflector 410 at number 2 at number 1, and then reflected to the concave circular array filter group at number 3, and so on, forming a ring-shaped reflection light path between the concave reflector and the circular array filter.

[0126] The above stability condition can be expressed by the formula: Cos(2θ) = 1-d / R, where 2θ is the angle between the reflected light of adjacent channels (i.e., in the concave circular array filter group, each concave filter has a reflection point, and the angle formed by the first reflection point and the next reflection point relative to the center of the concave circular array filter group along the light propagation path is, such as Fig.13 As shown in (b), A in the figure represents the straight-line distance between the concave circular array filter group and the reflection point on the filter), d is the straight-line distance between the center point of the concave reflector and the center point of the concave filter in the concave circular array filter group, and R is the curvature of the concave reflector.

[0127] Embodiment 5

[0128] The present embodiment provides a flow cytometer equipped with the annular spectroscopic detection module provided in the fourth embodiment. The flow cytometer is provided with installation positions for installing and fixing the collimated light path submodule, the spectroscopic submodule, the side scatter detection submodule and the fluorescence detection submodule. The annular spectroscopic detection module is installed on the flow cytometer to realize the installation and debugging of the collimated light path submodule, the side scatter detection submodule and the fluorescence detection submodule.

[0129] Those skilled in the art can install the annular spectroscopic detection module designed in the present application on a flow cytometer according to its structure and arrangement. The specific installation and fixing method of the various components of the annular spectroscopic detection module and the flow cytometer can be achieved by conventional structural fixing methods in the field. The flow cytometer can be a full-spectrum flow cytometer or a common flow cytometer.

[0130] The annular spectroscopic detection module configured in the flow cytometer provided in this embodiment is specifically described below by taking a 16-channel fluorescence detection submodule as an example.

[0131] The annular spectroscopic detection module in the full-spectrum flow cytometer mainly includes a collimated light path submodule, a first spectroscopic filter, a side scatter detection submodule and a fluorescence detection submodule. The collimated light path submodule collimates the incident light and then incidents it on the first spectroscopic filter, so that the incident light enters the side scatter detection submodule and the fluorescence detection submodule respectively. Among them, the fluorescence detection submodule has 16 channels.

[0132] Specifically, the fluorescence detection submodule includes a concave reflector 410, a concave circular array filter group 700, a circular array focusing lens group 430 and a circular array light detector group 440, such as Fig.15 . Among them, the concave circular array filter group 700 includes 16 concave filters 710 arranged in a circular array, the curvature of each concave filter is the same as the curvature of the concave reflector, and each concave filter will be coated (concave surface coated with reflective film) according to the wavelength of the fluorescence to be measured, so that the fluorescence of a specific wavelength passes through, while the fluorescence of other wavelengths is all reflected, and the reflective surfaces (concave surfaces) of the 16 concave filters need to be installed coplanarly; the circular array focusing lens group includes 16 spherical lenses arranged in an array, and both surfaces of each spherical lens are coated with anti-reflection medium film, and the spherical lens will image the fluorescence passing through the first spectroscopic filter on the target surface of the light detector; the circular array light detector group includes 16 light detectors arranged in a circular array, and the light detectors are set corresponding to the fluorescence wavelengths divided by the bandpass filter, and the light detectors are used to receive the fluorescence signal and convert the fluorescence signal into an electrical signal. The concave circular array filter group and the concave reflector are arranged coaxially. Each concave filter in the concave circular array filter group can transmit light in different wavelength ranges and reflect the reflected light to the concave reflector on the other side. The reflected light is focused by the concave reflector and then reflected to the next concave filter. Finally, the incident light forms 16 evenly distributed light spot reflection rings between the continuous concave filters and the concave reflector. The light passing through each concave filter is incident on the focusing lens and finally enters the light detector.

[0133] The schematic diagram of the 16-channel optical path simulation of the fluorescence detection submodule using the concave circular array filter group is shown in the figure. Fig.15The simulation diagram of the 16-channel optical path of the fluorescence detection submodule and the optical path of the concave reflector is shown in Fig.14 .

[0134] In this embodiment, the size of the fluorescent spot input by the collimated light path submodule is 4 mm, the diameter of the concave reflector is 25.8 mm, the curvature R=400, and the straight-line distance d between the concave reflector and the concave filter in the concave circular array filter group is 17.20653 mm. The light spots on the target surfaces of the above 16 light detectors are all smaller than 0.8 mm, and light detectors with a target surface larger than 0.8 mm can be selected.

[0135] In a specific embodiment, a white light source is connected to the collimated optical path submodule of the flow cytometer equipped with the above-mentioned 16-channel fluorescence detection submodule. After the white light source is split by the first spectroscopic filter, a part of the white light source enters the side scatter channel of the side scatter detection submodule, and the other part of the white light source is divided into 16 parts by the fluorescence detection submodule. The fluorescence wavelength range received by the light detector of each channel is shown in Tables 4 and 5 below, wherein channels 1 to 14 are visible fluorescence, and channels 15 to 16 are invisible infrared.

[0136] Table 4 Fluorescence bandwidth of visible fluorescence of channels 1 to 14 in the fluorescence detection submodule

[0137]

[0138] Table 5 The fluorescence bandwidth of the invisible infrared light of the 15th to 16th channels in the fluorescence detection submodule

[0139] aisle 15 16 Wavelength / nm 780 / 30 812 / 34

[0140] The light entering the side scatter channel of the side scatter detection submodule is purple light, and its fluorescence bandwidth is shown in Table 6 below.

[0141] Table 6 Fluorescence bandwidth of violet light in the side scattered channel

[0142] aisle Side channel Wavelength / nm 405 / 10

[0143] The tooling (pinhole aperture and side dispersion aperture) provided in the above-mentioned embodiment 3 is respectively installed on the light detector target surface of the circular array light detector group of the annular spectroscopic detection module on the flow cytometer, and on the target surface of the light detector of the side dispersion detection submodule. After the optical path is debugged, the visible fluorescence of each channel in the fluorescence detection submodule can be observed by the human eye through the pinhole aperture, the infrared light can be observed by the infrared observation instrument, and the purple light of the side dispersion channel in the side dispersion detection submodule can be observed by the human eye. The spectroscopic effect detected by debugging the tooling is consistent with the design result.

[0144] In summary, the present application provides an annular spectroscopic detection module with a completely new optical path design, a flow cytometer equipped with the annular spectroscopic detection module, and a debugging tool for the flow cytometer. The fluorescence detection submodule in the annular spectroscopic detection module in the present application utilizes the "light repeated focusing principle" and adopts a concave mirror and a circular array filter group to form an annular reflective light path, which can divide the white fluorescence into several fluorescence channels. During the multiple reflections of the fluorescence, the adjacent channels are only reflected once by the concave reflector, so the light energy loss is small, and the collimated light path in the fluorescence detection submodule in the present application is reflected once by the concave reflector. After focusing once, even if the input light spot of the collimated light path diverges, the reflected light spot can always be controlled within a certain range, so that the imaging size on the light detector of its light detection channel is almost unaffected by the fluorescence collimation and the number of detection channels; therefore, under the same number of channels, the annular spectroscopic detection module designed based on the "light repeated focusing principle" in this application has a more flexible channel configuration scheme, a simpler structure, a smaller volume and a smaller light energy loss compared with the fluorescence detection module of the existing flow cytometer, and the debugging of the optical path using the debugging tooling is simple, which can meet the laser channel expansion requirements of the current flow cytometer.

[0145] The technical features of the above embodiments may be arbitrarily combined (as long as there is no contradiction in the combination of these technical features). To make the description concise, not all possible combinations of the technical features in the above embodiments are described; these embodiments that are not explicitly written should also be considered to be within the scope of this specification.

[0146] The above is a relatively specific and detailed description of the present application through general description and specific embodiments. It should be understood that, based on the technical concept of the present application, several conventional adjustments or further innovations can be made to these specific embodiments; however, as long as they do not deviate from the technical concept of the present application, the technical solutions obtained by these conventional adjustments or further innovations also fall within the scope of protection of the claims of the present application.

Claims

1. A ring-shaped spectroscopic detection module, characterized in that: It includes a collimating light path submodule, a photon splitting submodule, a side scattering detection submodule and a fluorescence detection submodule; The collimating optical path submodule includes a light collimating mirror for collimating the input fluorescence into an output; The photon splitting module is used to receive the collimated emitted fluorescence and separate it into side scattered light and fluorescence; The side scattered light detection submodule comprises a filter and a focusing lens for allowing the side scattered light to pass through in sequence, and a light detector for receiving the light spot passing through the focusing lens; The fluorescence detection submodule comprises a concave reflector, a circular array filter group, a circular array focusing lens group and a circular array light detector group which are coaxially arranged in sequence; the circular array filter group comprises a plurality of filters arranged in a circular array, the reflection surfaces of the plurality of filters are oriented in the same direction and are coplanar, and each filter can transmit light of different wavelength ranges; the circular array focusing lens group comprises a plurality of focusing lenses arranged in a circular array, the circular array light detector group comprises a plurality of light detectors arranged in a circular array, and the filters, focusing lenses and light detectors correspond to each other to form a light detection channel; the fluorescence passes through the concave reflector and is incident on a filter, the filter reflects the reflected light back to the concave reflector and is reflected to the next filter, so that the incident light forms an annular reflection light path between the continuous filters and the concave reflector, and the light passing through each filter is incident on the corresponding focusing lens and is received by the target surface of the light detector.

2. The annular spectroscopic detection module according to claim 1, characterized in that: Each filter in the circular array filter group is equipped with a filter mounting seat, and each filter can be independently disassembled and assembled; The filters in the circular array filter group are bandpass filters, each of which has a different wavelength and bandwidth, and each of which is coated with a film layer according to the wavelength of the fluorescence to be measured, so that the fluorescence of the target wavelength passes through, while the fluorescence of the non-target wavelength is reflected; The circular array filter group is arranged at the exit end of the concave reflector, and the reflective surfaces of the filters in the circular array filter group face the concave reflector.

3. The annular spectroscopic detection module according to claim 1 or 2, characterized in that: Each focusing lens in the circular array focusing lens group is a plano-convex lens or a spherical lens with an anti-reflection film coated on the surface, and each of the plano-convex lens or the spherical lens has the same physical parameters; Each photodetector in the circular array photodetector group is arranged corresponding to the fluorescence wavelength divided by each filter in the circular array filter group, and the photodetectors in the circular array photodetector group are avalanche diodes.

4. The annular spectroscopic detection module according to claim 1, characterized in that: A stable adjustment relationship is formed between the concave reflector, the circular array filter group and the incident angle of the collimated light path, and the expression formula of the stable adjustment relationship is: Cos(2θ)=1-2d / R, wherein: 2θ is the angle between any two adjacent filters in the circular array filter group, d is the straight-line distance between the center point of the concave reflector and the filters in the circular array filter group, and R is the curvature of the concave reflector.

5. The annular spectroscopic detection module according to claim 1, characterized in that: The fluorescence input into the collimating optical path module is introduced through a quartz optical fiber, the fluorescence introduced by the quartz optical fiber is collected by a micro-light lens, and the fluorescence collected by the micro-light lens is collimated by the optical collimator; The light collimator is a doublet lens; The quartz optical fiber is a through-path optical fiber, the core diameter of the through-path optical fiber is 400um, and the NA at both ends is 0.22 respectively; The NA value of the low light lens is greater than 1.2, the half field of view is greater than 0.328 mm, and the magnification is less than 6.

1.

6. The annular spectroscopic detection module according to claim 3, characterized in that: The spectroscopic submodule comprises a first spectroscopic filter, and the first spectroscopic filter is located on the outgoing light path of the light collimator; The first spectroscopic filter is used to separate the incident light into side scattered light perpendicular to the incident light direction and fluorescence in the same direction as the incident light, the side scattered light detection submodule is used to receive the side scattered light, and the fluorescence detection submodule is used to receive the fluorescence; The first spectroscopic filter is a long-pass filter capable of deeply cutting off the wavelength of the excitation light, and the OD of the first spectroscopic filter is greater than 4.

7. The annular spectroscopic detection module according to claim 4, characterized in that: The spot image size on each filter in the circular array filter group is different, and the spot image size on each filter is sinusoidally distributed from small to large. The diameter of the maximum spot image is less than 5mm, and the diameter of the spot image focused on the target surface of each light detector is less than 0.8mm.

8. The annular spectroscopic detection module according to claim 1, characterized in that: The circular array filter group is replaced by a concave circular array filter group, wherein the concave circular array filter group is composed of a plurality of concave filters arranged in a circular array, each of the concave filters is mounted on a concave base, the curvature of the concave base is the same as the curvature of the concave filter, and the mounting surfaces of each of the concave filters are coplanar; The concave filters, focusing lenses and photodetectors correspond to each other to form a light detection channel; the fluorescence passes through the concave reflector and is incident on a concave filter, the concave filter reflects the reflected light back to the concave reflector and is reflected to the next concave filter, so that the incident light forms an annular reflection light path between the continuous concave filters and the concave reflectors, and the light passing through each concave filter is incident on the corresponding focusing lens and is received by the target surface of the photodetector.

9. The annular spectroscopic detection module according to claim 8, characterized in that: A stable adjustment relationship is formed between the concave reflector, the concave circular array filter group and the incident angle of the collimated light path, and the expression formula of the stable adjustment relationship is: Cos(2θ)=1-2d / R, wherein: 2θ is the angle formed by the first reflection point and the next reflection point along the light propagation path on the concave circular array filter group relative to the center of the concave circular array filter group, d is the straight-line distance between the center point of the concave reflector and the center point of the concave filter in the concave circular array filter group, and R is the curvature of the concave reflector.

10. A flow cytometer equipped with an annular spectroscopic detection module, characterized in that: The annular spectroscopic detection module is the annular spectroscopic detection module according to any one of claims 1 to 7, or the annular spectroscopic detection module is the annular spectroscopic detection module according to any one of claims 8 to 9.

11. A debugging tool for the flow cytometer according to claim 10, characterized in that: Including pinhole diaphragm and side diffuser diaphragm; The pinhole aperture comprises an annular plate, a plurality of observation pinhole structures are evenly arranged on the plate surface of the annular plate, the number of the observation pinhole structures is the same as the number of the light detectors in the circular array light detector group, the observation pinhole structure comprises a boss protruding outward from the plate surface of the annular plate, the boss is provided with a through observation pinhole along its axial direction, one observation pinhole corresponds to a target surface of the light detector, the inner diameter of the observation pinhole is 0.8 mm, the pinhole aperture is mounted on the target surface of the light detector of the circular array light detector group, after the optical path is debugged, the visible fluorescence of each light detection channel can be observed by human eyes through the pinhole aperture, and the infrared light of each light detection channel can be observed by an infrared observation instrument; The side dispersion aperture stop is a transparent or translucent cylindrical structure, and a through observation hole is provided along the axial direction of the side dispersion aperture stop, and the inner diameter of the observation hole is 0.8 mm. The side dispersion aperture stop is mounted on the target surface of the light detector of the side dispersion detection submodule, and the observation hole is connected with the target surface optical path of the light detector. After the optical path is debugged, the light energy of the side dispersion channel of the side dispersion detection submodule can be observed by the human eye through the side dispersion aperture stop.

Images