CN110146430B - Optical system of flow cytometer - Google Patents
Optical system of flow cytometer Download PDFInfo
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- CN110146430B CN110146430B CN201910459771.XA CN201910459771A CN110146430B CN 110146430 B CN110146430 B CN 110146430B CN 201910459771 A CN201910459771 A CN 201910459771A CN 110146430 B CN110146430 B CN 110146430B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N15/1436—Optical arrangements the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
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Abstract
The invention discloses a flow cytometer optical system, wherein a channel for a substance to be detected to pass through is arranged in a flow chamber; the excitation light source of the light source module forms misaligned focusing light spots in the channel; the light collecting module is used for collecting scattered light and/or fluorescence and outputting the scattered light and/or fluorescence after converging the scattered light and/or fluorescence; the forward detection module is used for receiving scattered light and detecting the scattered light; the fluorescence detection module comprises an optical signal transmission module, a light splitting module and a detection module, wherein the optical signal transmission module is arranged at any position which is not intersected with each other in the full aperture range of the optical lens and used for enabling an optical signal output by the light collecting module to deflect, the light splitting module is used for outputting the deflected optical signal, and the detection module is used for detecting the optical signal. By implementing the invention, a plurality of photoelectric sensors and light splitting devices can be arranged in the fluorescence detection module, the problem of degradation or failure of a measurement result caused by the tiny movement of the light collecting module when optical fiber transmission is adopted is solved, and the stability of a system is enhanced.
Description
Technical Field
The invention relates to the technical field of cell detection and analysis, in particular to a flow cytometer optical system.
Background
A flow cytometer is an instrument that can rapidly analyze the characteristics (e.g., size, refractive index, complexity of internal structures, etc.) of cells, or of some microparticles (e.g., polystyrene microspheres). The sample containing the cells is compressed and focused by the sheath fluid, enters the fluid pool and forms laminar flow after the cells are compressed on the sample streamline, and the cells pass through the laser spots one by one, scattered light generated when the cells pass through the laser spots is tested by using the front of the optical axis direction and the side direction of the optical axis of the detector, and specific fluorescence generated by the carried fluorescent dye is tested, so that the biophysical and biochemical properties of the cells or the tiny particles are tested.
With the innovation of technology, the requirements on the number of fluorescence channels which can be tested by the flow cytometry in biological research and clinical diagnosis are more and more increased, the single-laser flow cytometer cannot meet the requirements of the single-laser flow cytometer, so that the flow cytometer with a plurality of excitation lights can be developed, the number of fluorescence channels which can be measured by the flow cytometer is more and more increased, the flow cytometer consisting of the existing plurality of excitation lights can not be provided with a plurality of (for example, three) excitation lights for three-dimensional excitation (three focuses which are formed by three light sources in a flow chamber are not coincident and are separated) in order to avoid fluorescence crosstalk, and an objective lens is matched with an optical fiber to collect and transmit optical signals, but the scheme is limited by a scheme that the space position is limited (the distance between the three focuses is only about 80um to 200um, the distance is about 3.2mm to 8mm only assuming that a 40 times objective lens is adopted), and the optical signals generated by a plurality of lasers can not be collected by the existing flow cytometer, and the scheme can not be adapted to the testing scheme of the plurality of excitation lights.
Disclosure of Invention
In view of the above, the embodiment of the invention provides an optical system of a flow cytometer, which solves the technical problems that the existing flow cytometer composed of a plurality of excitation lights is limited in space position and cannot be provided with a plurality of photoelectric sensors and a light splitting device.
The technical scheme provided by the invention is as follows:
the embodiment of the invention provides a flow cytometer optical system, which comprises: the device comprises a flow chamber, a light source module, a light collection module, a forward detection module and a fluorescence detection module; a channel for passing a substance to be detected is arranged in the flow chamber, and the channel can transmit optical signals; at least two excitation light sources are arranged in the light source module, and the excitation light sources form misaligned focusing light spots in the channel; at least one optical lens is arranged in the light collecting module, and the optical lens is used for collecting scattered light and/or fluorescence generated by the irradiation of a substance to be detected through an excitation light source, converging the scattered light and/or fluorescence and outputting the converged scattered light and/or fluorescence; the forward detection module is used for receiving scattered light generated by the irradiation of a substance to be detected through the excitation light source, and detecting the scattered light to obtain biophysical information of the substance to be detected; the fluorescence detection module comprises an optical signal transmission module, a light splitting module and a detection module, wherein the optical signal transmission module is arranged at any position which is not intersected with each other in the full aperture range of the optical lens and used for enabling an optical signal output by the light collecting module to deflect, the light splitting module is used for outputting the deflected optical signal according to a specific wavelength, and the detection module is used for detecting the optical signal output by the light splitting module to obtain biophysical and biochemical information of a substance to be detected.
Optionally, the optical signal transmission module includes: any one of a mirror, a reflecting prism, or a refracting prism.
Optionally, the forward detection module includes: and the diaphragm is used for blocking the optical signal transmitted to the preset angle in the forward detection module.
Optionally, the diaphragm includes: the blocking strip is arranged in the aperture and used for blocking optical signals of a preset angle.
Optionally, the forward detection module includes: the optical device comprises a first optical filter and a forward detector, wherein the first optical filter is used for screening scattered light generated by a substance to be detected, and the forward detector is used for detecting a screened optical signal.
Optionally, the optical splitting module includes: and the second optical filter is used for separating fluorescent signals in the deflected optical signals.
Optionally, the optical splitting module includes: and the detection module comprises a plurality of photodetectors, and the lenses receive the separated optical signals and focus to form light spots, wherein the light spots are smaller than the photosensitive area of the photodetectors.
Optionally, the imaging distance of the optical lens is greater than or equal to the optical length of the optical lens to the photodetector.
Optionally, the image-side aperture angle of the optical lens is no greater than 5 degrees.
Optionally, the optical signal transmission module further includes: the optical fiber and the collimating lens are arranged at two ends of the optical fiber, and the optical fiber is used for transmitting the optical signals received by the collimating lens into the light splitting module.
The technical scheme provided by the invention has the following advantages:
According to the flow cytometer optical system provided by the embodiment of the invention, the optical signal transmission module is arranged in the fluorescence detection module of the system and used for deflecting the optical signals output by the optical collection module, and the optical signal transmission module is arranged at any position which is not intersected with each other in the full aperture range of the optical collection module, so that the optical signals output by the optical collection module can be dispersed, the separation of the optical signals generated by different excitation lights is realized, and a plurality of photoelectric sensors and a light splitting device are arranged. Compared with the scheme that a plurality of optical fibers are directly adopted to collect a plurality of optical signals and transmit the optical signals to a sensor for detection in the prior art, the optical system of the flow cytometer provided by the embodiment of the invention deflects the optical signals output by the optical collecting module through the arranged optical signal transmitting module, so that the deflected optical signals are received by the optical splitting module, the problem that the optical signals cannot be collected by the optical fibers when the optical paths are subjected to micro movement due to the limitation of the end faces of the optical fibers and the variation coefficient of measurement results is increased when the optical fibers are directly adopted for collection is avoided, and therefore, the problem that the optical signals cannot be collected even if the optical paths drift in a small range is avoided, the tolerable error of the system is increased, the problem that the measurement results are deteriorated or lose efficacy due to the micro movement of the optical collecting module when the optical fibers are adopted for transmission is overcome, and the stability of the system is enhanced. In addition, the diaphragm arranged in the optical system of the flow cytometer can block the light signal of a preset angle, separate the excitation light signal which is not scattered by the substance to be detected, improve the detection sensitivity of scattered light, and overcome the problem of larger forward scattered light detection noise of the existing flow cytometer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a block diagram of a flow cytometer optical system in accordance with an embodiment of the present invention;
FIG. 2 is a block diagram of a flow cytometer optical system in accordance with another embodiment of the present invention;
FIG. 3 is a block diagram of a flow cytometer optical system in accordance with another embodiment of the present invention;
FIG. 4 is a block diagram of the flow cytometer optical system in accordance with another embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; the two components can be directly connected or indirectly connected through an intermediate medium, or can be communicated inside the two components, or can be connected wirelessly or in a wired way. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
In addition, the technical features of the different embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
An embodiment of the present invention provides a flow cytometer optical system, as shown in fig. 1, including: a flow chamber 2, a light source module 3, a light collection module 4, a forward detection module 1 and a fluorescence detection module 5; a channel for passing a substance to be detected is arranged in the flow chamber 2, and the channel can transmit an optical signal; at least two excitation light sources 31 (three excitation light sources are shown in the figure) are arranged in the light source module 3, and the excitation light sources 31 form misaligned focusing light spots in the channel; at least one optical lens is arranged in the light collecting module 4 and is used for collecting scattered light and/or fluorescence generated by the irradiation of a substance to be detected through an excitation light source, and the scattered light and/or fluorescence are converged and then output; the forward detection module 1 is used for receiving scattered light generated by the irradiation of a substance to be detected through an excitation light source, and detecting the scattered light to obtain surface information of the substance to be detected; the fluorescence detection module 5 includes an optical signal transmission module 51, a light splitting module 52 and a detection module 53, where the optical signal transmission module 51 is disposed at any position within the full aperture range of the optical lens, and is used for deflecting the optical signal output by the optical collection module 4, the light splitting module 52 is used for outputting the deflected optical signal according to a specific wavelength, and the detection module 53 is used for detecting the optical signal output by the light splitting module, so as to obtain internal information of the substance to be detected.
According to the flow cytometer optical system provided by the embodiment of the invention, the optical signal transmission module is arranged in the fluorescence detection module of the system and used for deflecting the optical signals output by the optical collection module, and the optical signal transmission module is arranged at any position which is not intersected with each other in the full aperture range of the optical collection module, so that the optical signals output by the optical collection module can be dispersed, the separation of the optical signals generated by different excitation lights is realized, and a plurality of photoelectric sensors and a light splitting device are arranged. Compared with the scheme that a plurality of optical fibers are directly adopted to collect a plurality of optical signals and transmit the optical signals to a sensor for detection in the prior art, the optical system of the flow cytometer provided by the embodiment of the invention deflects the optical signals output by the optical collecting module through the arranged optical signal transmitting module, so that the deflected optical signals are received by the optical splitting module, the problem that the optical signals cannot be collected by the optical fibers when the optical fibers are subjected to tiny movement due to the limitation of the end faces of the optical fibers and the variation coefficient of a measurement result is increased when the optical fibers are directly subjected to optical fiber collection is avoided, and therefore, the problem that the optical signals cannot be collected even if the optical signal transmitting module is subjected to tiny movement is avoided, the tolerable error of the system is increased, the problem that the measurement result is deteriorated or fails due to the tiny movement of the optical collecting module when the optical fibers are adopted is overcome, and the stability of the system is enhanced.
Specifically, in the optical system of the flow cytometer provided by the embodiment of the present invention, the propagation direction of the optical signal emitted by the excitation light source 31 is perpendicular to the flow direction of the substance to be detected in the flow chamber 2. Alternatively, the substance to be detected may be cells or particles (e.g., polystyrene microspheres), or may be other substances, which is not limited in the present invention. The excitation light source 31 may include lasers having three wavelengths 488nm, 405nm, and 638 nm. Furthermore, the center line of the focused spot formed in the channel by the excitation light source 31 coincides with the flow line of the substance to be detected, which may be in the center of the channel of the flow cell 2.
In the optical system of the flow cytometer provided by the embodiment of the present invention, the light collecting module 4 may be an objective lens with positive optical power, and the field of view of the objective lens covers the focal points of all the excitation light sources 31 of the light source module in the micro-channel, and converges the optical signals after passing through the substance to be detected, so as to form an equal number of image points, or image at infinity. Specifically, the imaging distance of the objective lens is greater than or equal to the optical length of the objective lens to the photodetector in the detection module. Specifically, the objective lens has an image-side aperture angle of not more than 5 degrees. Alternatively, the objective lens may be an objective lens having a Numerical Aperture (NA) of 0.6 or more.
As an alternative implementation of the embodiment of the present invention, the optical signal transmission module 51 includes: any one of a mirror, a reflecting prism, or a refracting prism. The optical signal transmission module 51 may deflect all the optical signals output from the optical signal collection module 4 by an angle between 0 degrees and 180 degrees.
As an alternative implementation of the embodiment of the present invention, as shown in fig. 2, the forward detection module 1 includes: a diaphragm 11, a lens group 12, a first filter 13 and a forward detector 14.
Wherein the diaphragm 11 is used for blocking the optical signal transmitted to the preset angle in the forward direction detection module 1, in particular, the diaphragm 11 may block the optical signal from 0 to 2 degrees of the optical signal detection point. As shown in fig. 3, the diaphragm 11 may be constituted by an aperture, which may be an elliptical aperture, and a blocking bar provided in the aperture for blocking an optical signal of a preset angle. The diaphragm 11 may be disposed at an angle of 45 degrees in the forward direction detection module 14, and the clear aperture of the diaphragm 11 is circular, and the aperture angle thereof is 10 degrees. In addition, when the diaphragm 11 blocks the optical signal, the optical signal at a preset angle can be deflected by 90 degrees, and the deflected optical signal can be absorbed by adopting an optical trap. The diaphragm 11 arranged in the optical system of the flow cytometer can block the light signal of a preset angle, separate the excitation light signal which is not scattered by the substance to be detected, improve the detection sensitivity of scattered light and overcome the problem of larger forward scattered light detection noise of the existing flow cytometer.
The first filter 13 may be used to screen scattered light generated by the substance to be detected so that the desired wavelength of light is transmitted into the forward detection module 1. The forward detector 14 may detect the screened optical signal. The lens group 12 may be used to collect the scattered light entering the forward detection module 1, and transmit the scattered light to the forward detector 14 for detection, so as to obtain biophysical information of the substance to be detected, such as size, refractive index, etc.
As an alternative implementation of the embodiment of the present invention, the spectroscopic module 52 includes: the second optical filter is used for separating fluorescent signals in the deflected optical signals output by the optical signal transmission module, and the lens with positive focal power receives the separated fluorescent signals and focuses the fluorescent signals to form light spots. The photoelectric detector in the detection module 53 receives the light spot and analyzes the light spot to obtain biophysical and biochemical information of the substance to be detected, such as the complexity of the internal structure of the substance to be detected, the surface protein type, the expression level and the like. Specifically, the area of the light spot is smaller than the photosensitive area of the photodetector. The photodetector may employ an avalanche diode.
As an alternative implementation of the embodiment of the present invention, as shown in fig. 4, the optical signal transmission module 51 further includes: the optical fiber and the collimator lens are provided at both ends of the optical fiber, and thus the optical fiber 54 with the collimator lens may be used. When the reflecting mirror or prism in the optical signal transmission module deflects the optical signal output by the optical collecting module, the optical signal is transmitted to the optical fiber 54 with the collimating lens, and the optical fiber 54 is used for transmitting the received optical signal to the light splitting module 52.
According to the flow cytometer optical system provided by the embodiment of the invention, the optical signal transmission module is arranged in the fluorescence detection module of the system and used for deflecting the optical signals output by the optical collection module, and the optical signal transmission module is arranged at any position which is not intersected with each other in the full aperture range of the optical collection module, so that the optical signals output by the optical collection module can be dispersed, the separation of the optical signals generated by different excitation lights is realized, and a plurality of photoelectric sensors and a light splitting device are arranged. Compared with the scheme that a plurality of optical fibers are directly adopted to collect a plurality of optical signals and transmit the optical signals to a sensor for detection in the prior art, the flow cytometer optical system provided by the embodiment of the invention deflects the optical signals output by the optical collecting module through the arranged optical signal transmitting module, so that the deflected optical signals are received by the optical dividing module, the problem that the variation coefficient of a measurement result is increased because the optical signals cannot be collected by the optical fibers when the optical fibers are subjected to tiny movement due to the limitation of the end faces of the optical fibers when the optical fibers are directly collected is avoided, and the problem that the measurement result is deteriorated or invalid due to the tiny movement of the optical collecting module is solved, and therefore, the tolerable error of the system is increased even if the small-range drift of an optical path occurs in the flow cytometer optical system provided by the embodiment of the invention, and the stability of the system is enhanced. In addition, the diaphragm arranged in the optical system of the flow cytometer can block the light signal of a preset angle, separate the excitation light signal which is not scattered by the substance to be detected, improve the detection sensitivity of scattered light, and overcome the problem of larger forward scattered light detection noise of the existing flow cytometer.
Although embodiments of the present invention have been described in connection with the accompanying drawings, various modifications and variations may be made by those skilled in the art without departing from the spirit and scope of the invention, and such modifications and variations fall within the scope of the invention as defined by the appended claims.
Claims (9)
1. A flow cytometer optical system, comprising: the device comprises a flow chamber, a light source module, a light collection module, a forward detection module and a fluorescence detection module;
a channel for passing a substance to be detected is arranged in the flow chamber, and the channel can transmit optical signals;
At least two excitation light sources are arranged in the light source module, and the excitation light sources form misaligned focusing light spots in the channel;
At least one optical lens is arranged in the light collecting module, and the optical lens is used for collecting fluorescence generated by the irradiation of a substance to be detected through an excitation light source, converging the fluorescence and outputting the converged fluorescence;
The forward detection module is used for receiving scattered light generated by the irradiation of a substance to be detected through the excitation light source, and detecting the scattered light to obtain biophysical information of the substance to be detected;
the fluorescence detection module comprises an optical signal transmission module, a light splitting module and a detection module, wherein the optical signal transmission module comprises: any one of a reflecting mirror, a reflecting prism or a refracting prism is arranged at any position which is not intersected with each other in the full aperture range of the optical lens, and is used for enabling the optical signals output by the optical collecting module to deflect and enabling the optical signals output by the optical collecting module to disperse, so that separation of the optical signals generated by different excitation lights is achieved, the light splitting module is used for outputting the deflected optical signals according to specific wavelengths, and the detection module is used for detecting the optical signals output by the light splitting module to obtain biophysical and biochemical information of a substance to be detected.
2. The flow cytometer optical system of claim 1 wherein the forward detection module comprises: and the diaphragm is used for blocking the optical signal transmitted to the preset angle in the forward detection module.
3. The flow cytometer optical system of claim 2 wherein the aperture comprises: the blocking strip is arranged in the aperture and used for blocking optical signals of a preset angle.
4. The flow cytometer optical system of claim 1 wherein the forward detection module comprises: the optical device comprises a first optical filter and a forward detector, wherein the first optical filter is used for screening scattered light generated by a substance to be detected, and the forward detector is used for detecting a screened optical signal.
5. The flow cytometer optical system of claim 1 wherein the spectroscopic module comprises: and the second optical filter is used for separating fluorescent signals in the deflected optical signals.
6. The flow cytometer optical system of claim 5 wherein the spectroscopic module comprises: and the detection module comprises a plurality of photodetectors, and the lenses receive the separated optical signals and focus to form light spots, wherein the light spots are smaller than the photosensitive area of the photodetectors.
7. The flow cytometer optical system of claim 5 wherein the imaging distance of the optical lens is greater than or equal to the optical length of the optical lens to the photodetector.
8. The flow cytometer optical system of claim 7 wherein the optical lens has an image side aperture angle of no greater than 5 degrees.
9. The flow cytometer optical system of claim 1, wherein the optical signal transmission module further comprises: the optical fiber and the collimating lens are arranged at two ends of the optical fiber, and the optical fiber is used for transmitting the optical signals received by the collimating lens into the light splitting module.
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| CN112147044A (en) * | 2020-09-07 | 2020-12-29 | 桂林电子科技大学 | Spectral subdivision optical fiber collection and dispersive detection device for flow cytometer |
| CN112229781A (en) * | 2020-09-07 | 2021-01-15 | 桂林电子科技大学 | An Improved Spectral Subdivision Optical Fiber Distributed Detection Device for Flow Cytometer |
| WO2022164672A1 (en) * | 2021-01-27 | 2022-08-04 | Becton, Dickinson And Company | Flow cytometers including fiber optic light collectors, and methods of use thereof |
| CN113624666B (en) * | 2021-09-07 | 2022-09-23 | 清华大学 | Stream type imaging system based on dot matrix laser scanning |
| CN113899677A (en) * | 2021-09-17 | 2022-01-07 | 桂林优利特医疗电子有限公司 | Reflective light splitting module and light splitting method for flow cytometer detection |
| CN114486691A (en) * | 2022-02-16 | 2022-05-13 | 上海纬冉科技有限公司 | Portable energy detection equipment and debugging device thereof |
| CN115015178A (en) * | 2022-08-05 | 2022-09-06 | 天津迈科隆生物科技有限公司 | Optical detection device and blood analyzer |
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