A near-infrared two-zone microscopic imaging system and method based on laser scanning

Abstract

The present invention belongs to the field of biological fluorescence imaging technology, and provides a near-infrared second-zone microscopic imaging system and method based on laser scanning, wherein the method comprises: deflecting and combining laser beams of different wavelengths to obtain combined light; shaping the combined light to obtain shaped light; phase modulating the shaped light to generate a multi-focal illumination light field; relaying and two-dimensionally scanning the phase-modulated shaped light, so that the multi-focal illumination light field is two-dimensionally scanned in the focal plane of the microscope objective lens, exciting the sample to emit a fluorescence signal in the near-infrared second-zone band, and using a near-infrared camera to detect the fluorescence signal and image. The optical path system of this scheme is easy to integrate, and due to the longer excitation wavelength, deeper imaging can be achieved; the selection of near-infrared second-zone dyes is more flexible.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fluorescence imaging, and in particular relates to a near-infrared two-zone microscopic imaging system and method based on laser scanning. Background Art

[0002] Bioluminescence imaging technology has been widely used as an indispensable technical means in the field of biomedicine. It is a powerful tool for observing the morphology, structure and life phenomena of biological tissues. The most commonly used bioluminescence imaging technology is the laser scanning confocal microscope developed in the 1980s. Its characteristic is that it uses pinhole technology to eliminate the interference of light signals outside the focus on the image, thereby greatly improving the clarity and detail resolution of the image, and has a high axial contrast. Since the laser range used by the laser scanning confocal microscope is between 488nm and 647nm, it belongs to the visible light category, and biological tissues have a large scattering of visible light. In other words, the penetration depth of visible light in biological samples is shallow, and the deepest does not exceed a few hundred microns. It is difficult to collect information from thick specimens; in addition, because biological tissues have a large absorption of visible light, high-density visible light is more likely to cause phototoxicity and photobleaching when exciting biological samples.

[0003] In order to overcome these defects of laser scanning confocal microscope, Denk et al. of Cornell University in the United States proposed two-photon excitation fluorescence microscopy in the 1990s. It uses near-infrared lasers with high photon density to excite biological samples. Since biological tissues absorb less near-infrared light, phototoxicity to biological tissues is reduced and photobleaching is reduced; at the same time, biological tissues scatter less near-infrared light than visible light, which can easily penetrate deeper biological samples and is more suitable for observing thick samples. However, although two-photon laser fluorescence imaging technology uses near-infrared light sources and can achieve the observation of thick biological samples, the emission wavelength of the fluorescent dye used is still in the visible light range, and it still has absorption and scattering problems in biological tissues, making it difficult to observe deeper tissues.

[0004] In summary, existing bioluminescence imaging has at least the following problems: the near-infrared zone II microscopic imaging system has a complex system optical path and unsatisfactory imaging depth and imaging resolution; the two-dimensional scanning galvanometer has redundant movements during scanning, low scanning efficiency, and affects the system's temporal resolution; the near-infrared zone II microscopic imaging system has a single wavelength and low compatibility. Summary of the invention

[0005] The object of the present invention is to provide a near-infrared two-zone microscopic imaging system and method based on laser scanning, which can solve at least one of the technical problems of biological fluorescence imaging in the prior art.

[0006] The present invention provides a near-infrared two-zone microscopic imaging system based on laser scanning, comprising:

[0007] Multi-wavelength laser module, beam shaping module, multi-focus structured light generation module, beam relay and scanning module, imaging and detection module;

[0008] The multi-wavelength laser module is used to output laser beams of different wavelengths, and after beam deflection and beam combination, a combined beam is obtained;

[0009] The beam shaping module is used to shape the combined beam to obtain a shaped beam;

[0010] The multi-focus structured light generation module is used to perform phase modulation on the shaped beam, so as to finally generate a multi-focus illumination light field in the focal plane of the microscope objective in the imaging and detection module. The shaped beam after phase modulation returns to the beam shaping module, and after being reflected by the beam shaping module, it enters the beam relay and scanning module again;

[0011] The beam relay and scanning module is used to relay and two-dimensionally scan the beam, so that the multi-focus illumination light field is two-dimensionally scanned in the focal plane of the microscope objective;

[0012] The imaging and detection module is used to receive the incident light from the beam relay and scanning module, excite the sample to emit fluorescence signals in the second near-infrared band, and detect the fluorescence signals and image.

[0013] Preferably, the multi-wavelength laser module includes:

[0014] N laser groups, where N is an integer and N≥2:

[0015] Among them, the first laser group includes a first laser, a first band-pass filter, a first mirror, and a first dichroic mirror. After the laser emitted by the first laser passes through the first band-pass filter, it is reflected by the first mirror to the transmission surface of the first dichroic mirror for transmission to obtain a first-wavelength beam;

[0016] Each of the second to the (N - 1)th laser groups includes an nth laser, an nth band-pass filter, and an nth dichroic mirror. After the laser emitted by the nth laser passes through the nth band-pass filter, it is incident on the reflection surface of the (n - 1)th dichroic mirror for reflection to obtain an nth-wavelength beam, where n is an integer and 1 < n < N; the first-wavelength beam and the second-wavelength beam converge and are jointly incident on the reflection surface of the second dichroic mirror for reflection; and so on, the (N - 2)th-wavelength beam and the (N - 1)th-wavelength beam converge and are jointly incident on the reflection surface of the (N - 1)th dichroic mirror for reflection;

[0017] The Nth laser group includes an Nth laser and an Nth bandpass filter. The laser emitted by the Nth laser passes through the Nth bandpass filter and is incident on the transmission surface of the N-1th dichroic mirror for transmission, and then merges with the first wavelength light beam, the second wavelength light beam, ..., and the N-1th wavelength light beam and is incident on the reflection surface of the N-1th dichroic mirror for reflection to obtain the combined light beam.

[0018] Preferably, the beam shaping module comprises:

[0019] A laser beam expander, used for collimating and expanding the combined light beam;

[0020] A half-wave plate, used for adjusting the polarization direction of the combined light beam after beam expansion;

[0021] A broadband polarization beam splitter, used for separating the combined light passing through the half-wave plate into transmitted light with a specific polarization direction;

[0022] The non-polarization beam splitter is used to change the direction of the light beam from the multi-focus structured light generating module so that the light beam enters the light beam relay and scanning module.

[0023] Preferably, the multi-focal structured light generating module comprises: a spatial light modulator, configured to modulate the spatial phase distribution of the shaped light to generate the multi-focal illumination light field.

[0024] Preferably, the beam relay and scanning module comprises:

[0025] A first direction scanning unit, used for scanning the light beam in the first direction;

[0026] A second direction scanning unit, used for scanning the light beam in the second direction, the first direction and the second direction being orthogonal to each other;

[0027] A plurality of relay lenses are used for guiding and relaying the light beams.

[0028] Preferably, at least two relay lenses are arranged between the beam shaping module and the first direction scanning unit, at least two relay lenses are arranged between the first direction scanning unit and the second direction scanning unit, and at least two relay lenses are arranged between the second direction scanning unit and the imaging and detection module.

[0029] Preferably, the imaging and detection module further includes: a spatial filter; two relay lenses are arranged between the beam shaping module and the first direction scanning unit, and the spatial filter is arranged between the two relay lenses to filter out zero-order diffraction light.

[0030] Preferably, the beam relay and scanning module adopts a multi-focal array parallel scanning mode, and scans in each focal array in a "U" shape until the scanning of the focal array area is completed.

[0031] Preferably, the imaging and detection module comprises:

[0032] an excitation and detection unit, including the microscope objective lens, for focusing the incident light beam from the beam relay and scanning module to excite the sample to generate the fluorescence signal and detect the fluorescence signal;

[0033] The imaging unit comprises a lens group and a near-infrared camera. The fluorescence signal is imaged through the lens group, and the near-infrared camera records the fluorescence imaging of the sample.

[0034] The present invention also provides a near-infrared second-zone microscopic imaging method based on laser scanning, comprising the following steps:

[0035] S1, deflecting and combining laser beams of different wavelengths to obtain combined beam light;

[0036] S2, shaping the combined light beam to obtain shaped light;

[0037] S3, phase modulating the shaped light to ultimately generate a multi-focal illumination light field at a focal plane of a microscope objective;

[0038] S4, relaying and two-dimensionally scanning the phase-modulated shaped light, so that the multi-focus illumination light field is two-dimensionally scanned on the focal plane of the microscope objective lens;

[0039] S5, exciting the sample to emit a fluorescence signal in the near-infrared second band, and detecting the fluorescence signal and forming an image.

[0040] According to an embodiment of the present invention, a near-infrared second-zone microscopic imaging system and method based on laser scanning can achieve a deeper high-resolution imaging effect due to the longer excitation wavelength and deeper imaging penetration; by adopting a multi-wavelength laser module and its matching optical path, multi-wavelength output is achieved, so that it can be compatible with a variety of near-infrared second-zone fluorescent dyes, improve the compatibility and flexibility of the imaging system with fluorescent dyes, and meet the needs of different imaging scenarios; a multi-focus illumination light field is generated by a multi-focus structured light generation module, and the image resolution can be improved after image reconstruction; at the same time, in the two-dimensional scanning process of the light beam, a multi-focus array is used for parallel scanning, and each focus array is scanned in a "return" shape, so that the scanning efficiency and rate are greatly improved, and the time resolution of the system is significantly improved; compared with the existing laser confocal imaging technology, the microscopic imaging system of the embodiment of the present invention is more integrated and flexible, and the operation and control are more convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a functional principle diagram of a near-infrared two-zone microscopic imaging system based on laser scanning according to an embodiment of the present invention;

[0042] Figure 2 It is a schematic diagram of the scanning path of a single focus in a multi-focus array in a beam relay and scanning module of a near-infrared two-zone microscopic imaging system based on laser scanning according to an embodiment of the present invention.

[0043] Description of reference numerals:

[0044] 1-1550nm laser, 2-1300nm laser, 3-980nm laser, 4-first bandpass filter, 5-second bandpass filter, 6-third bandpass filter, 7-first reflector, 8-first dichroic mirror, 9-second dichroic mirror, 10-laser beam expander, 11-half-wave plate, 12-broadband polarization beam splitter cube, 13-non-polarization beam splitter cube, 14-spatial light modulator, 15-first relay lens, 1 6-spatial filter, 17-second reflecting mirror, 18-second relay lens, 19-X-axis scanning unit, 20-third relay lens, 21-fourth relay lens, 22-Y-axis scanning unit, 23-fifth relay lens, 24-sixth relay lens, 25-third dichroic mirror, 26-piezoelectric objective lens positioner, 27-microscope objective lens, 28-three-dimensional high-precision stage, 29-filter, 30-tube lens, 31-near-infrared camera. DETAILED DESCRIPTION

[0045] The specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0046] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0047] like Figure 1 As shown, an embodiment of the present invention provides a near-infrared two-zone microscopic imaging system based on laser scanning, including: a multi-wavelength laser module, a beam shaping module, a multi-focus structured light generating module, a beam relay and scanning module, and an imaging and detection module.

[0048] Among them, the multi-wavelength laser module is used to output laser beams of different wavelengths, and obtain combined light after beam deflection and beam combination; the beam shaping module is used to shape the combined light to obtain shaped light; the multi-focus structured light generation module is used to phase modulate the shaped light, so as to finally generate a multi-focus illumination light field in the focal plane of the microscope objective in the imaging and detection module, and the shaped light after phase modulation returns to the beam shaping module, and enters the beam relay and scanning module after reflection from the beam shaping module; the beam relay and scanning module is used to relay and two-dimensionally scan the light beam, so that the multi-focus illumination light field is two-dimensionally scanned in the focal plane of the microscope objective, so as to realize the fluorescence excitation of the multi-focus illumination light field at various positions on the sample surface; the imaging and detection module is used to receive the incident light from the beam relay and scanning module, excite the sample to emit a fluorescence signal in the near-infrared second band, and detect the fluorescence signal and image.

[0049] The following is a detailed description of the modules:

[0050] (1) Multi-wavelength laser module: used to output multi-wavelength laser beams to excite fluorescent dyes to emit fluorescence. In this embodiment, a laser module that outputs three different wavelengths of 1550nm, 1300nm, and 1550nm is used as an example for description, and the specific direction of the optical path is:

[0051] After the laser light emitted by the 1550 nm laser 1 passes through the first bandpass filter 4 , it is reflected by the first reflector 7 to the rear surface (ie, the transmission surface) of the first dichroic mirror 8 for transmission to obtain a light beam of the first wavelength.

[0052] The laser light emitted by the 1300 nm laser 2 passes through the second bandpass filter 5 and is incident on the front surface (reflection surface) of the first dichroic mirror 8 to obtain a light beam of a second wavelength.

[0053] After the laser light emitted by the 1550nm laser 1 is transmitted through the rear surface of the first dichroic mirror 8, it merges with the light beam emitted by the 1300nm laser 2 reflected by the first dichroic mirror 8 and they are incident on the rear surface (reflection surface) of the second dichroic mirror 9 together and reflected; that is, the first wavelength light beam and the second wavelength light beam merge and are incident on the rear surface (reflection surface) of the second dichroic mirror 9 together and are reflected.

[0054] The laser light emitted by the 980nm laser 3 is incident on the front surface (transmission surface) of the second dichroic mirror 9 through the third bandpass filter 6 and transmitted, and then merges with the light beams of the 1500nm laser and the 1300nm laser reflected by the rear surface (reflection surface) of the second dichroic mirror 9 to obtain a combined light beam of three wavelengths; then, the combined light beam enters the beam shaping module.

[0055] In this module, the functions of the main components are as follows:

[0056] 1550nm laser 1, 1300nm laser 2, 980nm laser 3: respectively used to output high-power single-mode lasers of corresponding wavelengths; multi-wavelength output, so as to be compatible with a variety of near-infrared second-zone fluorescent dyes in the sample, improve the compatibility of the system, and meet the needs of different imaging scenarios.

[0057] The first bandpass filter 4, the second bandpass filter 5, and the third bandpass filter 6 are all narrow bandpass filters used to improve the monochromaticity of the incident laser.

[0058] The first dichroic mirror 8 is a long-pass filter used for light beam deflection and light beam combination.

[0059] The second dichroic mirror 9 is a short-pass filter used for light beam deflection and light beam combination.

[0060] (2) Beam shaping module: used to shape the combined beam of multi-wavelength output to ensure the best phase modulation effect. During this period, the direction of the beam is specifically as follows: the combined beam passes through the laser beam expander 10 to improve its uniformity and parallelism, and then passes through the half-wave plate 11, the broadband polarization beam splitter 12 (in this embodiment, the broadband polarization beam splitter cube 12) and the non-polarization beam splitter 13 (in this embodiment, the 50:50 non-polarization beam splitter cube 13), and finally obtains the shaped light, which is incident on the multi-focus structured light generation module.

[0061] The functions of the main components in this module are as follows:

[0062] Laser beam expander 10: realizes collimation and beam expansion of incident laser. Half-wave plate 11: realizes adjustment of the polarization direction of incident laser. Broadband polarization beam splitter cube 12: separates linear polarized light (transmitted light) with horizontal polarization direction. 50:50 non-polarization beam splitter cube 13: is used to receive the light beam returned from the multi-focus structured light generation module and change its direction so that the light beam enters the beam relay and scanning module.

[0063] (3) Multi-focus structured light generation module: By phase modulating the shaped light, a preset multi-focus illumination light field is finally generated at the focal plane of the microscope objective 27. In this embodiment, the core component of the multi-focus structured light generation module is the spatial light modulator 14. The shaped light of the beam shaping module is incident on the spatial light modulator 14. By loading the pre-designed phase pattern file into the spatial light modulator 14, the spatial phase distribution of the shaped light is modulated and reflected. The reflected light returns to the 50:50 non-polarization beam splitter cube 13 along the original path and enters the beam relay and scanning module after being reflected by it.

[0064] In this module, the main components and their functions are as follows:

[0065] Spatial light modulator 14: specifically, it can be a reflective phase spatial light modulator, which is used to realize phase modulation of incident light and generate a multi-focal illumination light field, and the image resolution can be improved after image reconstruction.

[0066] (4) Beam relay and scanning module: realizes the relay and two-dimensional scanning of the light beam, so that the multi-focal illumination light field is two-dimensionally scanned in the focal plane of the microscope objective 27. The specific direction of the light path is as follows: the reflected light (i.e., the multi-focal illumination light field) of the 50:50 non-polarized beam splitter cube 13 passes through the first relay lens 15 and the spatial filter 16, is reflected by the second reflector 17, passes through the second relay lens 18, and is incident on the X-axis scanning unit 19; the reflected light of the X-axis scanning unit 19 passes through the third relay lens 20 and the fourth relay lens 21 in turn, and is incident on the Y-axis scanning unit 22; the reflected light of the Y-axis scanning unit 22 passes through the fifth relay lens 23 and the sixth relay lens 24 in turn, and enters the imaging and detection module. In this embodiment, the X-axis scanning unit 19 and the Y-axis scanning unit 22 are scanning galvanometers, respectively. Optionally, the X-axis scanning unit 19 and the Y-axis scanning unit 22 can also be acousto-optic deflectors.

[0067] In this module, the main components and their functions are as follows:

[0068] The first relay lens 15, the second relay lens 18, the third relay lens 20, the fourth relay lens 21, the fifth relay lens 23 and the sixth relay lens 24 are used for guiding and relaying the light beam. Since the common light beam relay uses the 4f system, a single 4f system needs to have two lenses; multiple 4f systems can also be combined, so the number of relay lenses in each group is an even number.

[0069] Spatial filter 16: After special design and processing, it filters out the zero-order in the diffraction beam and improves the quality of the final multi-focus structured light illumination light field.

[0070] X-axis scanning unit 19: realizes scanning of the incident light beam in the X direction.

[0071] Y-axis scanning unit 22: realizes scanning of the incident light beam in the Y direction, and the X-axis and the Y-axis are orthogonal to each other.

[0072] It should be noted that the X-axis scanning unit 19 and the Y-axis scanning unit 22 adopt a multi-focal array parallel scanning mode during the scanning process, that is, through voltage control, each focal array is scanned in a circuitous manner in a "U" shape until the scanning of the focal array area is completed. Specifically, the scanning of a single focal array in the multi-focal array is as follows: Figure 2As shown, the arrow direction indicates the path during the scanning process; since the multi-focal array is scanned in parallel as a whole, the scanning method of each single-focal array area in the dot matrix is ​​consistent, and each single-focal array area is scanned simultaneously. During the scanning process, the first scanning unit and the second scanning unit will perform continuous and effective alternating scanning, there is no redundant action, the scanning rate will be greatly improved, and the time resolution of the system will be greatly improved.

[0073] (5) Imaging and detection module: to realize the excitation and detection of fluorescence signals; wherein, the specific direction of the optical path is as follows: the outgoing light of the sixth relay lens 24 is incident on the third dichroic mirror 25 and then reflected; the reflected light passes through the piezoelectric objective lens positioner 26 and evenly fills the entrance pupil of the microscope objective lens 27; then, it enters the microscope objective lens 27 from the entrance pupil and is focused on the sample surface by it, thereby exciting the fluorescent probe marked inside the biological sample; meanwhile, the biological sample is placed on a three-dimensional high-precision translation stage 28; finally, the fluorescence signal excited in the biological sample returns to the original path through the microscope objective lens 27 and the piezoelectric objective lens positioner 26 and passes through the third dichroic mirror 25; then, the excitation light is further filtered out by the filter 29; finally, the fluorescence signal is imaged on the detection chip of the near-infrared camera 31 by the lens group 30 (in this embodiment, the sleeve lens 30), and the near-infrared camera 31 records the fluorescence image of the sample. As a planar array detector composed of multiple pixels, the near-infrared camera has spatial resolution capabilities that single-point detectors do not have. It also has the characteristics of high sensitivity and high signal-to-noise ratio. It has a high quantum efficiency in the near-infrared second band and can achieve high-quality detection of fluorescence signals.

[0074] In this module, the main components and their functions are as follows:

[0075] The third dichroic mirror 25 reflects the incident light and filters out a portion of the excitation light in the transmitted light;

[0076] Piezoelectric objective lens positioner 26: realizes scanning of the objective lens in the Z direction with high precision;

[0077] Microscope objective 27: used to focus the incident light beam to excite the fluorescent signal in the sample;

[0078] Three-dimensional high-precision translation stage 28: realizes precise movement of samples in three dimensions of XYZ;

[0079] Filter 29: further filters out the excitation light in the fluorescence signal to ensure the fluorescence image effect of the sample;

[0080] Tube lens 30: used for imaging;

[0081] Near infrared camera 31: used to detect fluorescence signals in the near infrared II region (1000-1700nm).

[0082] According to an embodiment of the present invention, a near-infrared second-zone microscopic imaging system based on laser scanning can achieve a deeper high-resolution imaging effect due to its longer excitation wavelength and deeper imaging penetration; by adopting a multi-wavelength laser module and its matching optical path, multi-wavelength output is achieved, so that it can be compatible with a variety of near-infrared second-zone fluorescent dyes, improve the compatibility and flexibility of the imaging system with fluorescent dyes, and meet the needs of different imaging scenarios; a multi-focus illumination light field is generated by a multi-focus structured light generation module to improve the efficiency of light energy utilization, and the image resolution can be improved after image reconstruction; at the same time, in the two-dimensional scanning of the light beam, multi-focus arrays are used for parallel scanning, and each focus array is scanned in a "return" shape, so that the scanning efficiency and rate are greatly improved, and the time resolution of the system is significantly improved; compared with the existing laser confocal imaging technology, the microscopic imaging system of the embodiment of the present invention is more integrated and flexible, and the operation and control are more convenient.

[0083] The embodiment of the present invention further provides a near-infrared second-zone microscopic imaging method based on laser scanning, the method is used in a near-infrared second-zone microscopic imaging system based on laser scanning, and comprises the following steps:

[0084] S1, deflecting and combining laser beams of different wavelengths to obtain combined beam light;

[0085] S2, shaping the combined light to obtain shaped light;

[0086] S3, phase modulating the shaped light to generate a multi-focal illumination light field;

[0087] S4, relaying and two-dimensionally scanning the phase-modulated shaped light, so that the multi-focus illumination light field is two-dimensionally scanned in the focal plane of the microscope objective;

[0088] S5, excites the sample to emit a fluorescence signal in the near-infrared second band, and detects the fluorescence signal and forms an image.

[0089] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A near-infrared two-zone microscopic imaging system based on laser scanning, characterized in that: include: Multi-wavelength laser module, beam shaping module, multi-focus structured light generation module, beam relay and scanning module, imaging and detection module; The multi-wavelength laser module includes a plurality of laser groups, each of which outputs laser beams of different wavelengths, and obtains combined beam light after beam deflection and beam combination; The beam shaping module is used to shape the combined light to obtain shaped light; The multi-focus structured light generation module includes a spatial light modulator, which is used to modulate the spatial phase distribution of the shaped light, so as to finally generate a multi-focus illumination light field at the focal plane of the microscope objective in the imaging and detection module, and the shaped light after phase modulation returns to the beam shaping module, and then enters the beam relay and scanning module after being reflected by the beam shaping module; The light beam relay and scanning module is used to relay and two-dimensionally scan the light beam, so that the multi-focus illumination light field is two-dimensionally scanned in the focal plane of the microscope objective lens; The imaging and detection module is used to receive incident light from the beam relay and scanning module through the microscope objective lens, stimulate the sample to emit a fluorescence signal in the near-infrared second band, and detect the fluorescence signal to form an image.

2. The near-infrared two-zone microscopic imaging system based on laser scanning according to claim 1, characterized in that: The multi-wavelength laser module includes three laser groups: The first laser group includes a 1550nm laser, a first bandpass filter, a first reflector and a first dichroic mirror. After the laser emitted by the 1550nm laser passes through the first bandpass filter, it is reflected by the first reflector to the transmission surface of the first dichroic mirror for transmission to obtain a first wavelength light beam. The second laser group includes a 1300nm laser, a second bandpass filter and a second dichroic mirror, wherein the laser light emitted by the 1300nm laser passes through the second bandpass filter and is incident on the reflection surface of the first dichroic mirror to obtain a light beam of a second wavelength; The first wavelength light beam and the second wavelength light beam merge and are incident on the reflection surface of the second dichroic mirror to be reflected; The third laser group includes a 980nm laser and a third bandpass filter. The laser light emitted by the 980nm laser is incident on the transmission surface of the second dichroic mirror through the third bandpass filter, and then merges with the first wavelength light beam and the second wavelength light beam reflected by the reflection surface of the second dichroic mirror to obtain the combined light of three wavelengths.

3. The near-infrared two-zone microscopic imaging system based on laser scanning according to claim 1, characterized in that: The beam shaping module comprises: A laser beam expander, used for collimating and expanding the combined light beam; A half-wave plate, used for adjusting the polarization direction of the combined light beam after beam expansion; A broadband polarization beam splitter, used for separating the combined light passing through the half-wave plate into transmitted light with a specific polarization direction; The non-polarization beam splitter is used to change the direction of the light beam from the multi-focus structured light generating module so that the light beam enters the light beam relay and scanning module.

4. The near-infrared two-zone microscopic imaging system based on laser scanning according to claim 1, characterized in that: The beam relay and scanning module comprises: A first direction scanning unit, used for scanning the light beam in the first direction; A second direction scanning unit, used for scanning the light beam in the second direction, the first direction and the second direction being orthogonal to each other; A plurality of relay lenses are used for guiding and relaying the light beams.

5. The near-infrared two-zone microscopic imaging system based on laser scanning according to claim 4, characterized in that: At least two relay lenses are arranged between the beam shaping module and the first direction scanning unit, at least two relay lenses are arranged between the first direction scanning unit and the second direction scanning unit, and at least two relay lenses are arranged between the second direction scanning unit and the imaging and detection module.

6. The near-infrared two-zone microscopic imaging system based on laser scanning according to claim 5, characterized in that: The beam relay and scanning module further includes: a spatial filter; Two relay lenses are arranged between the beam shaping module and the first direction scanning unit, and the spatial filter is arranged between the two relay lenses to filter out zero-order diffraction light.

7. The near-infrared two-zone microscopic imaging system based on laser scanning according to claim 4, characterized in that: The beam relay and scanning module adopts a multi-focal array parallel scanning mode, scanning in each focal array in a "U" shape until the scanning of the focal array area is completed.

8. The near-infrared two-zone microscopic imaging system based on laser scanning according to claim 1, characterized in that: The imaging and detection module comprises: an excitation and detection unit, including the microscope objective lens, for focusing the incident light beam from the beam relay and scanning module to excite the sample to generate the fluorescence signal and detect the fluorescence signal; The imaging unit comprises a lens group and a near-infrared camera. The fluorescence signal is imaged through the lens group, and the near-infrared camera records the fluorescence imaging of the sample.

9. A near-infrared two-zone microscopic imaging method based on laser scanning, characterized in that: The method is used on the near-infrared two-zone microscopic imaging system based on laser scanning as described in any one of claims 1 to 8, comprising the following steps: S1, deflecting and combining laser beams of different wavelengths to obtain combined beam light; S2, shaping the combined light beam to obtain shaped light; S3, phase modulating the shaped light to ultimately generate a multi-focal illumination light field at a focal plane of a microscope objective; S4, relaying and two-dimensionally scanning the phase-modulated shaped light, so that the multi-focus illumination light field is two-dimensionally scanned on the focal plane of the microscope objective lens; S5, exciting the sample to emit a fluorescence signal in the near-infrared second band, and detecting the fluorescence signal and forming an image.

Images