Flow cytometer and light path debugging method

Abstract

The invention provides a flow cytometer and a light path debugging method, the flow cytometer comprises a flow chamber, an illumination assembly, an objective lens, a first detection assembly and a second detection assembly, the illumination assembly comprises a laser, a first cylindrical mirror and a second cylindrical mirror, the laser, the first cylindrical mirror and the second cylindrical mirror are sequentially arranged from top to bottom from one side of the flow chamber in the vertical direction; the objective lens is arranged on one side of the flow chamber in the horizontal direction; the first detection assembly is used for detecting a lateral angle scattered light signal and a fluorescence signal from the objective lens; and the second detection assembly is used for collecting and detecting forward angle scattered light signals generated when the laser beams irradiate the to-be-detected particles or cells in the flow chamber. The laser beam is precisely shaped through the first cylindrical mirror and the second cylindrical mirror, it is ensured that the waist of the laser beam is located in the center of the flow chamber, particles or cells to be detected can be accurately irradiated, and compared with a traditional scheme that the laser beam is reflected into the flow chamber through a reflecting mirror, the laser beam pointing stability is better.

Description

Technical Field

[0001] The invention relates to the technical field of cell detection, and in particular to a flow cytometer and an optical path debugging method. Background Art

[0002] In the medical and biological fields, flow cytometers are usually used to analyze cells, DNA, bacteria and other tiny particles. Flow cytometers can analyze a large number of cells at high speed and can simultaneously detect multiple parameters of a cell, such as cell size, morphology, surface markers, DNA content, etc.

[0003] However, in the prior art, with respect to the illumination system of the flow cytometer, whether it is a traditional flow cytometer or a spectral flow cytometer, regardless of whether one or more lasers are configured, a reflector / dichroic filter is usually used to fold the optical path 90 degrees. The reflector / dichroic filter is mounted on a reflector frame, and the reflector frame can usually be fine-tuned in the horizontal and vertical directions. Although this can conveniently fine-tune the direction of the light beam, it will also cause the stability of the light beam pointing to deteriorate, and it is also greatly affected by the ambient temperature. Since the laser beam is a Gaussian beam, the energy is concentrated at the center of the beam, and the flow cytometer has very high requirements for the pointing stability of the laser beam, so the stability of the optical path of the flow cytometer has always been a problem that has troubled technicians and needs to be solved urgently. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide a flow cytometer and an optical path debugging method to solve the technical problems mentioned in the above background technology.

[0005] In one aspect, the present invention provides a flow cytometer, comprising:

[0006] Flow chamber;

[0007] An illumination assembly, the illumination assembly comprising a laser, a first cylindrical mirror and a second cylindrical mirror, wherein the laser, the first cylindrical mirror and the second cylindrical mirror are arranged in sequence from top to bottom on one side of the flow chamber in a vertical direction;

[0008] An objective lens, which is arranged at one side of the flow chamber in the horizontal direction and is used to collect side angle scattered light signals and fluorescence signals generated when the laser beam irradiates the particles or cells to be measured in the flow chamber;

[0009] A first detection component, the first detection component is used to detect the side angle scattered light signal and the fluorescence signal from the objective lens;

[0010] A second detection component is used to collect and detect forward angle scattered light signals generated when the laser beam irradiates the particles or cells to be detected in the flow chamber.

[0011] Further, in the flow cytometer, the first cylindrical mirror is used to shape the laser beam in the horizontal direction, and the second cylindrical mirror is used to shape the laser beam in the vertical direction, wherein the focal length of the first cylindrical mirror is greater than that of the second cylindrical mirror.

[0012] Further, in the flow cytometer, the first detection component includes a collimating lens, a dichroic filter, a plane mirror, a first detection array, and a second detection array. The collimating lens is used to receive the optical signal from the objective lens and adjust it into a collimated beam. The dichroic filter is disposed on one side of the collimating lens in the horizontal direction and is used to separate the collimated beam into transmitted light and reflected light according to wavelength. The plane mirror is disposed on one side of the dichroic filter in the vertical direction and is used to reflect the reflected light again to make it parallel to the transmitted light. The first detection array is used to receive the reflected light, and the second detection array is used to receive the transmitted light.

[0013] Further, in the flow cytometer, the first detection array includes a plurality of detection channels arranged in parallel. The first detection channel is configured to receive the reflected light from the plane mirror and separate the reflected light according to wavelength size, so that the reflected light within a preset wavelength range is used for detection, while the reflected light outside the preset wavelength range is reflected into the adjacent next set of detection channels, and so on.

[0014] Further, in the flow cytometer, each detection channel includes a concave mirror, and a band-pass filter, a focusing lens, and a detector sequentially arranged at intervals on one side of the concave mirror.

[0015] Further, in the flow cytometer, the optical path from the image-side principal plane of the collimating lens to the first concave mirror is the collimation distance. In each detection channel, the optical path from the concave mirror to the band-pass filter is half of the collimation distance, and the optical path from the band-pass filter in one detection channel to the concave mirror in the adjacent next detection channel is also half of the collimation distance.

[0016] Further, in the flow cytometer, in each detection channel, the concave curvature radius of the concave mirror is the same, and the size of the concave curvature radius is equal to the collimation distance.

[0017] Further, in the flow cytometer, the optical path from the band-pass filter in one detection channel to the focusing lens is greater than the optical path from the band-pass filter in the adjacent next detection channel to the focusing lens, so that the detectors in each detection channel can be aligned in the same plane.

[0018] Further, for the flow cytometer, the second detection component includes a light blocking strip, a first positive lens, a second positive lens, and a forward detector, and the light blocking strip, the first positive lens, the second positive lens, and the forward detector are sequentially arranged from top to bottom on the other side in the vertical direction of the flow chamber.

[0019] On the other hand, the present invention also provides an optical path debugging method for the flow cytometer in the above technical solution, and the method includes:

[0020] First, debug the internal optical path of the first detection component;

[0021] A small hole located on the image side focal plane of the objective lens is arranged in front of the collimating lens of the first detection component, and an observation device is arranged on the side of the flow chamber far from the objective lens. The observation device is used to observe the inside of the flow chamber and can see the image of the small hole formed by the objective lens at the center of the flow chamber and the laser beam inside the flow chamber;

[0022] Adjust the position of the objective lens or the first detection component in the horizontal direction until the image formed by the small hole is located at the center of the internal flow channel of the flow chamber;

[0023] Adjust the position of the laser or the first detection component in the vertical direction until the center height of the laser beam at the center position of the flow chamber is equal to the center height of the small hole image;

[0024] Finally, debug the optical path of the second detection component.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] The laser beam is precisely shaped by the first cylindrical lens and the second cylindrical lens to ensure that the beam waist of the laser beam is located at the center of the flow chamber and can accurately irradiate the particles or cells to be measured. Compared with the traditional scheme of reflecting the laser beam into the flow chamber by a mirror, it has better beam pointing stability. Description of the Drawings

[0027] Figure 1 It is a schematic diagram of the flow cytometer in the present invention;

[0028] Figure 2 It is a schematic diagram of the cooperation relationship between the illumination component and the second detection component in the present invention;

[0029] Figure 3 It is a schematic diagram of the cooperation relationship between the objective lens and the first detection component in the present invention;

[0030] Figure 4 It is a schematic diagram of the collimated light beam being separated into transmitted light and reflected light in the present invention;

[0031] Figure 5 It is a schematic diagram of the first detection array in the present invention;

[0032] Figure 6 Schematic diagram of the second detection array in the present invention;

[0033] Description of main component symbols:

[0034] 01, laser; 02, laser beam; 03, first cylindrical lens; 04, second cylindrical lens; 05, flow cell; 06, lateral angle scattering light signal and fluorescence signal; 07, objective lens; 08, forward angle scattering light signal; 10, second detection component; 11, light blocking strip; 12, first positive lens; 13, second positive lens; 14, forward detector; 20, first detection component; 21, collimating lens; 22, dichroic filter; 23, plane mirror; M01 - M20, concave mirror; F01 - F20, band - pass filter; S01 - S20, attenuation sheet; L01 - L20, focusing lens; D01 - D20, detector.

[0035] The following specific embodiments will further illustrate the present invention in conjunction with the above - mentioned drawings. Specific embodiments

[0036] For ease of understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0037] It should be noted that when an element is referred to as being "fixedly provided on" another element, it can be directly on the other element or there may also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0039] Please refer to Figures 1 to 6, the flow cytometer in the first embodiment of the present invention includes a flow cell 05, an illumination assembly, an objective lens 07, a first detection assembly 20, and a second detection assembly 10. The illumination assembly includes a laser 01, a first cylindrical lens 03, and a second cylindrical lens 04. The laser 01, the first cylindrical lens 03, and the second cylindrical lens 04 are sequentially arranged from top to bottom on one side in the vertical direction of the flow cell 05. The objective lens 07 is arranged on one side in the horizontal direction of the flow cell 05. The objective lens 07 is used to collect the side scatter light signal and the fluorescence signal 06 generated when the laser beam 02 irradiates the particles or cells to be measured in the flow cell 05. The first detection assembly 20 is used to detect the side scatter light signal and the fluorescence signal 06 from the objective lens 07. The second detection assembly 10 is used to collect and detect the forward scatter light signal 08 generated when the laser beam 02 irradiates the particles or cells to be measured in the flow cell 05.

[0040] The laser beam 02 is precisely shaped by the first cylindrical lens 03 and the second cylindrical lens 04 to ensure that the beam waist of the laser beam 02 is located at the center of the flow cell 05 and can accurately irradiate the particles or cells to be measured. Compared with the traditional scheme of reflecting the laser beam 02 into the flow cell 05 using a mirror, it has better beam pointing stability.

[0041] In this embodiment, the first cylindrical lens 03 is used to shape the laser beam in the horizontal direction, and the second cylindrical lens 04 is used to shape the laser beam in the vertical direction. Among them, the focal length of the first cylindrical lens 03 is greater than the focal length of the second cylindrical lens 04. It can be understood that the longer focal length of the first cylindrical lens 03 makes the beam shaping in the horizontal direction relatively gentle and suitable for forming a uniform beam distribution. The shorter focal length of the second cylindrical lens 04 makes the beam shaping in the vertical direction more significant and suitable for forming a concentrated beam distribution. Through this combination, the laser beam 02 can be shaped in the horizontal and vertical directions respectively, so as to form an ideal beam waist (the thinnest part of the beam) at the center of the flow cell 05, ensuring that the laser beam 02 can accurately irradiate the particles or cells to be measured.

[0042] In this embodiment, for the forward scatter light signal 08, the propagation direction forms a small angle, such as about 10°, with the laser transmission direction. For the side scatter light signal and the fluorescence signal 06, the propagation direction forms a large angle, such as about 90°, with the laser transmission direction.

[0043] Refer to Figure 3, the first detection component 20 includes a collimating lens 21, a dichroic filter 22, a plane mirror 23, a first detection array, and a second detection array. The collimating lens 21 is configured to receive an optical signal from the objective lens 07 and adjust it into a collimated light beam. The dichroic filter 22 is disposed on one side of the collimating lens 21 in the horizontal direction, and the dichroic filter 22 is configured to separate the collimated light beam into transmitted light and reflected light according to wavelength. The plane mirror 23 is disposed on one side of the dichroic filter 22 in the vertical direction, and the plane mirror 23 is configured to reflect the reflected light again to make it parallel to the transmitted light. The first detection array is configured to receive the reflected light, and the second detection array is configured to receive the transmitted light.

[0044] By using a single dichroic filter 22 to first divide the collimated light beam into two parts, i.e., transmitted light and reflected light, and then separately performing spectral separation on these two parts of the spectrum by the first detection array and the second detection array, the number of detection channels is doubled within a limited collimation distance, meeting the optical path design of multiple detection channels.

[0045] In this embodiment, the dichroic filter 22 is placed at a 45° angle, that is, the incident angle of light is 45°. The included angle between the reflected light reflected by the dichroic filter 22 and the transmitted light is 90°, that is, the beam direction is deflected by 90°. Similarly, in this embodiment, the plane mirror 23 is also placed at a 45° angle. The reflected light reflected by the dichroic filter 22 is reflected again by the plane mirror 23, and the reflected light is deflected by 90° again, so as to be parallel to the transmitted light. Among them, regarding the placement angles of the dichroic filter 22 and the plane mirror 23, this embodiment is only an example rather than a limitation, and can be flexibly adjusted in practical applications as long as the beam reflected by the plane mirror 23 is parallel to the beam transmitted through the dichroic filter 22.

[0046] In addition, it should be noted that the dichroic filter 22 in this embodiment includes but is not limited to a long-pass dichroic filter 22, and can also be a short-pass dichroic filter 22. For the long-pass dichroic filter 22, light with a wavelength greater than the starting response wavelength will pass through, and light with a wavelength less than the starting response wavelength will be reflected. On the contrary, for the short-pass dichroic filter 22, light with a wavelength less than the starting response wavelength will pass through, and light with a wavelength greater than the starting response wavelength will be reflected.

[0047] Refer to Figure 5, the first detection array includes a plurality of detection channels arranged in parallel. The first detection channel is configured to receive the reflected light from the planar mirror 23 and separate the reflected light according to the wavelength magnitude, so that the reflected light within a preset wavelength range is used for detection, while the reflected light outside the preset wavelength range is reflected into the adjacent next set of detection channels, and so on. Specifically, the detection channel includes a concave mirror, and a band-pass filter, a focusing lens, and a detector sequentially and spaced apart on one side of the concave mirror. In this embodiment, the first detection array includes concave mirrors M01 to M10, band-pass filters F01 to F10, focusing lenses L01 to L10, and detectors D01 to D10. Among them, the concave mirror M01 is located on one side of the planar mirror 23 in the horizontal direction, and is used to reflect all the light rays emitted from the planar mirror 23 onto the band-pass filter F01 for separation. The light with a wavelength within its bandwidth passes through the band-pass filter F01 and the focusing lens L01 and enters the detector D01 for detection. The light with a wavelength outside its bandwidth is reflected by the band-pass filter F01 to the mirror M02, and then is continuously reflected by the mirror M02 into the adjacent next band-pass filter F02. The band-pass filter F02 performs the same steps as the above band-pass filter F01, and so on.

[0048] In this embodiment, the second detection array has the same structure as the first detection array. Specifically, refer to Figure 6 , the second detection array includes concave mirrors M11 to M20, band-pass filters F11 to F20, focusing lenses L11 to L20, and detectors D11 to D20. Among them, the concave mirror M11 is located on one side of the dichroic filter 22 in the horizontal direction, and is used to reflect all the light rays emitted from the dichroic filter 22 onto the band-pass filter F11 for separation. The light with a wavelength within its bandwidth passes through the band-pass filter F11 and the focusing lens L11 and enters the detector D11 for detection. The light with a wavelength outside its bandwidth is reflected by the band-pass filter F11 to the mirror M12, and then is continuously reflected by the mirror M12 into the adjacent next band-pass filter F12. The band-pass filter F12 performs the same steps as the above band-pass filter F11, and so on.

[0049] It can be seen that in this embodiment, the spectral separation method adopts the continuous reflection method, which reduces the number of times light passes through the band-pass filter. In each detection channel, the number of times light passes through the band-pass filter is only once. Compared with transmission two or more times, the light energy loss is greatly reduced, and the detection sensitivity of the system is improved. The reasons are as follows: The transmittance within the bandwidth of the band-pass filter can generally ensure being greater than 95%, the reflectance outside the bandwidth can generally ensure being greater than 99.99%, and the reflectance of the mirror can generally ensure being greater than 99.5%. Secondly, a concave mirror is used for relay to extend the limited collimation distance, which can theoretically be extended indefinitely, that is, there is no upper limit to the number of channels. However, with the accumulation of optical aberration and manufacturing tolerance, the aberration of the subsequent channels will become larger and larger, resulting in the spot size focused on the photosensitive area of the detector exceeding the range of the photosensitive area, which limits the upper limit of the number of channels.

[0050] It should be noted that in this embodiment, the concave mirrors M01 and M11 are located at the primary image plane position, that is, the light rays emitted by the objective lens 07 are focused for the first time at this position. Similarly, if it is the second focus, it is the secondary image plane position, and if it is the third focus, it is the tertiary image plane position, and so on. In this embodiment, the concave mirrors M03 and M13 are located at the secondary image plane position, the concave mirrors M05 and M15 are located at the tertiary image plane position, the concave mirrors M07 and M17 are located at the quaternary image plane position, and the concave mirrors M09 and M19 are located at the quinary image plane position.

[0051] The optical path from the image-side principal plane of the collimating lens 21 to the primary image plane position is the collimation distance. The optical path from the concave mirror M01 to the band-pass filter F01 is half of the collimation distance, and the optical path from the band-pass filter F01 to the next concave mirror M02 is also half of the collimation distance, and so on.

[0052] The function of the concave mirrors M01~M20 is to extend the collimation distance, and the size of their concave curvature radius will affect the position of the subsequent image plane.

[0053] In one implementation manner of this embodiment, the concave curvature radii of the concave mirrors M01~M20 are the same. At this time, the size of the concave curvature radius is equal to the collimation distance or within a certain tolerance range thereof.

[0054] In another implementation manner of this embodiment, the concave curvature radii of the concave mirrors M01~M20 can also be different. Taking the concave mirrors M01~M10 as an example, as the optical path extends backward, the subsequent concave mirrors increase or decrease their respective concave curvature radii according to the aberration adaptability to compensate for the accumulation of optical aberration and ensure that the light beam can be accurately focused within the photosensitive area of the detector. This design can effectively improve the detection accuracy and stability of the system.

[0055] Furthermore, in this embodiment, the concave mirrors M01 to M20 and the band-pass filters F01 to F20 are placed at a certain angle, and the incident angle of light on the concave mirrors and the band-pass filters is in the range of 5 to 20°. The concave mirrors M01 to M10 are arranged in an array, and all the concave centers are located in the same plane. The band-pass filters F01 to F10 are arranged in an array and are located in the same plane. The concave mirrors M11 to M20 are arranged in an array, and all the concave centers are located in the same plane. The band-pass filters F11 to F20 are arranged in an array and are located in the same plane.

[0056] Optionally, the first detection array further includes attenuation sheets, and the attenuation sheets are arranged between the band-pass filters and the focusing lenses. The main function of the attenuation sheets is to reduce the intensity of light when the intensity of light is high and the detector is saturated and cannot work properly. There are also related settings of attenuation sheets in the second detection array. Specifically, the first detection array includes attenuation sheets S01 to S10, and the second detection array includes attenuation sheets S11 to S20.

[0057] Specifically, in this embodiment, the light beam passing through the band-pass filter F01 is focused by the focusing lens L01 onto the photosensitive area of the detector D01 after passing through the attenuation sheet S01, which is detection channel one;

[0058] The light beam passing through the band-pass filter F02 is focused by the focusing lens L02 onto the photosensitive area of the detector D02 after passing through the attenuation sheet S02, which is detection channel two;

[0059] The light beam passing through the band-pass filter F03 is focused by the focusing lens L03 onto the photosensitive area of the detector D03 after passing through the attenuation sheet S03, which is detection channel three;

[0060] The light beam passing through the band-pass filter F04 is focused by the focusing lens L04 onto the photosensitive area of the detector D04 after passing through the attenuation sheet S04, which is detection channel four;

[0061] The light beam passing through the band-pass filter F05 is focused by the focusing lens L05 onto the photosensitive area of the detector D05 after passing through the attenuation sheet S05, which is detection channel five;

[0062] The light beam passing through the band-pass filter F06 is focused by the focusing lens L06 onto the photosensitive area of the detector D06 after passing through the attenuation sheet S06, which is detection channel six;

[0063] The light beam passing through the band-pass filter F07 is focused by the focusing lens L07 onto the photosensitive area of the detector D07 after passing through the attenuation sheet S07, which is detection channel seven;

[0064] The light beam passing through the band-pass filter F08 is attenuated by the attenuation filter S08 and then focused by the focusing lens L08 into the photosensitive area of the detector D08, which is Detection Channel Eight;

[0065] The light beam passing through the band-pass filter F09 is attenuated by the attenuation filter S09 and then focused by the focusing lens L09 into the photosensitive area of the detector D09, which is Detection Channel Nine;

[0066] The light beam passing through the band-pass filter F10 is attenuated by the attenuation filter S10 and then focused by the focusing lens L10 into the photosensitive area of the detector D10, which is Detection Channel Ten;

[0067] The light beam passing through the band-pass filter F11 is attenuated by the attenuation filter S11 and then focused by the focusing lens L11 into the photosensitive area of the detector D11, which is Detection Channel Eleven;

[0068] The light beam passing through the band-pass filter F12 is attenuated by the attenuation filter S12 and then focused by the focusing lens L12 into the photosensitive area of the detector D12, which is Detection Channel Twelve;

[0069] The light beam passing through the band-pass filter F13 is attenuated by the attenuation filter S13 and then focused by the focusing lens L13 into the photosensitive area of the detector D13, which is Detection Channel Thirteen;

[0070] The light beam passing through the band-pass filter F14 is attenuated by the attenuation filter S14 and then focused by the focusing lens L14 into the photosensitive area of the detector D14, which is Detection Channel Fourteen;

[0071] The light beam passing through the band-pass filter F15 is attenuated by the attenuation filter S15 and then focused by the focusing lens L15 into the photosensitive area of the detector D15, which is Detection Channel Fifteen;

[0072] The light beam passing through the band-pass filter F16 is attenuated by the attenuation filter S16 and then focused by the focusing lens L16 into the photosensitive area of the detector D16, which is Detection Channel Sixteen;

[0073] The light beam passing through the band-pass filter F17 is attenuated by the attenuation filter S17 and then focused by the focusing lens L17 into the photosensitive area of the detector D17, which is Detection Channel Seventeen;

[0074] The light beam passing through the band-pass filter F18 is attenuated by the attenuation filter S18 and then focused by the focusing lens L18 into the photosensitive area of the detector D18, which is Detection Channel Eighteen;

[0075] The light beam passing through the band-pass filter F19 is attenuated by the attenuation filter S19 and then focused by the focusing lens L19 into the photosensitive area of the detector D19, which is Detection Channel Nineteen;

[0076] The light beam passing through the bandpass filter F20 is focused by the focusing lens L20 into the photosensitive area of ​​the detector D20 after passing through the attenuation plate S20. This is the detection channel 20.

[0077] In this embodiment, taking the first detection array as an example, the optical path from the bandpass filter F01 to the focusing lens L01 is greater than or equal to half of the collimation distance, ensuring that the light beam does not exceed the effective aperture of the focusing lens.

[0078] In addition, the optical path from the bandpass filter in one detection channel to the focusing lens is greater than the optical path from the bandpass filter in the next adjacent detection channel to the focusing lens, so that the detectors in each detection channel can be aligned in the same plane. Taking the first detection array as an example, the optical path from the bandpass filter F01 to the focusing lens L01 is greater than the optical path from the bandpass filter F02 to the focusing lens L02, and the optical path from the bandpass filter F02 to the focusing lens L02 is greater than the optical path from the bandpass filter F03 to the focusing lens L03, and so on. Specifically, the values ​​that decrease in sequence can be calculated based on the collimation distance and the incident angle of the bandpass filter. The purpose of this design is to make multiple detectors D01 to D10 located in the same plane, so that it is convenient to weld or connect multiple detectors on the same circuit board, and the photocurrent can be directly processed. Compared with the switching method, the anti-interference ability is stronger and the structure is simpler.

[0079] It is worth mentioning that as the number of detection channels increases, the distance from the bandpass filter to the focusing lens is constantly shortening, which will limit the upper limit of the number of detection channels. Therefore, the number of detection channels is mainly affected by the collimation distance, the incident angle of the bandpass filter and the size of the detector's photosensitive area. The longer the collimation distance, the smaller the incident angle of the bandpass filter and the larger the size of the detector's photosensitive area, the greater the number of detection channels will be.

[0080] See also Figure 2 In this embodiment, the second detection component 10 includes a light shielding strip 11, a first positive lens 12, a second positive lens 13 and a forward detector 14, and the light shielding strip 11, the first positive lens 12, the second positive lens 13 and the forward detector 14 are arranged in sequence from top to bottom from the other side of the flow chamber 05 in the vertical direction. Among them, the light shielding strip 11 is used to block the background signal of the laser (i.e., the laser beam 02 that is not scattered by the particles or cells to be measured), prevent the laser beam 02 from directly entering the forward detector 14, avoid interference with the forward angle scattered light signal 08, improve the signal-to-noise ratio, and ensure the detection accuracy of the forward angle scattered light signal 08. The first positive lens 12 is used to collect the forward angle scattered light signal 08, the second positive lens 13 is used to focus the collected forward angle scattered light signal 08 in the photosensitive area of ​​the forward detector 14, and the forward detector 14 is used for photoelectric conversion.

[0081] In summary, for the flow cytometer in the above embodiments of the present invention, the first cylindrical lens 03 and the second cylindrical lens 04 are used to precisely shape the laser beam 02, ensuring that the beam waist of the laser beam 02 is located at the center of the flow chamber 05, enabling accurate irradiation of the particles or cells to be measured. Compared with the conventional solution of using a mirror to reflect the laser beam 02 into the flow chamber 05, it has better beam pointing stability.

[0082] The second embodiment of the present invention further provides an optical path debugging method for the flow cytometer described in the above technical solution. The method includes:

[0083] First, debug the internal optical path of the first detection component;

[0084] A small hole located on the focal plane of the object mirror side is set in front of the collimating lens of the first detection component, and an observation device is set on the side of the flow chamber away from the objective lens. The observation device is used to observe the inside of the flow chamber and can see the image of the small hole formed by the objective lens at the center of the flow chamber and the laser beam inside the flow chamber;

[0085] Adjust the position of the objective lens or the first detection component in the horizontal direction until the image formed by the small hole is located at the center of the internal flow channel of the flow chamber;

[0086] Adjust the position of the laser or the first detection component in the vertical direction until the center height of the laser beam at the center position of the flow chamber is equal to the center height of the small hole image;

[0087] Finally, debug the optical path of the second detection component (fine-tune in the horizontal or vertical direction).

[0088] It should be noted that in this embodiment, the observation device is specifically a microscope system.

[0089] In addition, in step four, if they are not of equal height, the reason needs to be checked first to see whether it is caused by the height deviation of the small hole or the height deviation of the laser beam itself, and then corresponding adjustments are made. For example, if the height of the first detection component is too low, resulting in a low height of the small hole, the height of the image formed by the small hole through the objective lens will be too high. In this case, the overall height of the first detection component should be adjusted, rather than the height of the laser beam.

[0090] Furthermore, in this embodiment, after the step of finally debugging the optical path of the second detection component, the method further includes:

[0091] Step six: Use standard microspheres to calibrate the optical path, and fine-tune the optical paths of the illumination component, the objective lens, the first detection component, and the second detection component to further improve the system performance.

[0092] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0093] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. A flow cytometer, characterized in that: include: Flow chamber; An illumination assembly, the illumination assembly comprising a laser, a first cylindrical mirror and a second cylindrical mirror, wherein the laser, the first cylindrical mirror and the second cylindrical mirror are arranged in sequence from top to bottom on one side of the flow chamber in a vertical direction; An objective lens, which is arranged at one side of the flow chamber in the horizontal direction and is used to collect side angle scattered light signals and fluorescence signals generated when the laser beam irradiates the particles or cells to be measured in the flow chamber; A first detection component, the first detection component is used to detect the side angle scattered light signal and the fluorescence signal from the objective lens; A second detection component is used to collect and detect forward angle scattered light signals generated when the laser beam irradiates the particles or cells to be detected in the flow chamber.

2. The flow cytometer according to claim 1, characterized in that The first cylindrical mirror is used to shape the laser beam in the horizontal direction, and the second cylindrical mirror is used to shape the laser beam in the vertical direction, wherein the focal length of the first cylindrical mirror is greater than the focal length of the second cylindrical mirror.

3. The flow cytometer according to claim 1, characterized in that The first detection component includes a collimating lens, a dichroic filter, a plane mirror, a first detection array and a second detection array. The collimating lens is used to receive the light signal from the objective lens and adjust it into a collimated light beam; the dichroic filter is arranged on one side of the collimating lens in the horizontal direction, and the dichroic filter is used to separate the collimated light beam into transmitted light and reflected light according to wavelength; the plane mirror is arranged on one side of the dichroic filter in the vertical direction, and the plane mirror is used to reflect the reflected light again to make it parallel to the transmitted light; the first detection array is used to receive the reflected light, and the second detection array is used to receive the transmitted light.

4. The flow cytometer according to claim 3, characterized in that The first detection array includes a plurality of detection channels arranged in parallel, and the first detection channel is configured to receive reflected light from the plane reflector and separate the reflected light according to wavelength, so that the reflected light within a preset wavelength range is used for detection, and the reflected light outside the preset wavelength range is reflected to the next adjacent group of detection channels, and so on.

5. The flow cytometer according to claim 4, characterized in that The detection channel comprises a concave reflector, and a bandpass filter, a focusing lens and a detector which are sequentially and spaced apart and arranged on one side of the concave reflector.

6. The flow cytometer according to claim 5, characterized in that The optical path from the image-side principal surface of the collimating lens to the first concave reflector is the collimation distance. In each detection channel, the optical path from the concave reflector to the bandpass filter is half of the collimation distance, and the optical path from the bandpass filter in one detection channel to the concave reflector in the next adjacent detection channel is also half of the collimation distance.

7. The flow cytometer according to claim 6, characterized in that In each detection channel, the concave curvature radius of the concave reflector is the same, and the size of the concave curvature radius is equal to the collimation distance.

8. The flow cytometer according to claim 5, characterized in that The optical path from the bandpass filter to the focusing lens in one detection channel is greater than the optical path from the bandpass filter to the focusing lens in the next adjacent detection channel, so that the detectors in each detection channel can be aligned in the same plane.

9. The flow cytometer according to claim 1, characterized in that: The second detection assembly includes a light shielding strip, a first positive lens, a second positive lens and a forward detector, which are arranged in sequence from top to bottom from the other side of the flow chamber in the vertical direction.

10. An optical path debugging method, applied to the flow cytometer according to any one of claims 1 to 9, characterized in that: The method comprises: First, debug the internal optical path of the first detection component; A small hole located on the focal plane of the objective lens is arranged in front of the collimating lens of the first detection assembly, and an observation device is arranged on the side of the flow chamber away from the objective lens, wherein the observation device is used to observe the interior of the flow chamber, and can see the image of the small hole formed by the objective lens at the center of the flow chamber and the laser beam inside the flow chamber; Adjust the position of the objective lens or the first detection assembly in the horizontal direction until the image formed by the small hole is located at the center of the flow channel inside the flow chamber; Adjust the position of the laser or the first detection assembly in the vertical direction until the center height of the laser beam at the center of the flow chamber is equal to the center height of the pinhole image; Finally, debug the optical path of the second detection component.

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