An upconversion structured light illumination three-dimensional super-resolution imaging system and method

Abstract

The application discloses an up-conversion structured light illumination three-dimensional super-resolution imaging system and method, and belongs to the field of microscopic imaging. Light emitted by a laser light source is transmitted into a Fourier lens group to become diffraction light spots of various orders after phase control by a reflective spatial light modulator, and light of diffraction orders of +1 and +2 is filtered out by a light filtering pinhole. The light of diffraction orders of +1 and +2 is irradiated to a sample on an objective table by a microscope objective, and the sample generates a fluorescence band after being excited. The fluorescence is focused to the target surface of a first camera to form an image after being focused by a focusing assembly after passing through the microscope objective. A second super-lens modulator performs axial zoom layer scanning on the fluorescence, and a second camera performs focal plane imaging to lock the best focal plane to adjust the focal length of the first camera. The focal plane searching is more stable, and the introduced interference factors are less. Three-dimensional structured illumination light is formed by interference of the diffraction light of +1 and +2 orders, and three-dimensional super-resolution imaging with a lateral resolution of 100 nm and an axial resolution of 300 nm is realized.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging, and in particular to an upconversion structured light illumination three-dimensional super-resolution imaging system and method. Background Technology

[0002] Structured light illumination fluorescence microscopy is a super-resolution microscopy technique widely used in the biomedical field. With increasing focus on fine three-dimensional structures and rapid dynamic activities in life science research, achieving faster three-dimensional super-resolution imaging of live cells has become a key research focus in structured light illumination fluorescence microscopy and the entire field of super-resolution imaging.

[0003] Currently, structured illumination microscopy primarily achieves 3D imaging through axial layer-by-layer scanning, with the axial scanning speed determining the system's 3D imaging speed. There are two main methods for achieving axial layer-by-layer scanning: the first uses mechanical components to move a 3D nanoscale displacement stage, moving the sample and placing different layers on the objective lens's focal plane; the second involves adding a variable-focus lens to the imaging optical path, typically using a mechanical variable-focus lens group or a liquid lens group. These 3D imaging methods have the following drawbacks: 1) Axial mechanical scanning introduces jitter errors, leading to imaging artifacts, and the scanning direction cannot be guaranteed to be strictly parallel to the optical axis; 2) Long response time and slow scanning speed make them unsuitable for observing rapid biological activities; 3) Liquid variable-focus lens groups introduce significant aberrations; 4) Axial movement of the sample stage or microscope objective can cause vibration and backlash, resulting in focus drift during long-term imaging.

[0004] Meanwhile, structured illumination microscopy often uses visible light sources. However, visible light suffers from severe scattering and shallow penetration in biological tissue samples, limiting the depth of three-dimensional imaging. To improve the depth of three-dimensional imaging and the quality of super-resolution imaging, methods often involve increasing the intensity of the illumination light. This, in turn, leads to problems such as phototoxicity and photobleaching. Some researchers have proposed directly increasing the excitation wavelength, which can increase the depth of illumination, but traditional excitation modes result in longer wavelengths of emitted light, directly causing a decrease in resolution.

[0005] Therefore, given the existing problems with structured illumination micro-imaging technology, there is an urgent need for a fast three-dimensional super-resolution imaging device and method. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide an upconversion structured light illumination three-dimensional super-resolution imaging system that can effectively improve the speed of three-dimensional super-resolution imaging and extend the illumination and three-dimensional imaging depth.

[0007] In order to overcome the shortcomings of the prior art, the second objective of this invention is to provide an upconversion structured light illumination three-dimensional super-resolution imaging method that can effectively improve the speed of three-dimensional super-resolution imaging and extend the illumination and three-dimensional imaging depth.

[0008] One of the objectives of this invention is achieved through the following technical solution:

[0009] The upconversion structured light illumination 3D super-resolution imaging system includes a structured light illumination module, a microscopic imaging module, and an autofocus module.

[0010] The structured light illumination module includes a laser source, a reflective spatial light modulator, a Fourier lens group, and a filter aperture. The light emitted by the laser source is phase-modulated by the reflective spatial light modulator and then enters the Fourier lens group to become diffraction spots of various orders before exiting. The filter aperture filters out the ±1st and ±2nd order diffraction light.

[0011] The microscopic imaging module includes a stage, a microscope objective, a focusing assembly, and a first camera. The ±1 and ±2 order diffraction light is irradiated onto the sample on the stage by the microscope objective. After the sample is excited, it generates a fluorescence band. The fluorescence passes through the microscope objective and is focused by the focusing assembly onto the target surface of the first camera for imaging.

[0012] The autofocus module includes a second superlens modulator and a second camera. The second superlens modulator performs axial zoom layer scanning of the fluorescence, and the second camera performs focal plane imaging to lock the optimal focal plane to adjust the focal length of the first camera.

[0013] Furthermore, the structured light illumination module also includes a rotating half-wave plate, which is located between the laser source and the reflective spatial light modulator. The rotating half-wave plate reduces the light intensity of the 0th order diffraction spot of the reflective spatial light modulator to the weakest level and increases the light intensity of the ±1st and ±2nd order diffraction spots.

[0014] Furthermore, the structured light illumination module also includes a polarizing beam splitter, which is located between the laser source and the reflective spatial light modulator. The light emitted by the laser source passes through the polarizing beam splitter and enters the reflective spatial light modulator. The light modulated by the reflective spatial light modulator is reflected by the polarizing beam splitter and then enters the Fourier lens group.

[0015] Furthermore, the structured light illumination module also includes a segmented polarization modulation half-wave plate, which is located between the Fourier lens group and the filter aperture. The segmented polarization modulation half-wave plate modulates the diffraction spots of each order into having different polarization directions.

[0016] Furthermore, the segmented polarization modulation half-wave plate includes multiple sector waveplates with different fast axis directions.

[0017] Furthermore, the microscopic imaging module also includes a first dichroic mirror, which is located between the microscope objective and the focusing assembly and faces the filter aperture. The light filtered out by the filter aperture is reflected by the first dichroic mirror to the microscope objective, and the fluorescence of the microscope objective is irradiated to the focusing assembly after passing through the first dichroic mirror.

[0018] Furthermore, the microscopic imaging module also includes a second dichroic mirror, which is located between the microscope objective and the focusing assembly and faces the autofocus module. The fluorescence passing through the microscope objective is partially reflected by the second dichroic mirror to the second superlens modulator.

[0019] Furthermore, the microscopic imaging module also includes a multi-channel filter, which is located between the microscope objective and the focusing assembly, and filters out excitation light in the near-infrared band.

[0020] Furthermore, the focusing assembly includes a second lens group and a first superlens modulator, wherein the second lens group is a 4f system and the first superlens modulator is located on the conjugate surface of the back focal plane of the microscope objective.

[0021] Furthermore, the first superlens modulator has a single-layer or multi-layer surface of a micro-nano structure array, which can modulate the optical wavefront at subwavelength spatial resolution and is configured to modulate the incident light to achieve control over the focal length range.

[0022] The second objective of this invention is achieved by the following technical solution:

[0023] A three-dimensional super-resolution imaging method with upconversion structured light illumination, implemented based on any of the above-mentioned three-dimensional super-resolution imaging systems with upconversion structured light illumination, includes the following steps:

[0024] Sample pretreatment: Upconversion nanoparticles were used as fluorescent staining materials to perform upconversion fluorescent staining on the samples;

[0025] Forming illumination light: Near-infrared laser light source is used as illumination light source, dual-period spatial light modulator stripes are used, and the ±1st and ±2nd order diffraction lights are used to form three-dimensional structured illumination light through interference;

[0026] Excitation fluorescence: Illumination light shines on the sample and emits fluorescence in the visible light band;

[0027] Autofocus: Using a near-infrared light source for illumination, a three-dimensional zoom layer scan is performed using a second superlens modulator to lock the optimal focal plane position of the first camera and achieve focal length adjustment of the first camera;

[0028] Automatic focusing: Modulates the incident fluorescence to control the focal length range of the first camera;

[0029] Imaging: The fluorescence is focused onto the target surface of the first camera after focusing;

[0030] 3D reconstruction: Fluorescence images of samples illuminated by structured light of different phases and directions are acquired, and all acquired raw images are post-processed to reconstruct the super-resolution 3D structure.

[0031] Compared with existing technologies, the upconversion structured light illumination three-dimensional super-resolution imaging system of the present invention has the following advantages:

[0032] (1) Near-infrared laser light source is used as illumination source. Near-infrared illumination light has a greater penetration depth of biological tissue, lower illumination power, and less phototoxicity, making it more suitable for long-term three-dimensional super-resolution imaging of living cells.

[0033] (2) By using dual-period spatial light modulator stripes and interfering with ±1 and ±2 order diffraction lights to form three-dimensional structure illumination light, three-dimensional super-resolution imaging with a lateral resolution of about 100 nm and an axial resolution of about 300 nm can be achieved.

[0034] (3) Using a method based on superlens modulation for three-dimensional zoom layer scanning, compared with the traditional structured light illumination super-resolution system, the present invention has the advantages of fast three-dimensional layer scanning speed, high system stability, compact structure and fewer three-dimensional imaging artifacts, which is in line with the trend of miniaturization and lightweighting of modern optical systems.

[0035] (4) The autofocus module shares the near-infrared light source of the structured light illumination module, without the need to introduce an additional focal plane detection light source. The autofocus module has a more compact structure and a simpler optical path. Compared with the mechanical structure focusing method, the focal plane finding method based on superlens modulation is more stable and introduces fewer interference factors. Attached Figure Description

[0036] Figure 1 This is the optical path diagram of the upconversion structured light illumination three-dimensional super-resolution imaging system of the present invention;

[0037] Figure 2 A dual-period fringe pattern for a spatial light modulator;

[0038] Figure 3 This is a cross-sectional view of the two-periodic stripes of a spatial light modulator after Fourier transform.

[0039] Figure 4 A schematic diagram of the spectral extension of the 0th order diffracted light;

[0040] Figure 5 A schematic diagram of the spectral extension of ±1st order diffracted light;

[0041] Figure 6 A schematic diagram of the spectral extension of ±2nd order diffracted light;

[0042] Figure 7 A schematic diagram of the spectral extension of ±3rd order diffracted light;

[0043] Figure 8 A schematic diagram of the spectral extension of ±4th order diffracted light;

[0044] Figure 9 The spectrum of the final super-resolution result obtained by stitching together all spectral components.

[0045] In the diagram: 10. Structured light illumination module; 11. Laser source; 12. Collimating beam expander; 13. Polarizing beam splitter; 14. Rotating half-wave plate; 15. Reflective spatial light modulator; 16. Fourier lens group; 17. Segmented polarization modulation half-wave plate; 18. Filter aperture; 19. First lens group; 20. Microscopic imaging module; 21. Stage; 22. Microscopic objective; 23. First dichroic mirror; 24. Second dichroic mirror; 25. Multi-channel filter; 26. Focusing assembly; 261. Second lens group; 262. First superlens modulator; 27. Tube mirror; 28. First camera; 30. Autofocus module; 31. Single-channel filter; 32. Second superlens modulator; 33. Second tube mirror; 34. Second camera. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or it can be fixed through another intermediate component. When a component is said to be "connected to" another component, it can be directly connected to the other component or it may be fixed through another intermediate component. When a component is said to be "set on" another component, it can be set directly on the other component or it may be set through another intermediate component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] like Figure 1 As shown, the upconversion structured light illumination three-dimensional super-resolution imaging system of the present invention includes a structured light illumination module 10, a microscopic imaging module 20, and an autofocus module 30.

[0050] The structured light illumination module 10 includes a laser source 11, a collimating beam expander 12, a polarizing beam splitter 13, a rotating half-wave plate 14, a reflective spatial light modulator 15, a Fourier lens group 16, a segmented polarization modulation half-wave plate 17, a filter aperture 18, and a first lens group 19.

[0051] The laser source 11 is a near-infrared laser, specifically a 975nm laser. Near-infrared illumination light has a greater penetration depth into biological tissues, lower illumination power, and less phototoxicity. Therefore, the upconversion structured light illumination super-resolution microscopy system is more suitable for long-term three-dimensional super-resolution imaging of live cells.

[0052] The collimating beam expander 12 collimates the light from the laser source 11.

[0053] The polarizing beam splitter 13 is used to allow light entering from one direction to pass through and light entering from the other direction to be refracted. The collimated light after being collimated by the collimating beam expander 12 enters the rotating half-wave plate 14 through the polarizing beam splitter 13.

[0054] The reflective spatial light modulator 15 alters the phase and direction of the structured illumination light, performing lateral and axial spectral shifts and broadening to achieve three-dimensional super-resolution imaging. The principle is as follows: Figure 4 As shown.

[0055] The light modulated by the reflective spatial light modulator 15 passes through the rotating half-wave plate 14, is reflected by the polarizing beam splitter 13, and then enters the Fourier lens group 16. After the light field transformation by the Fourier lens group 16, it is transformed into diffraction spots of various orders and emitted, such as... Figure 3 As shown. By rotating the half-wave plate 4, the light intensity of the 0th order diffraction spot of the reflective spatial light modulator 15 can be minimized to maximize the light intensity of the ±1st and ±2nd order diffraction spots.

[0056] The segmented polarization modulation half-wave plate 17 is composed of fan-shaped waveplates with different fast axis directions, offering advantages such as simple structure and good stability. Diffracted light of each order is modulated into different polarization directions by passing through different fan-shaped waveplates and focused at the filter aperture 18. The filter aperture 18 filters out ±1st and ±2nd order diffraction light, which exits through the first lens group 19. Specifically, although the segmented polarization modulation half-wave plate 17 has advantages such as simple structure and good stability, it cannot pass through the 0th order diffraction spot; therefore, the traditional three-dimensional structured light super-resolution imaging method using 0th and ±1st order diffraction interference cannot be used. In existing technologies, after employing the segmented polarization modulation half-wave plate 17, only ±1st order diffraction light is used for interference to form two-dimensional structured illumination light, thus only achieving two-dimensional super-resolution imaging.

[0057] In this application, the structured light illumination module 10 employs dual-period spatial light modulator stripes, such as... Figure 2 As shown, ±1st and ±2nd order diffraction light are used to interfere on the sample surface to form illumination light with a three-dimensional finite structure. The phase and direction of the structure illumination light are changed by a reflective spatial light modulator 15 to perform lateral and axial spectral shifting and expansion, thereby realizing three-dimensional super-resolution imaging.

[0058] The three-dimensional structured illumination light is formed by the interference of ±1st and ±2nd order diffracted beams, exhibiting a certain periodic distribution in both the transverse and axial directions, and can be divided into 9 spectral components (m represents the spectral order). For example... Figures 4 to 9 The figures shown are schematic diagrams of the spectrum spread represented by the nine spectral components. Figure 4 The observable spectral range for wide-field microscopy (m=0); Figure 5 These are the ±1 level spectral components in this scheme; Figure 6 These are the ±2 level spectral components in this scheme; Figure 7 These are the ±3 level spectral components in this scheme; Figure 8 These are the ±4 level spectral components in this scheme. Figure 9 This is the spectrum of the final super-resolution result obtained by stitching together all spectral components in this scheme. The horizontal and vertical axes represent the spatial frequencies in the lateral and axial directions, respectively. Kxy and Kz represent the maximum spatial frequencies of the illumination light in the lateral and axial directions, respectively, characterizing the system's ability to improve lateral and axial resolution.

[0059] In the structured light illumination module 10, the laser source 11, collimating beam expander 12, polarizing beam splitter 13, rotating half-wave plate 14, and reflective spatial light modulator 15 are arranged sequentially and located on the first straight line. The polarizing beam splitter 13, Fourier lens group 16, segmented polarization modulation half-wave plate 17, filter aperture 18, and first lens group 19 are located on the second straight line, which is perpendicular to the first straight line.

[0060] The microscopic imaging module 20 is used to form a fluorescence image based on the fluorescence acquired by the structured light illumination module 10. The microscopic imaging module 20 includes a stage 21, a microscope objective 22, a first dichroic mirror 23, a second dichroic mirror 24, a multi-channel filter 25, a focusing assembly 26, a tube mirror 27, and a first camera 28, arranged sequentially. The stage 21, microscope objective 22, first dichroic mirror 23, second dichroic mirror 24, multi-channel filter 25, focusing assembly 26, tube mirror 27, and first camera 28 are located on a third straight line, which is parallel to the first straight line.

[0061] The stage 21 is used to hold the sample. The sample is treated with upconversion nanoparticles as fluorescent staining materials for upconversion nanofluorescent probe treatment. The illumination light eventually interferes on the sample surface to form striped structured light, which excites the upconversion nanofluorescent probe and emits fluorescence in the visible light band. The fluorescence is acquired by the microscopic imaging module 20 through the imaging optical path. The stage 21 includes a two-dimensional electric displacement stage in the XY direction with sample clamps.

[0062] The microscope objective 22 is reused in the structured light illumination module 10 and the microscope imaging module 20, both focusing the illumination light onto the sample surface and collecting the fluorescence emitted by the sample.

[0063] The first dichroic mirror 23 is located between the microscope objective 22 and the second dichroic mirror 24. The first dichroic mirror 23 faces the first lens group 19. The light emitted from the first lens group 19 is reflected by the first dichroic mirror 23 to the microscope objective 22. The fluorescence of the microscope objective 22 passes through the first dichroic mirror 23 and then illuminates the second dichroic mirror 24.

[0064] The second dichroic mirror 24 is located between the first dichroic mirror 23 and the multi-channel filter 25, and the second dichroic mirror 24 is directly facing the autofocus module 30. The fluorescence passing through the microscope objective 22 is partially reflected by the second dichroic mirror 24 to the single-channel filter 31.

[0065] The multi-channel filter 25 is located between the second dichroic mirror 24 and the focusing assembly 26, and the multi-channel filter 25 filters out the excitation light in the near-infrared band.

[0066] The focusing assembly 26 includes a second lens group 261 and a first superlens modulator 262. The second lens group 261 is a 4f system, positioning the first superlens modulator 262 on the conjugate surface of the rear focal plane of the microscope objective 22, ensuring that the magnification of the optical system remains essentially constant during zooming. The first superlens modulator 262 is a single-layer or multi-layer surface with a micro / nano structure array, capable of modulating the optical wavefront at subwavelength spatial resolution. It is configured to modulate incident light, thereby controlling the focal length range. Specifically, both the substrate layer and the nanostructure layer of the first superlens modulator 262 are transparent materials with complete light transmission properties. The focal length control methods of the first superlens modulator 262 include, but are not limited to: (a) controlling the focal length by changing the relative angle of the phase-complementary double-layer nanocylindrical array structure; (b) controlling the focal length by obtaining a phase shift through a bias voltage; (c) controlling the focal length by changing the conductivity of the substrate material of the superlens modulator; and (d) changing the focal position through mechanical modulation methods such as electrostatic driving, rotation, and stretching. The first superlens modulator 262 is polarization insensitive and has a consistent modulation effect on incident light with different polarization directions. It can achieve high-efficiency phase control for arbitrary polarization. For incident light with different polarization states, the first superlens modulator 262 can achieve focusing with the focal position unchanged.

[0067] After focusing by the first superlens modulator 262, the fluorescence passes through the tube lens 27 and enters the target surface of the first camera 28 for imaging. The system acquires fluorescence images generated by structured light illuminating the sample at different phases and directions. The computer performs algorithmic post-processing on all acquired raw images to reconstruct the super-resolution three-dimensional structure.

[0068] The autofocus module 30 includes a single-channel filter 31, a second superlens modulator 32, a second tube mirror 33, and a second camera 34 arranged sequentially. The single-channel filter 31, the second superlens modulator 32, the second tube mirror 33, and the second camera 34 are located on a fourth straight line, which is perpendicular to the third straight line.

[0069] A single-channel filter 31 is located on one side of the second dichroic mirror 24. The single-channel filter 31 allows only near-infrared illumination light to pass through. The second superlens modulator 32 performs axial zoom scanning, and the second tube mirror 33 and the second camera 34 achieve focal plane imaging. The autofocus module, in conjunction with a hill-climbing algorithm, finds and locks the optimal focal plane. Based on the feedback of the optimal focal plane position, the first superlens modulator 262 is adjusted to achieve clear focusing of the first camera 28. The focal length control methods of the second superlens modulator 32 include, but are not limited to: (a) controlling the focal length by changing the relative angle of the phase-complementary double-layer nanocylindrical array structure; (b) controlling the focal length by obtaining a phase shift through a bias voltage; (c) controlling the focal length by changing the conductivity of the superlens modulator substrate material; and (d) changing the focal position through mechanical modulation methods such as electrostatic driving, rotation, and stretching.

[0070] When using an upconversion structured light illumination 3D super-resolution imaging system, the light emitted from the laser source 11 is phase-modulated by the reflective spatial light modulator 15 and then enters the Fourier lens group 16 to become diffraction spots of various orders. The filter aperture 18 filters out the ±1st and ±2nd order diffraction light. The ±1st and ±2nd order diffraction light is irradiated onto the sample on the stage 21 by the microscope objective 22. After the sample is excited, it generates a fluorescence band. The fluorescence is focused onto the target surface of the first camera 28 by the focusing assembly 26 after passing through the microscope objective 22. The second superlens modulator 32 performs axial zoom layer scanning of the fluorescence, and the second camera 34 performs focal plane imaging to lock the optimal focal plane to adjust the focal length of the first camera 28.

[0071] The upconversion structured light illumination 3D super-resolution imaging system of this invention uses a near-infrared laser source 11 as the illumination source. Near-infrared illumination light has a greater penetration depth into biological tissues, lower illumination power, and less phototoxicity, making it more suitable for long-term 3D super-resolution imaging of live cells. It employs dual-period spatial light modulator fringes and uses ±1st and ±2nd order diffraction light to form 3D structured illumination light through interference, achieving 3D super-resolution imaging with a lateral resolution of approximately 100nm and an axial resolution of approximately 300nm. It utilizes a superlens modulation-based method for 3D zoom tomography. Compared with traditional structured light illumination super-resolution systems, this invention has advantages such as faster 3D tomography speed, higher system stability, compact structure, and fewer 3D imaging artifacts, conforming to the trend of miniaturization and lightweighting in modern optical systems. The autofocus module 30 shares the near-infrared light source of the structured light illumination module 10, eliminating the need for an additional focal plane detection source, resulting in a more compact structure and simpler optical path for the autofocus module 30. Compared to mechanical focusing methods, the superlens modulation-based focal plane finding method is more stable and introduces fewer interference factors.

[0072] This invention also relates to an upconversion structured light illumination three-dimensional super-resolution imaging method, implemented based on the above-mentioned upconversion structured light illumination three-dimensional super-resolution imaging system, comprising the following steps:

[0073] Sample pretreatment: Upconversion nanoparticles were used as fluorescent staining materials to perform upconversion fluorescent staining on the samples;

[0074] Forming illumination light: Near-infrared laser light source is used as illumination light source, dual-period spatial light modulator stripes are used, and the ±1st and ±2nd order diffraction lights are used to form three-dimensional structured illumination light through interference;

[0075] Excitation fluorescence: Illumination light shines on the sample and emits fluorescence in the visible light band;

[0076] Autofocus: Using a near-infrared light source for illumination, a three-dimensional zoom layer scan is performed using a second superlens modulator to lock the optimal focal plane position of the first camera and achieve focal length adjustment of the first camera;

[0077] Automatic focusing: Modulates the incident fluorescence to control the focal length range of the first camera;

[0078] Imaging: The fluorescence is focused onto the target surface of the first camera after focusing;

[0079] 3D reconstruction: Fluorescence images of samples illuminated by structured light of different phases and directions are acquired, and all acquired raw images are post-processed to reconstruct the super-resolution 3D structure.

[0080] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These are all equivalent modifications and improvements made to the above embodiments based on the essential technology of the present invention, and all of these fall within the protection scope of the present invention.

Claims

1. An upconversion structured light illumination three-dimensional super-resolution imaging system, comprising a structured light illumination module, a microscopic imaging module, and an autofocus module, characterized in that: The structured light illumination module includes a laser source, a reflective spatial light modulator, a Fourier lens group, and a filter aperture. The light emitted by the laser source is phase-modulated by the reflective spatial light modulator and then enters the Fourier lens group to become diffraction spots of various orders before exiting. The filter aperture filters out the ±1st and ±2nd order diffraction light. The microscopic imaging module includes a stage, a microscope objective, a focusing assembly, and a first camera. The ±1st and ±2nd order diffraction light rays are irradiated onto the sample on the stage by the microscope objective. After the sample is excited, it generates a fluorescence band. The fluorescence passes through the microscope objective and is focused onto the target surface of the first camera by the focusing assembly. The focusing assembly includes a second lens group and a first superlens modulator. The first superlens modulator has a single-layer or multi-layer surface of a micro-nano structure array, which can modulate the optical wavefront at subwavelength spatial resolution. It is configured to modulate the incident light rays to control the focal length range. The second lens group is a 4f system, so that the first superlens modulator is located on the conjugate surface of the back focal plane of the microscope objective. The microscopic imaging module further includes a first dichroic mirror, which is located between the microscope objective and the focusing assembly and faces the filter aperture. The light filtered out by the filter aperture is reflected by the first dichroic mirror to the microscope objective, and the fluorescence of the microscope objective is irradiated to the focusing assembly after passing through the first dichroic mirror. The autofocus module includes a second superlens modulator and a second camera. The second superlens modulator performs axial zoom layer scanning of the fluorescence, and the second camera performs focal plane imaging to lock the optimal focal plane in order to adjust the focal length of the first camera. The microscopic imaging module further includes a second dichroic mirror, which is located between the microscope objective and the focusing assembly and faces the autofocus module. Fluorescence from the microscope objective is partially reflected by the second dichroic mirror to the second superlens modulator.

2. The upconversion structured light illumination three-dimensional super-resolution imaging system according to claim 1, characterized in that: The structured light illumination module also includes a rotating half-wave plate, which is located between the laser source and the reflective spatial light modulator. The rotating half-wave plate reduces the light intensity of the 0th order diffraction spot of the reflective spatial light modulator to the weakest level and increases the light intensity of the ±1st and ±2nd order diffraction spots.

3. The upconversion structured light illumination three-dimensional super-resolution imaging system according to claim 1, characterized in that: The structured light illumination module also includes a polarizing beam splitter, which is located between the laser source and the reflective spatial light modulator. The light emitted by the laser source passes through the polarizing beam splitter and enters the reflective spatial light modulator. The light modulated by the reflective spatial light modulator is reflected by the polarizing beam splitter and then enters the Fourier lens group.

4. The upconversion structured light illumination three-dimensional super-resolution imaging system according to claim 1, characterized in that: The structured light illumination module also includes a segmented polarization modulation half-wave plate, which is located between the Fourier lens group and the filter aperture. The segmented polarization modulation half-wave plate modulates the diffraction spots of each order into having different polarization directions.

5. The upconversion structured light illumination three-dimensional super-resolution imaging system according to claim 4, characterized in that: The segmented polarization modulation half-wave plate includes multiple sector waveplates with different fast axis directions.

6. The upconversion structured light illumination three-dimensional super-resolution imaging system according to claim 1, characterized in that: The microscopic imaging module also includes a multi-channel filter, which is located between the microscope objective and the focusing assembly. The multi-channel filter filters out excitation light in the near-infrared band.

7. A three-dimensional super-resolution imaging method with upconversion structured light illumination, implemented based on the three-dimensional super-resolution imaging system with upconversion structured light illumination as described in any one of claims 1-6, characterized in that, Includes the following steps: Sample pretreatment: Upconversion nanoparticles were used as fluorescent staining materials to perform upconversion fluorescent staining on the samples; Forming illumination light: Near-infrared laser light source is used as illumination light source, dual-period spatial light modulator stripes are used, and the ±1st and ±2nd order diffraction lights are used to form three-dimensional structured illumination light through interference; Excitation fluorescence: Illumination light shines on the sample and emits fluorescence in the visible light band; Autofocus: Using a near-infrared light source for illumination, a three-dimensional zoom layer scan is performed using a second superlens modulator to lock the optimal focal plane position of the first camera and achieve focal length adjustment of the first camera; Automatic focusing: Modulates the incident fluorescence to control the focal length range of the first camera; Imaging: The fluorescence is focused onto the target surface of the first camera after focusing; 3D reconstruction: Fluorescence images of samples illuminated by structured light of different phases and directions are acquired, and all acquired raw images are post-processed to reconstruct the super-resolution 3D structure.

Images