Optical scattering signal negative feedback detection device and method for flow cytometry

Abstract

The invention provides an optical scattering signal negative feedback detection device and an optical scattering signal negative feedback detection method for a flow cytometer, which are applied to the technical field of cell detection and analysis equipment.The optical scattering signal negative feedback detection device and the optical scattering signal negative feedback detection method are characterized in that a forward scattering light path is used for shunting particle scattering light signals and laser direct light signals excited by a laser light source in a flow chamber; according to the invention, particle scattered light signals and laser direct light signals are separated, the split particle scattered light signals are collected, and the laser direct light signals are eliminated, so that the isolation degree of forward scattering direct laser is improved, stray light is efficiently processed, the forward scattering detection limit is improved, and cell particles with smaller sizes can be detected; and the separated laser direct light signal can be used for detecting a feedback mechanism of a light path state in real time to realize real-time detection of a system light path, so that the stability and the reliability of a product are ensured.

Description

Technical Field

[0001] The present application relates to the technical field of cell detection and analysis equipment, and in particular to a negative feedback detection device and method for an optical scattering signal of a flow cytometer. Background Art

[0002] Flow cytometry has a wide range of applications in biomedical research, clinical diagnosis and treatment. As cells pass through a flow cytometer, they pass through a laser beam one by one and are detected by a detector by scattering and emitting fluorescence. Based on the cell's characteristics such as size, complexity and fluorescence intensity, flow cytometry can provide information about cell number, size, expression of surface markers, cell cycle, etc.

[0003] Forward scatter is considered low-angle scatter and is roughly proportional to the diameter of the cell. Therefore, forward scatter is useful for distinguishing specific cell subpopulations from other subpopulations based on their size. Since forward scatter is usually measured along the same path as the beam propagation, the refracted high-intensity original laser beam needs to be isolated from being detected by the detector.

[0004] Based on this, a new technical solution is needed. Summary of the invention

[0005] In view of this, the embodiments of the present specification provide a negative feedback detection device and method for an optical scattering signal of a flow cytometer.

[0006] The embodiments of this specification provide the following technical solutions:

[0007] An embodiment of the present specification provides an optical scattering signal negative feedback detection device for a flow cytometer, comprising a forward scattering light path, wherein the forward scattering light path is used to shunt particle scattering light signals and laser direct light signals excited by a laser light source in a flow chamber, and collect the shunted particle scattering light signals.

[0008] Preferably, the forward scattering light path includes laser light emitted by a laser light source, a sheath flow device, an achromatic light-collecting lens, and a reflector with a hole;

[0009] The laser is focused on the center of the flow channel of the sheath flow device, and the laser irradiates the cell particles flowing through the center of the flow channel of the sheath flow device, and becomes scattered light and direct light, and the scattered light is formed around the direct light along the propagation direction of the direct light;

[0010] The achromatic light-collecting lens collimates the scattered light and the direct light;

[0011] The holes of the perforated reflector are used to transmit collimated direct light, and the reflecting surface of the perforated reflector is used to reflect collimated scattered light, so that the perforated reflector propagates the direct light and the reflected light in different directions to realize the diversion of the direct light and the reflected light.

[0012] Preferably, the forward scattering light path further includes a filter, a light collecting lens and a photodetector;

[0013] The optical filter filters the reflected scattered light;

[0014] The light-collecting lens collects the filtered scattered light;

[0015] The photoelectric detector detects the received scattered light.

[0016] Preferably, the forward scattering light path further includes a converging lens and a camera;

[0017] The converging lens converges the collimated direct light;

[0018] The camera images the light spot focused on the center of the sheath flow channel according to the converged direct light;

[0019] The detection device uses a camera to perform real-time imaging monitoring of the position of the direct light spot passing through the perforated reflector and feedback to adjust the laser to ensure that the center of the light spot is always located in the center of the sheath flow device flow channel.

[0020] Preferably, the forward scattering light path further comprises an attenuation plate; the attenuation plate is located between the perforated reflector and the converging lens;

[0021] The attenuation plate attenuates the direct light passing through the perforated reflector, and the converging lens converges the attenuated direct light;

[0022] There is an acute angle between the attenuation sheet and the passing direct light, so that the attenuation sheet is an inclined attenuation sheet.

[0023] Preferably, an illumination light source is placed between the sheath flow device and the laser, so that the camera can photograph the flow channel wall, record the position of the flow channel wall and calculate and calibrate the flow channel center.

[0024] Preferably, the focal length of the achromatic light-collecting lens is 25-30 mm, and the numerical aperture is 0.35-0.45; and / or, the size of the perforated reflector is 40*30 mm, and the hole size is 8*5 mm; and / or, the optical density value of the attenuation plate is 0.8-1.2; and / or, the focal length of the converging lens is 50-70 mm.

[0025] Preferably, the bandwidth of the filter is 10±5 nm; and / or the focal length of the light-collecting lens is 50-70 mm; and / or the photodetector is a photodiode.

[0026] The embodiments of the present specification also provide a method for detecting negative feedback of an optical scattering signal for a flow cytometer, which uses any of the above-mentioned negative feedback detection devices for an optical scattering signal for a flow cytometer, including: using a forward scattering light path to shunt a particle scattering light signal excited by a laser light source in a flow chamber and a laser direct light signal, and collecting the shunted particle scattering light signal.

[0027] Preferably, the detection method further comprises: calibrating the flow channel center of the sheath flow device by means of a camera, monitoring the center of the light spot in the flow channel, and if the center of the light spot deviates from the center of the flow channel, adjusting the laser position and re-monitoring the center of the light spot in the flow channel.

[0028] Compared with the prior art, the at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects:

[0029] The present application uses a forward scattering optical path to shunt the particle scattering light signal and the laser direct light signal excited by the laser light source in the flow chamber, so as to separate the particle scattering light signal and the laser direct light signal, and collect the shunted particle scattering light signal, and exclude the laser direct light signal, which is beneficial to improve the isolation degree of the forward scattered direct laser, efficiently process stray light, improve the forward scattering detection limit, and detect smaller cell particles. The separated laser direct light signal can be used for the feedback mechanism of real-time detection of the optical path state to realize real-time detection of the system optical path, thereby ensuring the stability and reliability of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. 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 paying creative work.

[0031] Figure 1 It is a conventional forward light collection device for flow cytometry;

[0032] Figure 2 Schematic diagram of cell particle distribution and laser cross-section distribution;

[0033] Figure 3 This is a schematic diagram of the forward scattering light path of this application;

[0034] Figure 4 This is a feedback adjustment flow chart of the forward scattering device of the present application.

[0035] Figure numerals: 1. sheath flow meter; 2. achromatic light-collecting lens; 3. reflector with hole; 4. inclined attenuation plate; 5. converging lens; 6. filter; 7. light-collecting lens; 8. camera; 9. photodetector; 10. cell; 11. shielding handle; 12. forward scattered light; 13. light-collecting system; 14. detector. DETAILED DESCRIPTION

[0036] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0037] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0038] It should be noted that various aspects of the embodiments within the scope of the appended claims are described below. It should be apparent that the aspects described herein can be embodied in a wide variety of forms, and any specific structure and / or function described herein is merely illustrative. Based on the present application, it should be understood by those skilled in the art that an aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspect described herein can be used to implement the device and / or practice the method. In addition, other structures and / or functionalities other than one or more of the aspects described herein can be used to implement this device and / or practice this method.

[0039] It should also be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present application. The drawings only show components related to the present application rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0040] Additionally, in the following description, specific details are provided to facilitate a thorough understanding of the examples. However, it will be understood by those skilled in the art that the examples can be practiced without these specific details.

[0041] Currently Figure 1As shown, forward scatter detection is usually performed by adding a beam shield 11 behind the flow chamber to block the non-scattered, high-intensity original laser beam along the optical path of the laser beam, and the optical path includes the laser, the cell 10, the shield 11, the forward scattered light 12, the light collection system 13 and the detector 14. The disadvantage is that although the shield 11 can block most of the incident laser, there is still scattered light that can enter the detector 14.

[0042] When particles pass through the excitation light spot, the inhomogeneity of the excitation light spot energy causes particles of the same size to receive different intensities of scattered light. The spot incident on the cell 10 can be shaped into a flat-top spot through laser shaping technology to improve the uniformity of the spot energy. The inhomogeneity of the spot energy incident on the particle will increase the system CV value. Factors affecting the system CV include the radius r of the cell particle, the position K of the particle, the position of the excitation light spot, and the energy distribution y(x) of the excitation light spot. You can use Figure 2 The relationship between CV and CV is used to characterize the CV value of the system. The cell particles exist in the axial flow interval of detta. Among them, CV represents the coefficient of variation, x represents the value of the random variable, and y represents the corresponding probability density.

[0043] The scattering signal intensity U excited by the particle can be expressed as:

[0044]

[0045] Considering the distribution of cell particles in the axial flow interval, the system CV value can be calculated. It can be seen that when the spot energy distribution y(x) coincides with the center of the axial flow interval detta, that is, the coincidence of the spot center and the axial flow center is the best condition to ensure the minimum system CV value. Unfortunately, most of the current product designs cannot provide real-time feedback on whether the spot center and the axial flow center coincide.

[0046] A Chinese patent document with publication number CN117740654A discloses an optical collection system for flow cytometry, which includes a laser excitation module, an optical shaping module, a beam combiner, a sheath flow device, a forward collection module and a lateral collection module. The forward collection module is arranged at the rear side of the sheath flow device, the optical axis of the collection optical system in the forward collection module is the same as the emission direction of the laser beam in the sheath flow device, the optical axis of the collection optical system in the forward collection module is perpendicular to the particle flow direction of the laser beam in the sheath flow device, the laser beam is Gaussian distributed in the direction perpendicular to the optical axis of the collection optical system in the forward collection module, and in the sheath flow device, the particles to be measured are restricted to flow within a range of width detta; the optical axis of the collection optical system of the lateral collection module passes through the center of the sheath flow device, and the optical axis of the collection optical system of the lateral collection module is perpendicular to the emission direction of the laser beam in the sheath flow device.

[0047] The optical collection system optimizes the collection efficiency of the forward scattered light of the particles to be measured through the forward collection module, keeps the product of the intensity of particle laser irradiation and the collection efficiency of the lateral collection system consistent within the flow range, and reduces the system CV caused by fluctuations in laser beam intensity.

[0048] The prior art also discloses an optical engine for a flow cytometer, a flow cytometer system, and a method of use, which improves the efficiency of shielding stray light by optimizing the geometric shape of a forward light shielding handle, thereby improving system performance.

[0049] In view of this, the applicant has conducted in-depth research and improvement exploration on flow cytometers and detection of optical scattering signals, and found that: the design of the optical collection system for flow cytometry can theoretically reduce the system CV, but there is a problem of incomplete isolation of the forward scattered direct laser. Although the optical engine, flow cytometer system and method of use for flow cytometer can greatly reduce the interference of direct laser, the system also lacks a feedback mechanism that can detect the state of the optical path in real time.

[0050] Based on this, the technical solutions provided by the embodiments of the present application are described below in conjunction with the accompanying drawings.

[0051] The embodiments of this specification provide a negative feedback detection device for optical scattering signals of a flow cytometer, such as Figure 3 As shown, it includes a forward scattering light path, which is used to shunt the particle scattering light signal and the laser direct light signal excited by the laser light source in the flow chamber to separate the particle scattering light signal and the laser direct light signal, and collect the shunted particle scattering light signal. Before shunting, the particle scattering light signal is located around the laser direct light signal. The laser light source is a 488 light source. That is, the forward light path is to collect the particle scattering light signal excited by the 488 light source in the flow chamber, and to exclude the laser direct light signal.

[0052] In one embodiment, if Figure 3 As shown, the forward scattering light path includes the laser emitted by the laser light source, the sheath flow device 1, the achromatic light receiving lens 2 and the holed reflector 3.

[0053] The laser is focused on the center of the flow channel of the sheath flow device 1, and the laser irradiates the cell particles flowing through the center of the flow channel of the sheath flow device 1, and becomes scattered light and direct light. The scattered light is formed around the direct light along the propagation direction of the direct light. In the forward scattered light path, the laser is focused on the center of the sheath flow device 1 to irradiate the cell particles flowing through. The sheath flow device 1 allows the sample cell particles to be irradiated by the laser in turn, and the fluctuation range of the cell particles is detta, where detta represents the axial flow interval.

[0054] The achromatic light-collecting lens 2 collimates the scattered light and the direct light; the hole of the perforated reflector 3 is used to transmit the collimated direct light, and the reflecting surface of the perforated reflector 3 is used to reflect the collimated scattered light, so that the perforated reflector 3 propagates the direct light and the reflected light in different directions to realize the diversion of the direct light and the reflected light, that is, to separate the direct light and the reflected light, and the propagation directions of the diverted direct light and the reflected light can be vertical.

[0055] Among them, the achromatic light-collecting lens 2 collimates the scattered light and the direct laser; the hole is the small hole in the middle of the perforated reflector 3, which can transmit the direct laser; the hole passes through the reflector along the propagation direction of the direct light to form the perforated reflector 3, so that the hole direction of the perforated reflector 3 is consistent with the propagation direction of the direct light.

[0056] In one embodiment, if Figure 3 As shown, the forward scattered light path also includes a filter 6, a light collecting lens 7 and a photodetector 9; the filter 6 filters the reflected scattered light; the light collecting lens 7 collects the filtered scattered light; and the photodetector 9 detects the collected scattered light. The filter 6 prevents the forward scattered light from being collected outside the wavelength.

[0057] In one embodiment, if Figure 3 As shown, the forward scattering light path also includes a converging lens 5 and a camera 8; the converging lens 5 converges the collimated direct light; the camera 8 images the light spot focused on the center of the sheath flow device 1 flow channel according to the converged direct light; the detection device monitors the position of the direct light spot passing through the perforated reflector 3 in real time through the camera 8 and adjusts the laser by feedback to ensure that the center of the light spot is always located at the center of the sheath flow device 1 flow channel. In other words, the detection device also includes a feedback regulator, which monitors the position of the direct light spot passing through the perforated reflector 3 in real time through the camera 8 and adjusts the laser by feedback to ensure that the center of the light spot is always located at the center of the sheath flow device 1 flow channel. The converging lens 5 focuses the collimated laser and images the light spot focused on the center of the sheath flow device 1.

[0058] In one embodiment, if Figure 3 As shown, the forward scattering light path also includes an attenuation plate; the attenuation plate is located between the perforated reflector 3 and the converging lens 5; the attenuation plate attenuates the direct light passing through the perforated reflector 3, and the converging lens 5 converges the attenuated direct light; there is an acute angle between the attenuation plate and the direct light passing through, so that the attenuation plate is an inclined attenuation plate 4. The inclined attenuation plate 4 prevents the direct laser from being too strong to damage the camera 8, and at the same time prevents the direct laser from being reflected back to the sheath flow device 1 along the original path.

[0059] In one embodiment, an illumination light source is placed between the sheath flow device 1 and the laser, so that the camera 8 can photograph the flow channel wall, record the position of the flow channel wall and calculate and calibrate the flow channel center.

[0060] The present application isolates the direct laser by opening a hole in the reflector to prevent it from entering the photodetector 9, and at the same time, performs real-time imaging monitoring of the position of the direct laser spot passing through the reflector and feedback-adjusts the laser to ensure that the center of the spot is always located at the center of the flow channel of the sheath flow device 1. For example, an illumination light source is placed between the sheath flow device 1 and the laser, and the camera 8 photographs the flow channel wall, records the position of the flow channel wall, and calculates and calibrates the flow channel center.

[0061] In one embodiment, the focal length of the achromatic light-collecting lens 2 is 25-30 mm, and the numerical aperture is 0.35-0.45; and / or, the size of the perforated reflector 3 is 40*30 mm, and the hole size is 8*5 mm; and / or, the optical density (OD, Optical Density) value of the attenuation plate is 0.8-1.2; and / or, the focal length of the converging lens 5 is 50-70 mm.

[0062] In one embodiment, the bandwidth of the filter 6 is 10±5 nm; and / or the focal length of the light-collecting lens 7 is 50-70 mm; and / or the photodetector 9 is a photodiode.

[0063] The embodiments of this specification also disclose a method for detecting negative feedback of an optical scattering signal for a flow cytometer, using any of the above embodiments of the optical scattering signal negative feedback detection device for a flow cytometer, such as Figure 3 and Figure 4 As shown, it includes: using a forward scattering light path to split the particle scattering light signal excited by the laser light source in the flow chamber and the laser direct light signal, and collecting the split particle scattering light signal.

[0064] In one embodiment, the detection method further includes: calibrating the flow channel center of the sheath flow device by using the camera 8, monitoring the center of the light spot in the flow channel, and if the center of the light spot deviates from the center of the flow channel, adjusting the laser position and re-monitoring the center of the light spot in the flow channel.

[0065] The present application not only isolates the direct laser to the greatest extent, but also can monitor the center of the flow channel and the center of the light spot in real time, thereby increasing the system's ability to detect tiny particles and improving the stability of the system.

[0066] Compared with the prior art, this application is a more efficient solution for handling stray light. This application addresses the problem of lack of a feedback mechanism for real-time detection of the optical path status, achieves real-time detection of the system optical path, and ensures the stability and reliability of the product; addresses the problem of incomplete isolation of forward scattered direct laser light, improves the degree of isolation of forward scattered direct laser light, and improves the forward scattering detection limit, which can detect smaller cell particles; addresses the problem of cumbersome optical path calibration process, provides a method for automatic optical path calibration, and automates optical path calibration, greatly improving production integration efficiency.

[0067] In this specification, the same or similar parts between the various embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the embodiments described later, the description is relatively simple, and the relevant parts can be referred to the partial description of the previous embodiments.

[0068] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. An optical scattering signal negative feedback detection device for flow cytometer, characterized in that: It comprises a forward scattering light path, which is used to shunt the particle scattering light signal and the laser direct light signal excited by the laser light source in the flow chamber, and collect the shunted particle scattering light signal.

2. The optical scattering signal negative feedback detection device for flow cytometer according to claim 1, characterized in that: The forward scattering optical path includes laser light emitted by a laser light source, a sheath flow device, an achromatic light-collecting lens, and a reflector with a hole; The laser is focused on the center of the flow channel of the sheath flow device, and the laser irradiates the cell particles flowing through the center of the flow channel of the sheath flow device, and becomes scattered light and direct light, and the scattered light is formed around the direct light along the propagation direction of the direct light; The achromatic light-collecting lens collimates the scattered light and the direct light; The holes of the perforated reflector are used to transmit collimated direct light, and the reflecting surface of the perforated reflector is used to reflect collimated scattered light, so that the perforated reflector propagates the direct light and the reflected light in different directions to realize the diversion of the direct light and the reflected light.

3. The optical scattering signal negative feedback detection device for flow cytometer according to claim 2, characterized in that: The forward scattering light path also includes a filter, a light collecting lens and a photodetector; The optical filter filters the reflected scattered light; The light-collecting lens collects the filtered scattered light; The photoelectric detector detects the received scattered light.

4. The optical scattering signal negative feedback detection device for flow cytometer according to claim 2, characterized in that: The forward scattering light path also includes a converging lens and a camera; The converging lens converges the collimated direct light; The camera images the light spot focused on the center of the sheath flow channel according to the converged direct light; The detection device uses a camera to perform real-time imaging monitoring of the position of the direct light spot passing through the perforated reflector and feedback to adjust the laser to ensure that the center of the light spot is always located in the center of the sheath flow device flow channel.

5. The optical scattering signal negative feedback detection device for flow cytometer according to claim 4, characterized in that: The forward scattering optical path further includes an attenuation plate; the attenuation plate is located between the perforated reflector and the converging lens; The attenuation plate attenuates the direct light passing through the perforated reflector, and the converging lens converges the attenuated direct light; There is an acute angle between the attenuation sheet and the passing direct light, so that the attenuation sheet is an inclined attenuation sheet.

6. The optical scattering signal negative feedback detection device for flow cytometer according to claim 4, characterized in that: An illumination light source is placed between the sheath flow device and the laser, so that the camera can photograph the flow channel wall, record the position of the flow channel wall and calculate and calibrate the flow channel center.

7. The optical scattering signal negative feedback detection device for flow cytometer according to claim 5, characterized in that: The focal length of the achromatic light-collecting lens is 25-30 mm, and the numerical aperture is 0.35-0.45; and / or, the size of the perforated reflector is 40*30 mm, and the hole size is 8*5 mm; and / or, the optical density value of the attenuation plate is 0.8-1.2; and / or, the focal length of the converging lens is 50-70 mm.

8. The optical scattering signal negative feedback detection device for flow cytometer according to claim 3, characterized in that: The bandwidth of the filter is 10±5 nm; and / or the focal length of the light-collecting lens is 50-70 mm; and / or the photodetector is a photodiode.

9. A method for detecting negative feedback of optical scattering signals in a flow cytometer, characterized in that: The optical scattering signal negative feedback detection device for a flow cytometer according to any one of claims 1 to 8 comprises: a forward scattering light path for shunting particle scattering light signals and laser direct light signals excited by a laser light source in a flow chamber, and collecting the shunted particle scattering light signals.

10. The method for detecting negative feedback of optical scattering signals for flow cytometer according to claim 9, characterized in that: The detection method also includes: calibrating the flow channel center of the sheath flow device through a camera, monitoring the center of the light spot in the flow channel, and if the center of the light spot deviates from the center of the flow channel, adjusting the laser position and re-monitoring the center of the light spot in the flow channel.

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