Multi-laser spectrum flow cytometry optical system

Abstract

The invention provides a multi-laser spectrum flow cytometer optical system, which comprises an illumination module, the illumination module comprises a plurality of sub-laser paths, a beam combining light path and a flat-topped shaping assembly, a laser and a beam expanding assembly are arranged on the path of the sub-laser paths, the beam expanding assembly is used for expanding a laser beam emitted by the laser, and the laser beam is transmitted to the flat-topped shaping assembly. The wavelengths of the laser beams emitted by the laser devices on all the sub laser paths are different; a dichroic laser beam combining mirror and a reflecting mirror are arranged on the path of the beam combining light path, the dichroic laser beam combining mirror is located between the reflecting mirror and the flat-topped shaping assembly, the dichroic laser beam combining mirror and the reflecting mirror respectively correspond to one sub-laser path, and beam-expanded laser beams emitted by the multiple sub-laser paths are combined to obtain a combined laser beam; and the flat-top shaping assembly is used for converting the combined laser beams into flat-top contour laser beams, so that the accuracy, sensitivity and reliability of detection are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of spectral flow cytometers, and in particular to an optical system of a multi-laser spectral flow cytometer. Background Art

[0002] A spectral flow cytometer (Full Spectrum Flow Cytometry) is a high-performance cell analysis instrument developed based on the traditional flow cytometer. The spectral flow cytometer is usually equipped with one or more lasers. The laser beam emitted by the laser is shaped and then used to detect the particles or cells to be measured at the detection position, which usually refers to the position of the central flow channel inside the flow chamber. The particles or cells to be measured are pre-treated with immune reactions, staining, etc., and are coated with fluorescent dyes that can be excited by lasers. When the particles or cells to be measured flow through the detection position and are irradiated by the laser, scattered light signals and fluorescence signals will be generated. These light signals are then captured by a photodetector and converted into electrical signals, and then processed and analyzed by data analysis software. Among them, the scattered light signals are mainly used to detect physical parameters such as the size and shape of cells, while the fluorescence signals are used to detect biological characteristics such as the expression level of markers on the cell surface. Refer to the patent document CN207717594.

[0003] In a spectral flow cytometer, the laser beam needs to accurately irradiate each cell flowing through the detection position. If the direction of the laser beam is unstable, the light spot irradiated on the cell will change, resulting in fluctuations and errors in the detection signal. At the same time, the direction stability of the laser beam also directly affects the sensitivity of the detection system. If the direction of the laser beam is unstable, even if the fluorescence signal emitted by the cell is very weak, it may not be accurately detected due to the offset of the light spot position. In addition, when performing multi-parameter analysis, each parameter needs to be detected by a specific laser beam. If the direction of the laser beam is unstable, there will be deviations in the detection results between different parameters, thus affecting the reliability and accuracy of the data.

[0004] The existing laser beam is a Gaussian beam, and its energy is mainly concentrated in the center position of the beam, while the energy at the edge is relatively low, greatly reducing the detection accuracy, sensitivity and reliability. Summary of the Invention

[0005] The purpose of the present invention is to provide an optical system of a multi-laser spectral flow cytometer in view of the deficiencies in the prior art, which has good stability of the illumination system and improves the detection accuracy, sensitivity and reliability.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A multi-laser spectral flow cytometer optical system, including an illumination module, and the illumination module includes:

[0008] Multiple sub-laser paths, on the paths of the sub-laser paths, a laser and a beam expander assembly are provided, the beam expander assembly is used to expand the laser beam emitted by the laser to obtain an expanded laser beam, and the laser beams emitted by the lasers on each sub-laser path have different wavelengths;

[0009] A beam combining optical path, on the path of the beam combining optical path, a dichroic laser beam combiner and a reflector are provided, the dichroic laser beam combiner is located between the reflector and the flat-top shaping component, the dichroic laser beam combiner and the reflector each correspond to a sub-laser path respectively, and the expanded laser beams emitted by multiple sub-laser paths are combined to obtain a combined laser beam;

[0010] A flat-top shaping component, the flat-top shaping component is used to convert the combined laser beam into a flat-top profile laser beam.

[0011] As a preferred embodiment, the flat-top shaping component includes:

[0012] A diffractive optical device;

[0013] An achromatic positive lens, the achromatic positive lens is located between the diffractive optical device and the flow cell;

[0014] The combined laser beam changes its energy distribution through the diffractive optical device, and then forms a flat-top profile laser beam after being focused by the achromatic positive lens.

[0015] As a preferred embodiment, the reflector, the dichroic laser beam combiner, the diffractive optical device, and the achromatic positive lens are kept on a straight line.

[0016] As a preferred embodiment, the dichroic laser beam combiner reflects the expanded laser beam at an angle of 45° relative to its corresponding sub-laser path;

[0017] The reflector reflects the expanded laser beam at an angle of 45° relative to its corresponding sub-laser path.

[0018] As a preferred embodiment, the sub-laser path is perpendicular to the beam combining optical path.

[0019] As a preferred embodiment, the dichroic laser beam combiner includes a long-pass lens, and the wavelengths of the laser beams emitted by the lasers decrease in the direction from the reflector to the flat-top shaping component;

[0020] Alternatively, the dichroic laser beam combiner includes a short-pass lens, and the wavelengths of the laser beams emitted by the lasers increase in the direction from the reflector to the flat-top shaping component;

[0021] Alternatively, the dichroic laser beam combiner includes a long-pass lens and a short-pass lens.

[0022] As a preferred embodiment, the beam expander assembly includes a negative lens and a positive lens, and the negative lens is located between the laser and the positive lens;

[0023] Alternatively, the beam expander assembly includes a first positive lens and a second positive lens, the focal length of the first positive lens is shorter than that of the second positive lens, and the first positive lens is located between the laser and the second positive lens.

[0024] As a preferred embodiment, it further includes:

[0025] A forward angular astigmatism detection device, and the forward angular astigmatism detection device and the flat-top shaping assembly are located on opposite sides of the flow cell;

[0026] The flat-top profile laser beam generates a forward angular astigmatism signal after being focused by the flow cell, and the forward angular astigmatism signal is collected by the forward angular astigmatism detection device and subjected to photoelectric conversion.

[0027] As a preferred embodiment, the forward angular astigmatism detection device includes a light blocking strip, a third positive lens, a band-pass filter, a fourth positive lens, and a forward detector.

[0028] As a preferred embodiment, the flat-top profile laser beam generates a lateral angular scattering light signal and a fluorescence signal after being focused by the flow cell, and the multi-laser spectral flow cytometry optical system further includes:

[0029] Optical fibers, the number of the optical fibers is equal to that of the lasers and they are in one-to-one correspondence;

[0030] An objective lens, which is used to introduce the lateral angular scattering light and fluorescence generated by different lasers into the corresponding optical fibers.

[0031] Compared with the prior art, the technical solution has the following advantages:

[0032] The beam expander assembly expands the passing laser beam to obtain an expanded laser beam, so as to meet the requirements of the flat-top shaping assembly for the size of the input beam. The expanded laser beams emitted by multiple sub-laser paths are combined by a dichroic laser combiner and a reflector to obtain a combined laser beam. The flat-top shaping assembly changes the energy distribution of the passing combined laser beam, etc., so as to transform and obtain a flat-top profile laser beam. The flat-top profile laser beam maintains a constant irradiance value in the cross section. Compared with the existing Gaussian beam, it has the characteristic of uniform energy distribution, greatly reduces the requirements for the fluctuation of the illumination beam, and also has better performance during high-speed testing, improving the accuracy, sensitivity and reliability of detection, and also improving the detection efficiency of the instrument.

[0033] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. Description of the Drawings

[0034] Figure 1 It is a schematic structural diagram of the optical system of the multi-laser spectral flow cytometer described in the present invention;

[0035] Figure 2 It is a coordinate diagram of the relationship between relative irradiance at y and x positions;

[0036] Figure 3 It is a coordinate diagram of the relationship between relative irradiance at x and y positions;

[0037] Figure 4 It is a schematic diagram of the cooperation between the objective lens and the flow cell described in the present invention;

[0038] Figure 5 It is Figure 4 an enlarged schematic diagram of A in

[0039] Figure 6 It is Figure 4 an enlarged schematic diagram of B in

[0040] Figure 7 It is a schematic diagram of the image space position in the optical system of the multi-laser spectral flow cytometer described in the present invention. Detailed Embodiments

[0041] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and other obvious variations can be conceived by those skilled in the art. The basic principles defined in the following description can be applied to other embodiments, variations, improvements, equivalent schemes, and other technical schemes that do not depart from the spirit and scope of the present invention.

[0042] As Figure 1 shown, the optical system of the multi-laser spectral flow cytometer includes an illumination module 10, and the illumination module 10 includes:

[0043] Multiple sub-laser paths, on which a laser 11, 21, 31 and a beam expander assembly are provided. The beam expander assembly is used to expand the laser beams 12, 22, 32 emitted by the lasers 11, 21, 31 to obtain expanded laser beams 15, 25, 35. The laser beams 12, 22, 32 emitted by the lasers 11, 21, 31 on each sub-laser path have different wavelengths.

[0044] One beam combining path, on which a dichroic laser combiner 26, 36 and a reflector 16 are provided. The dichroic laser combiners 26, 36 are located between the reflector 16 and the flat-top shaping component 101. The dichroic laser combiners 26, 36 and the reflector 16 respectively correspond to one sub-laser path, and the expanded laser beams 15, 25, 35 emitted by multiple sub-laser paths are combined to obtain a combined laser beam 100.

[0045] A flat-top shaping component, which is used to convert the combined laser beam 100 into a flat-top profile laser beam 103.

[0046] The beam expander assembly expands the passing laser beams 12, 22, 32 to obtain expanded laser beams 15, 25, 35 to meet the requirements of the flat-top shaping component for the input beam size. The expanded laser beams 15, 25, 35 emitted by multiple sub-laser paths pass through the dichroic laser combiners 26, 36 and are combined by the reflector 16 to obtain a combined laser beam 100. The flat-top shaping component changes the energy distribution of the passing combined laser beam 100, etc., to convert it into a flat-top profile laser beam 103. The flat-top profile laser beam 103 maintains a constant irradiance value in the cross-section, and compared with the existing Gaussian beam, it has the characteristic of uniform energy distribution, greatly reducing the requirements for the fluctuation of the illumination beam, and also having better performance in high-speed testing, improving the accuracy, sensitivity and reliability of detection.

[0047] As Figure 1 shown, the illumination module 10 is located on the left side of the flow cell 110, and multiple sub-laser paths are arranged horizontally, and each sub-laser path is arranged vertically, which can effectively utilize the limited space on the left side of the flow cell 110.

[0048] On each sub-laser path, the number of the lasers 11, 21, 31 and the beam expander assembly is one. The lasers 11, 21, 31 are located on the front side of the beam expander assembly, and the lasers 11, 21, 31 irradiate towards the beam expander assembly.

[0049] The beam expander assembly expands the passing laser beams 12, 22, 32. Beam expansion refers to changing the diameter and divergence angle of the laser beam so that the laser beam maintains a more stable diameter during propagation, or expands to the required size at a specific position as needed to meet the size requirements of the input beam for the flat-top shaping assembly.

[0050] In one embodiment, the beam expander assembly includes a negative lens 13, 23, 33 and a positive lens 14, 24, 34. The negative lens 13, 23, 33 is located between the lasers 11, 21, 31 and the positive lens 14, 24, 34.

[0051] The focal lengths of the negative lenses 13, 23, 33 are negative values, which function to diverge the light beam. The focal lengths of the positive lenses 14, 24, 34 are positive values, which function to converge the light beam.

[0052] The foci of the negative lenses 13, 23, 33 and the positive lenses 14, 24, 34 are located at the same position, which is the key to achieving the beam expansion function. When the laser beams 12, 22, 32 first pass through the negative lenses 13, 23, 33, the laser beams 12, 22, 32 will diverge and form an enlarged virtual focus, which is located on the side of the negative lenses 13, 23, 33 away from the positive lenses 14, 24, 34. Then, after the diverging laser beam passes through the positive lenses 14, 24, 34, the positive lenses 14, 24, 34 will attempt to refocus the diverging laser beam. In this process, the focal position of the positive lenses 14, 24, 34 becomes crucial. If the focal point of the positive lenses 14, 24, 34 matches the position of the virtual focus of the negative lenses 13, 23, 33 (i.e., they coincide or are very close in space), then the positive lenses 14, 24, 34 can effectively converge the diverging laser beam and form a laser beam with a larger diameter and a smaller divergence angle at the output end, that is, the expanded laser beams 15, 25, 35.

[0053] The beam expansion multiple is the ratio of the focal length of the positive lenses 14, 24, 34 to the focal length of the negative lenses 13, 23, 33. This ratio determines the degree of diameter expansion of the laser beams 12, 22, 32 after passing through the beam expander assembly. The focal length of a concave lens refers to the distance from the optical center of the lens to the virtual focus, and the focal length of a convex lens refers to the distance from the optical center of the lens to the focus.

[0054] In another embodiment, the beam expander assembly includes a first positive lens (not shown) and a second positive lens (not shown). The focal length of the first positive lens is shorter than that of the second positive lens. The first positive lens is located between the lasers 11, 21, 31 and the second positive lens.

[0055] Both the first positive lens and the second positive lens are convex lenses. The laser beams 12, 22, and 32 first pass through the first positive lens with a shorter focal length, and they will be slightly converged. Then the laser beams 12, 22, and 32 pass through the second positive lens with a longer focal length. Since the focal length of the second positive lens is longer, it means that the laser beams 12, 22, and 32 are allowed to propagate with a larger diameter without significant focusing, that is, the expanded laser beams 15, 25, and 35 are formed.

[0056] Similarly, the beam expansion multiple is the ratio of the focal length of the second positive lens to the focal length of the first positive lens.

[0057] It should be noted that the flat-top shaping component has strict requirements on the size of the input laser beam, which is why the laser beam emitted by the laser needs to be expanded.

[0058] In addition, the diameter of the laser beam may vary in actual applications. This variation may be caused by factors such as the manufacturing precision of the laser, changes in the working environment, or performance drift after long-term use. Therefore, the size of the laser beam emitted by the laser has a certain positive and negative tolerance range. In order to ensure that the flat-top shaping component can work properly and produce the required beam shape, it is necessary to precisely control and adjust the laser beam generated by the laser. By matching beam expansion components with different magnification factors, the size of the laser beam output by the laser can be adjusted to meet the requirements of the flat-top shaping component for the input beam size.

[0059] As Figure 1 shown, the beam combining optical path is located on the left side of the flow cell 110, the beam combining optical path is located behind the beam expansion component, and the beam combining optical path, the flat-top shaping component, and the flow cell 110 are kept on the same straight line. Among them, the beam combining optical path is perpendicular to multiple sub-laser paths respectively.

[0060] On the path of the beam combining optical path, dichroic laser beam combiners 26, 36 and a mirror 16 are provided. The dichroic laser beam combiners 26, 36 and the mirror 16 each correspond to a sub-laser path respectively, and are used to reflect or project the expanded laser beams 15, 25, 35 formed by the corresponding sub-laser paths onto a common optical path to combine them into a combined laser beam 100.

[0061] Taking three sub-laser paths as an example, the three sub-laser paths are divided into a left sub-laser path, a middle sub-laser path, and a right sub-laser path from left to right. Among them, the left sub-laser path is located on the outermost side relative to the flow cell 110. The number of the mirrors 16 is one, the mirror 16 corresponds to the left sub-laser path, and the number of the dichroic laser beam combiners 26, 36 is two, corresponding to the middle sub-laser path and the right sub-laser path respectively.

[0062] The mirror 16 reflects the expanded laser beam 15 at an angle of 45° relative to its corresponding sub-laser path. The dichroic laser beam combiners 26 and 36 reflect the expanded laser beams 25 and 35 at an angle of 45° relative to their corresponding sub-laser paths. It can be seen that the expanded laser beams 15, 25, and 35 are irradiated towards the flat-top shaping component. Among them, after the expanded laser beam 15 passes through the two dichroic laser beam combiners 26 and 36 corresponding to the middle and right sub-laser paths in sequence, and after the expanded laser beam 25 passes through the dichroic laser beam combiner 36 corresponding to the right sub-laser path, the combined laser beam 100 is obtained.

[0063] In one embodiment, the dichroic laser beam combiners 26 and 36 include long-pass lenses, and the wavelengths of the laser beams emitted by the lasers 11, 21, and 31 decrease in the direction from the mirror 16 to the flat-top shaping component.

[0064] The dichroic laser beam combiners 26 and 36, also known as beam combiners or beam splitters, are optical elements with special optical coatings. It can selectively reflect or transmit light of a specific wavelength. In laser beam combination, the dichroic laser beam combiners 26 and 36 are used to combine laser beams of different wavelengths into one beam. The long-pass lens allows light longer than a certain specific wavelength to pass through, while light shorter than that wavelength is reflected.

[0065] Due to the characteristics of the long-pass lens, the expanded laser beam 15 formed in the left sub-laser path, after being reflected by the mirror 16, passes through the two dichroic laser beam combiners 26 and 36 corresponding to the middle and right sub-laser paths in sequence and will not be interfered by the expanded laser beams 25 and 35. Similarly, the expanded laser beam 25 formed in the middle sub-laser path, after being reflected by its corresponding dichroic laser beam combiner 26, passes through the dichroic laser beam combiner 36 corresponding to the right sub-laser path and will not be interfered by the expanded laser beam 35.

[0066] For example, the wavelength of the laser in the left sub-laser path is 638 nm, the wavelength of the laser in the middle sub-laser path is 488 nm, and the wavelength of the laser in the right sub-laser path is 405 nm.

[0067] In another embodiment, the dichroic laser beam combiners 26 and 36 include short-pass lenses, and the wavelengths of the laser beams emitted by the lasers 11, 21, and 31 increase in the direction from the mirror 16 to the flat-top shaping component.

[0068] The short-pass lens allows light shorter than the selected wavelength to pass through, while light longer than that wavelength is reflected or attenuated.

[0069] Due to the characteristics of the short-wave pass lens, the expanded laser beam 15 formed by the left sub-laser path, after being reflected by the mirror 16, sequentially passes through the two dichroic laser beam combiners 26 and 36 corresponding to the middle and right sub-laser paths, and will not be interfered by the expanded laser beams 25 and 35. The expanded laser beam 25 formed by the middle sub-laser path, after being reflected by its corresponding dichroic laser beam combiner 26, passes through the dichroic laser beam combiner 36 corresponding to the right sub-laser path, and will not be interfered by the expanded laser beam 35.

[0070] In another embodiment, the dichroic laser beam combiners 26 and 36 include a long-wave pass lens and a short-wave pass lens, that is, by adjusting the positions of the lasers 11, 21, and 31 of different wavelength types, the dichroic laser beam combiners 26 and 36 including both the long-wave pass lens and the short-wave pass lens can be selected for use.

[0071] As Figure 1 shown, the flat-top shaping component includes:

[0072] Diffractive optical device 101;

[0073] An achromatic positive lens 102, and the achromatic positive lens 102 is located between the diffractive optical device 101 and the flow cell 110;

[0074] The combined laser beam 100 changes its energy distribution through the diffractive optical device 101, and then forms a flat-top profile laser beam 103 after being focused by the achromatic positive lens 102.

[0075] The mirror 16, the dichroic laser beam combiners 26 and 36, the diffractive optical device 101, and the achromatic positive lens 102 are kept in a straight line, wherein one outwardly convex side of the achromatic positive lens 102 corresponds to the diffractive optical device 101, and the other outwardly convex side of the achromatic positive lens 102 corresponds to the flow cell 110, so that the flat-top profile laser beam 103 formed after being focused by the achromatic positive lens 102 is focused on the detection position of the flow cell 110 in a lateral manner.

[0076] The combined laser beam 100 is a Gaussian laser beam. A diffractive optical element (DOE), which is an optical element based on the principle of light diffraction and belongs to the phase element, has a microstructure embedded inside. These microstructures can change the phase and amplitude distribution of the input laser beam (i.e., the combined laser beam 100). The laser beam processed by the diffractive optical element 101 is then focused by an achromatic lens 102. The achromatic lens 102 can correct chromatic aberration to ensure that light rays of different wavelengths can be focused on the same plane, thereby improving the focusing effect. This flat-top profile laser beam 103 has characteristics such as uniform energy distribution and clear edges.

[0077] Further explanation: The flat-top shaping component shapes the combined laser beam 100 (i.e., the Gaussian laser beam) in the horizontal direction and converts it into a flat-top profile laser beam 103. This conversion helps to ensure that when the cell passes through the detection area of the flow cytometer, the laser beam uniformly irradiates the cell in the horizontal direction, thereby improving the accuracy and stability of the measurement.

[0078] However, in the vertical direction, the flow cytometer may not require a flat-top laser beam. Because the size of the cell in the vertical direction is relatively small, and usually such uniform irradiation of the cell is not required. Therefore, the flat-top shaping device maintains the original shape of the Gaussian laser beam in the vertical direction, which helps to provide an appropriate irradiation intensity in the vertical direction while avoiding unnecessary energy waste.

[0079] Reference Figure 2 , the X-axis represents the vertical direction, the Y-axis represents the horizontal direction, and its irradiance distribution curve is relatively flat in the horizontal direction, indicating uniform intensity distribution, that is, the flat-top profile laser beam 103. Reference Figure 3 , its irradiance distribution curve shows a curve with a high center and a low edge, that is, a high-speed laser beam.

[0080] As Figure 1 shown, the central position of the flow chamber 110 is precisely placed on the focal plane of the achromatic positive lens 102. This means that the main function of the achromatic positive lens 102 is to focus the laser beam to a very small point, which is usually located on the focal plane. When the central position of the flow chamber 110 coincides with this focus, the laser beam can irradiate the cells flowing through the flow chamber in the best focused state. This precise focusing helps to improve the sensitivity and resolution of the measurement because a smaller light spot means more concentrated laser energy, which can more precisely detect the scattered light and fluorescence signals of the cells.

[0081] The flow cell 110 is one of the key components in a flow cytometer. It provides a tiny channel through which the cell suspension flows at a certain speed. The cross-section of the internal flow channel of the flow cell 110 is usually rectangular, which facilitates the irradiation of the laser beam and the detection of signals. In addition to the rectangular cross-section of the internal flow channel, the cross-section of the outer contour of the flow cell 110 is also often designed to be rectangular. This design not only facilitates processing and installation but also helps to maintain the stability and rigidity of the flow cell, reducing errors caused by vibration or external interference during the measurement process. When the laser beam irradiates the central position of the flow cell through the focusing lens, since the flow cell is located on the focal plane, the laser beam can form a very small light spot, thus achieving precise irradiation of the cells. At the same time, due to the rectangular cross-section of the internal flow channel of the flow cell, the laser beam can uniformly irradiate the cells flowing through this cross-section, further improving the accuracy and stability of the measurement.

[0082] The sample to be tested contains particles or cells. Under the action of the liquid flow system of the flow cytometer, the sample flow and the sheath flow form a laminar flow relationship. The sample flow is focused at the center of the flow cell 110 and flows along the center line of the internal flow channel of the flow cell 110. When it flows through the detection position, it is irradiated by the laser beam, generating scattered light signals and fluorescence signals. At the detection position of the flow cell 110, laser beams of different wavelengths have a certain interval in the vertical direction, generally ranging from dozens of micrometers to hundreds of micrometers. The particles or cells to be tested are successively irradiated by multiple laser beams of different wavelengths, and both scattered light signals and fluorescence signals will be generated.

[0083] The scattered light signals are divided into forward scatter light signals and side scatter light signals, and their wavelengths are equal to the laser wavelength. The energy of the forward scatter light 111 is mainly concentrated in the range of small angles in the laser transmission direction, such as within ±10 degrees. The energy of the side scatter light and fluorescence 112 is mainly concentrated in the range of about 90 degrees to the laser transmission direction. Refer to Figure 1 .

[0084] Such as Figure 1 shown, the multi-laser spectral flow cytometer optical system further includes:

[0085] A forward scatter light detection device 120, and the forward scatter light detection device 120 and the flat-top shaping component are located on opposite sides of the flow cell 110;

[0086] The flat-top profile laser beam 103 generates a forward scatter light signal after being focused by the flow cell 110, and the forward scatter light signal is collected by the forward scatter light detection device 120 and subjected to photoelectric conversion.

[0087] The forward angle light scattering detection device 120 is located on the right side of the flow chamber 110, and includes a light blocking strip 121, a third positive lens 122, a band-pass filter 123, a fourth positive lens 124, and a forward detector 125. The light blocking strip 121, the third positive lens 122, the band-pass filter 123, the fourth positive lens 124, and the forward detector 125 are arranged at intervals in sequence from left to right.

[0088] The light blocking strip 121 is used to block the background signal of the laser to avoid interfering with the forward angle light scattering signal, mainly reflected in reducing background noise, optimizing signal detection, etc. The forward angle light scattering signal is an important basis for the flow cytometer to detect cell size, shape and other characteristics. If the background noise is too high, it will cover or interfere with the forward angle light scattering signal, resulting in inaccurate detection results. The use of the light blocking strip can ensure that the forward angle light scattering signal can be clearly detected, thereby improving the accuracy and reliability of the detection.

[0089] The third positive lens 122 is used to collect the forward angle light scattering signal. The band-pass filter 123 is used to screen the wavelength of the forward angle light scattering signal, usually only allowing one laser wavelength to pass through, such as a wavelength of 488 nm. The fourth positive lens 124 is used to focus the screened forward angle light scattering signal on the photosensitive area of the forward detector 125, and the forward detector 125 is used for photoelectric conversion.

[0090] As Figure 1 、 Figures 4 to 6 shown, after the flat-top profile laser beam 103 is focused by the flow chamber 110, a lateral angle light scattering signal and a fluorescence signal are generated. The optical system of the multi-laser spectral flow cytometer further includes:

[0091] Optical fibers 151, 152, 153, the number of the optical fibers 151, 152, 153 is equal to the number of the lasers 11, 21, 31, and they correspond one by one;

[0092] An objective lens 130, which is used to guide the lateral angle light scattering and fluorescence generated by different lasers 11, 21, 31 into the corresponding optical fibers 151, 152, 153.

[0093] The lateral angle light scattering signal and the fluorescence signal are collected by the objective lens 130. Lasers of different wavelengths will generate lateral angle light scattering and fluorescence 112. The lateral angle light scattering and fluorescence collected by the objective lens are called the spectral to be measured or the light beam to be measured. The objective lens 130 guides the lateral angle light scattering and fluorescence generated by different lasers into the corresponding optical fibers. The number of the optical fibers changes with the change of the number of the lasers.

[0094] At the detection point position, laser beams of different wavelengths are designed to have a certain interval in the vertical direction. The objective lens is used to focus the optical signals from the sample onto its image plane focal plane. Since the laser beams of different wavelengths have an interval in the vertical direction, the image points formed by them on the objective lens image plane focal plane will also have corresponding intervals. This interval ensures that the lateral scattered light and fluorescence from the laser of different wavelengths are separated in physical space and have very little crosstalk with each other.

[0095] Specifically, the laser beam emitted by the laser 11 finally irradiates at the position of the first field of view 131, the laser beam emitted by the laser 21 finally irradiates at the position of the second field of view 132, and the laser beam emitted by the laser 31 finally irradiates at the position of the third field of view 133. After the first field of view 131, the second field of view 132, and the third field of view 133 are imaged by the objective lens 130, the image-side positions are at 131', 132', and 133' respectively. The optical fibers 151, 152, and 153, the positions of the end faces 141, 142, and 143 are located at 131', 132', and 133' respectively. In this way, the lateral scattered light and fluorescence generated by the laser of different wavelengths are separated and have very little crosstalk with each other.

[0096] The optical fiber is a multi-mode optical fiber. Multiple optical fibers are arranged in a "one" shape in the vertical direction to form a multi-core optical fiber connector 140, which is encapsulated in the structure for convenient installation. The other end of the optical fiber generally uses conventional connectors such as FC or SMA905, etc., and they are separated from each other, which are called single-core optical fiber connectors.

[0097] The spectrum to be measured is transmitted in the optical fiber. Each single-core optical fiber connector is connected to a wavelength division multiplexer and a multi-channel detection device. In this way, the spectrum to be measured is transmitted to the wavelength division multiplexer and the multi-channel detection device through the optical fiber.

[0098] The wavelength division multiplexer and the multi-channel detection device include a collimating lens, a dichroic filter, a plane mirror, a concave mirror (array), a band-pass filter (array), an attenuation sheet, a condenser lens, and a detector arranged in sequence along the optical path.

[0099] The illumination module 10, the forward-angle scattered light detection device 120, the objective lens 130, and the flow cell 110 can be integrated in the same mainframe.

[0100] In summary, the beam expander assembly expands the incident laser beams 12, 22, and 32 to obtain the expanded laser beams 15, 25, and 35, so as to meet the requirements of the flat-top shaping assembly for the input beam size. The expanded laser beams 15, 25, and 35 emitted from multiple sub-laser paths are combined by dichroic laser combiners 26 and 36 and a reflector 16 to obtain the combined laser beam 100. The flat-top shaping assembly changes the energy distribution of the incident combined laser beam 100, etc., to transform and obtain the flat-top profile laser beam 103. The flat-top profile laser beam 103 maintains a constant irradiance value in the cross-section, has the characteristic of uniform energy distribution compared with the existing Gaussian beam, greatly reduces the requirements for the fluctuation of the illumination beam, and also has better performance during high-speed testing, improving the accuracy, sensitivity, and reliability of detection, as well as the detection efficiency of the instrument.

[0101] The embodiments described above are only used to illustrate the technical ideas and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The patent scope of the present invention cannot be limited only by these embodiments. That is, any equivalent changes or modifications made according to the spirit disclosed by the present invention still fall within the patent scope of the present invention.

Claims

1. A multi-laser spectral flow cytometer optical system, comprising an illumination module (10), characterized in that: The lighting module (10) comprises: A plurality of sub-laser paths, wherein lasers (11, 21, 31) and beam expansion components are arranged on the paths of the sub-laser optical paths, wherein the beam expansion components are used to expand laser beams (12, 22, 32) emitted by the lasers (11, 21, 31) to obtain expanded laser beams (15, 25, 35), and the laser beams (12, 22, 32) emitted by the lasers (11, 21, 31) on each sub-laser path have different wavelengths; A beam combining optical path, wherein a dichroic laser beam combining mirror (26, 36) and a reflecting mirror (16) are arranged on the path of the beam combining optical path, wherein the dichroic laser beam combining mirror (26, 36) is located between the reflecting mirror (16) and the flat top shaping component (101), wherein the dichroic laser beam combining mirror (26, 36) and the reflecting mirror (16) each correspond to a sub-laser path respectively, and the expanded laser beams (15, 25, 35) emitted by the plurality of sub-laser paths are combined to obtain a combined laser beam (100); A flat top shaping component is provided, wherein the flat top shaping component is used to convert a combined laser beam (100) into a flat top profile laser beam (103).

2. The multi-laser spectral flow cytometer optical system according to claim 1, characterized in that: The flat top shaping assembly comprises: Diffractive optical device (101); an achromatic positive lens (102), wherein the achromatic positive lens (102) is located between the diffractive optical device (101) and the flow chamber (110); The combined laser beam (100) changes its energy distribution through the diffraction optical device (101), and is then focused by the achromatic positive lens (102) to form a flat-top profile laser beam (103).

3. The multi-laser spectral flow cytometer optical system according to claim 2, characterized in that: The reflecting mirror (16), the dichroic laser beam combining mirrors (26, 36), the diffractive optical device (101), and the achromatic positive lens (102) are kept in a straight line.

4. The multi-laser spectral flow cytometer optical system according to claim 1, characterized in that: The dichroic laser beam combining mirror (26, 36) reflects the expanded laser beam (25, 35) at an angle of 45° relative to its corresponding sub-laser path; The reflector (16) reflects the expanded laser beam (15) at an angle of 45° relative to its corresponding sub-laser path.

5. The multi-laser spectral flow cytometer optical system according to claim 1, characterized in that: The sub-laser path is perpendicular to the combined beam path.

6. The multi-laser spectral flow cytometer optical system according to claim 1, characterized in that: The dichroic laser beam combining mirror (26, 36) comprises a long-wave pass lens, and the wavelength of the laser beam emitted by the laser (11, 21, 31) decreases in a direction from the reflector (16) to the flat-top shaping component; Alternatively, the dichroic laser beam combiner (26, 36) comprises a short-wave pass lens, and the wavelength of the laser beam emitted by the laser (11, 21, 31) increases in a direction from the reflector (16) to the flat-top shaping component; Alternatively, the dichroic laser beam combiner (26, 36) includes a long-wave pass lens and a short-wave pass lens.

7. The multi-laser spectral flow cytometer optical system according to claim 1, characterized in that: The beam expansion assembly comprises a negative lens (13, 23, 33) and a positive lens (14, 24, 34), wherein the negative lens (13, 23, 33) is located between the laser (11, 21, 31) and the positive lens (14, 24, 34); Alternatively, the beam expansion assembly includes a first positive lens and a second positive lens, the focal length of the first positive lens is shorter than the focal length of the second positive lens, and the first positive lens is located between the laser (11, 21, 31) and the second positive lens.

8. The multi-laser spectral flow cytometer optical system according to claim 1, characterized in that: Also includes: A forward angular scattered light detection device (120), wherein the forward angular scattered light detection device (120) and the flat top shaping component are located on two opposite sides of the flow chamber (110); The flat-top profile laser beam (103) is focused by the flow chamber (110) to generate a forward angular scattered light signal, and the forward angular scattered light signal is collected by the forward angular scattered light detection device (120) and subjected to photoelectric conversion.

9. The multi-laser spectral flow cytometer optical system according to claim 8, characterized in that: The forward angular scattered light detection device (120) comprises a light blocking strip (121), a third positive lens (122), a bandpass filter (123), a fourth positive lens (124) and a forward detector (125).

10. The multi-laser spectral flow cytometer optical system according to claim 1, characterized in that: The flat-top profile laser beam (103) generates a side angle scattered light signal and a fluorescence signal after being focused by the flow chamber (110). The multi-laser spectral flow cytometer optical system further comprises: Optical fibers (151, 152, 153), the number of the optical fibers (151, 152, 153) and the number of the lasers (11, 21, 31) being equal and corresponding one to one; An objective lens (130) is used to guide the side angle scattered light and fluorescence generated by different lasers (11, 21, 31) into the corresponding optical fibers (151, 152, 153).

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