A three-dimensional isotropic single-objective lens super-resolution imaging method and device based on high-order nonlinear fluorescence

Abstract

The present invention discloses a three-dimensional isotropic single-objective lens super-resolution imaging method and device based on high-order nonlinear fluorescence. The present invention constructs a microscopic imaging device consisting of an excitation light generation module, a dual-focus modulation module, a multi-photon microscopic scanning module, a reflector sample module, and a photoelectric detection module. Under incident laser excitation, a three-dimensional sidelobe-free isotropic super / high-resolution microscopy technology with no photobleaching, low phototoxicity, low complexity, low cost, and ease of use is achieved.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy, specifically to a three-dimensional isotropic single-objective super-resolution imaging method and apparatus based on high-order nonlinear fluorescence. Background Technology

[0002] For a long time, far-field optical fluorescence microscopy has been the most commonly used observation tool in life sciences due to its advantages such as non-contact, non-destructive nature, and ability to probe the interior of samples. However, due to the optical diffraction limit, the light spot is elongated along the optical axis, resulting in a much lower axial resolution than lateral resolution in traditional fluorescence microscopy. This means that its three-dimensional resolution is not isotropic, thus affecting the quality of three-dimensional imaging. Three-dimensional uniform super-resolution imaging is of great significance to many interdisciplinary fields such as biomedicine and micro-nano optics. Therefore, many researchers have conducted extensive research on three-dimensional isotropic optical super-resolution methods.

[0003] Some researchers have proposed using a dual-objective 4Pi microscope to increase the point spread function (NPF) scale and thus improve resolution by increasing the receiving angle of the objective lens (equivalent to increasing the NA of the objective lens). However, the dual-objective 4Pi microscope compresses the NPF axially without improving lateral resolution, resulting in an imbalance between axial and lateral resolution and severe image distortion. The sidelobe effect is significant, with longitudinal sidelobes accounting for approximately 40%–45% of the main lobe, and even exceeding 70% in experiments. Even with techniques such as pinhole filtering and deconvolution, the sidelobe intensity remains around 30%. The dual-objective 4Pi microscope is highly demanding on the sample; because the interference main lobe cannot be moved, it can only be used to image thin, transparent samples. Alignment requirements are extremely stringent, the system's optical path is complex, and even minor perturbations can significantly affect the imaging results. Therefore, if the shortcomings of the dual-objective 4Pi microscope can be overcome and a low-cost, simple, sidelobe-free three-dimensional isotropic microscopy method can be developed, it will be of great significance to enhancing my country's international influence in basic research and instrument development in super-resolution microscopy. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide a three-dimensional isotropic single-objective super-resolution imaging method and apparatus based on high-order nonlinear fluorescence.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A three-dimensional isotropic single-lens super-resolution imaging method based on high-order nonlinear fluorescence, the method comprising the following steps:

[0007] The S1 laser emits a stable laser beam. This laser beam is filtered out by a filter to remove other wavelengths of laser light. After being collimated and expanded by a double convex lens and filtered and shaped by a pinhole filter, it enters a dual-focus modulation system. The spatial light modulator performs wavefront modulation to obtain two focal points distributed along the axial direction.

[0008] The two focal points of S2 exit at the objective lens end. Due to the effect of the mirror, the incident light field at the front focal point and the reflected light field at the rear focal point converge at the front focal point, forming an alternating bright and dark interference light field on the mirror surface.

[0009] S3 places the sample labeled with the fluorescent probe on the surface of the mirror, scans it point by point in the X, Y, and Z directions using an interference light field, and then uses a photodetector to detect the fluorescence signal corresponding to each point. Combined with nonlinear effects, a three-dimensional isotropic high / super-resolution fluorescence image without sidelobes can be obtained.

[0010] It should be noted that the reflector is placed at the midpoint of the front and rear focal points distributed along the optical axis. The incident light field from the front focal point of the objective lens and the reflected light field from the rear focal point of the objective lens converge at the front focal point. The position of the interference light field distribution changes with the relative position of the front focal point and the reflector. By changing the distance between the two focal points, the axial movement of the interference light field is ensured, thereby achieving axial high / super-resolution imaging.

[0011] It should be noted that the laser of the present invention includes at least one or more of continuous near-infrared light (wavelength: 700nm-1100nm), femtosecond near-infrared light (wavelength: 700nm-1100nm), and continuous visible light (wavelength: 400nm-700nm).

[0012] It should be noted that the reflector of the present invention includes at least one of aluminum-coated plane reflector, silver-coated plane reflector, and gold-coated plane reflector and has a half-wave loss (π phase difference) on the light field.

[0013] It should be noted that the interference light field is symmetrically distributed along the central spot (main lobe), and the intensity of the longitudinal spots (side lobes) perpendicular to the optical axis decreases step by step. Among them, the axial half-width at half maximum (HWHM) of the main lobe of the interference light field is significantly compressed, and its HWHM is 4-7 times higher than that of the spot after the laser of the excitation wavelength is focused by the objective lens. The intensity of the first-order side lobe is half that of the main lobe of the interference light field, and the intensity of the side lobe decreases as the order increases.

[0014] It should be noted that by using the high / super-compressed full width at half maximum (FWHM) of the main lobe, layer-by-layer scanning in the Z direction is achieved, enabling continuous axial high / super-resolution imaging at different distances from the mirror surface.

[0015] It should be noted that the fluorescent probe contains rare-earth-doped upconversion nanoparticles with a nonlinear order n≥4, which can be matched with its activated ion Tm by continuous / femtosecond near-infrared light. 3+ The excited state absorbs energy, Nd 3+ or Yb 3+ As a sensitizer co-doped upconversion nanoparticle, it can be excited by lasers with wavelengths of 980nm, 795nm, and 730nm to generate a four-photon fluorescence radiation process, eliminating sidelobes of the interference light field and improving lateral resolution, breaking through the diffraction limit, and realizing three-dimensional sidelobe-free high-resolution imaging.

[0016] It should be noted that the fluorescent probe may also be one of quantum dots or organic dyes.

[0017] It should be noted that the fluorescent probe can also be avalanche fluorescent nanoprobe to generate photon avalanche upconversion fluorescence, producing a higher-order nonlinear effect n≥28, in order to obtain a higher-order nonlinear effect, eliminate sidelobes of the interference light field and improve lateral resolution, break through the diffraction limit, and realize three-dimensional sidelobe-free isotropic ultra-high resolution imaging.

[0018] This invention also provides a device for three-dimensional isotropic single-objective super-resolution imaging based on high-order nonlinear fluorescence. The device includes an excitation light generation module, a dual-focus modulation module, a multiphoton microscopy scanning module, a mirror sample module, and a photodetector module. The laser generation module generates continuous near-infrared light, femtosecond near-infrared light, and continuous visible light as excitation light. After shaping, the excitation light enters the dual-focus modulation module for wavefront modulation to obtain two focal points distributed along the optical axis. These focal points are then focused onto the mirror sample module via the multiphoton microscopy scanning module to generate dual-focus interference. The nonlinearity of the high-order fluorescence probe is used to obtain a sidelobe-free isotropic central spot, which scans different regions of the sample. The relevant fluorescence signals are collected by the photodetector module.

[0019] It should be noted that the system also includes filters, a collimating and expanding system, a half-wave plate, and a polarizer, arranged sequentially along the optical axis of the incident beam emitted by the laser in the laser generation module. The laser generates stable, single-peaked laser light, which is filtered out by the filters to remove stray incident light. The laser then enters the collimating and expanding system to enlarge the excitation spot size and improve the excitation light power utilization. The incident laser power is adjusted by the half-wave plate and polarizer to match the absorption of the corresponding activated ion excited state in the sample.

[0020] It should be noted that the dual-focus modulation module includes a mirror and a spatial light modulator placed along the direction of laser beam propagation. The mirror reflects the collimated beam incident from the laser generation module to the spatial light modulator, and its modulation focal plane is optically conjugate to the rear focal plane of the objective lens. By inputting different phase modulations, two focal points with different distances along the optical axis are obtained, which are used to modulate the subsequent single-objective lens interference imaging light field.

[0021] It should be noted that the multiphoton microscopy scanning module includes a scanning galvanometer, a scanning lens, a tube mirror, a high-reflection, low-transmission dichroic mirror, and an objective lens arranged sequentially along the laser beam propagation direction. The scanning galvanometer controls the optical path deflection of the laser beam to perform two-dimensional scanning of the sample. The scanning lens and field lens focus and collimate the laser beam emitted from the scanning galvanometer. The high-reflection, low-transmission dichroic mirror can reflect near-infrared excitation light and transmit sample fluorescence, and is used to separate the excitation light and fluorescence. The incident light field from the dual-focus modulation module is focused onto the reflector sample module by the objective lens.

[0022] It should be noted that the reflector sample module consists of, from top to bottom, a glass / silicon substrate, a silver / gold / aluminum plating layer, a silicon dioxide protective layer, a sample labeled with a fluorescent probe, and a coverslip. The two focal points, distributed along the optical axis and obtained through wavefront modulation, converge at the objective lens exit end due to the effect of the reflector. This causes the incident light field at the front focal point and the reflected light field at the rear focal point to converge at the front focal point, forming an alternating bright and dark interference light field on the mirror surface. This eliminates the side lobes of the interference light field through a highly nonlinear effect and improves the lateral resolution of the central main lobe, ultimately resulting in a three-dimensional, side-lobe-free, isotropic interference light field.

[0023] It should be noted that the photoelectric detection module includes a focusing lens and a photodetector placed coaxially in sequence. The focusing lens and photodetector are positioned in the forward direction of the fluorescence collected by the objective lens. The sample emits fluorescence in various directions under the excitation of the incident laser. The objective lens collects a portion of the fluorescence signal. The signal passes through a high-reflection, low-transmission dichroic mirror and the focusing lens, and is received by the photodetector. After receiving a detection signal, the photodetector sends the signal to the computer. The computer controls the rotation of the scanning galvanometer through a rotating device, moves the focused spot to scan the next pixel, thereby obtaining a two-dimensional laser scanning super-resolution fluorescence image. A motor is provided on one side of the stage to drive the stage to move along the Z-axis. Through computer control, Z-axis scanning can be performed based on the above, and finally a three-dimensional laser scanning super-resolution fluorescence image can be obtained.

[0024] It should be noted that, in this invention, in addition to using an XY scanning galvanometer paired with a Z-axis displacement stage to achieve laser point scanning, an XYZ three-axis displacement stage can also be used to achieve three-dimensional imaging. The XYZ displacement stage is linked with the input and output signals through a data acquisition card. By moving the sample in three-dimensional space, three-dimensional scanning of the sample is achieved. The signals collected in each scan are received by a photodetector. After receiving each detection signal, the photodetector sends the signal to the computer, ultimately obtaining a three-dimensional laser scanning super-resolution fluorescence image.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. Compared with the traditional dual-objective 4Pi microscope, this invention introduces a spatial light modulator and a silver / gold / aluminum coated reflector, which achieves the compression of the axial point spread function in a simple single-objective system. The system is simple, has low construction cost, and does not require strict objective alignment conditions.

[0027] 2. Compared with traditional dual-objective 4Pi microscopes, this invention eliminates the side lobes of the interference light field, solving the artifact problem of traditional interference light field imaging. Without using complex deconvolution algorithms or introducing additional filter pinholes, this invention combines ultra-high-order fluorescent nonlinear materials to ensure that the interference light field retains only the axially compressed central spot, greatly improving the signal-to-noise ratio of the imaging.

[0028] 3. Compared with traditional dual-objective 4Pi microscopes, this invention achieves axial movement of the interference center spot. Due to the deterministic position of the interference field center spot, traditional dual-objective 4Pi microscopes can only be used to image thin, transparent samples. This invention, by adjusting the phase image of the input spatial light modulator, changes the distance between the two focal points (the relative position of the front focal point and the mirror), thus achieving axial movement of the interference field center spot, making it possible to image thick samples; due to the reflection effect of the mirror, there is no need for fluorescence collection on the back of the sample, and high / super-resolution imaging can also be performed on opaque samples.

[0029] 4. Compared with traditional far-field optical microscopes, this invention combines the mirror double-focus effect with an ultra-high-order nonlinear fluorescent probe, which greatly compresses the point spread function size in the lateral and axial directions, eliminates the influence of side lobes, and realizes true three-dimensional super-resolution isotropic imaging. Attached Figure Description

[0030] Figure 1 This refers to the dual-focus phase modulation board used in Example 1 for wavefront modulation;

[0031] Figure 2 This is a simulation diagram of the movement of the interference optical field achieved by modulating the two focal points at different distances in Example 1;

[0032] Figure 3The images show simulated light fields of confocal microscopy and bifocal interferometry based on high-order fluorescence nonlinearity at different nonlinear orders in Example 1.

[0033] Figure 4 This is a schematic diagram of the microscopic imaging device in Example 2;

[0034] Figure 5 This is a schematic diagram of the reflector sample module in Example 2. Detailed Implementation

[0035] The present invention will be further described below. It should be noted that the following embodiments are based on the present technical solution and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to these embodiments.

[0036] This invention relates to a three-dimensional isotropic single-lens super-resolution imaging method based on high-order nonlinear fluorescence, the method comprising the following steps:

[0037] The S1 laser emits a stable laser beam. This laser beam is filtered out by a filter to remove other wavelengths of laser light. After being collimated and expanded by a double convex lens and filtered and shaped by a pinhole filter, it enters a dual-focus modulation system. The spatial light modulator performs wavefront modulation to obtain two focal points distributed along the optical axis.

[0038] The two focal points of S2 exit at the objective lens end. Due to the effect of the mirror, the incident light field at the front focal point and the reflected light field at the rear focal point converge at the front focal point, forming an alternating bright and dark interference light field on the mirror surface.

[0039] S3 places the sample labeled with the fluorescent probe on the surface of the mirror, scans it point by point in the X, Y, and Z directions using an interference light field, and then uses a photodetector to detect the fluorescence signal corresponding to each point. Combined with nonlinear effects, a three-dimensional isotropic high / super-resolution fluorescence image without sidelobes can be obtained.

[0040] Furthermore, the reflector of the present invention is placed at the midpoint of the front and rear focal points distributed along the optical axis. The incident light field of the objective lens exiting the front focal point and the reflected light field of the objective lens exiting the rear focal point converge at the front focal point position. The position of the interference light field distribution changes with the relative position of the front focal point and the reflector. By changing the distance between the two focal points, the axial movement of the interference light field is ensured, thereby achieving axial high / super-resolution imaging.

[0041] Furthermore, the laser of the present invention includes at least one or more of continuous near-infrared light (wavelength: 700nm-1100nm), femtosecond near-infrared light (wavelength: 700nm-1100nm), and continuous visible light (wavelength: 400nm-700nm).

[0042] Furthermore, the reflector of the present invention comprises at least one of an aluminum-coated plane reflector, a silver-coated plane reflector, and a gold-coated plane reflector, and has a half-wave loss (π phase difference) on the light field.

[0043] Furthermore, the interference light field of the present invention is symmetrically distributed along the central spot (main lobe), and the intensity of the longitudinal spots (side lobes) perpendicular to the optical axis gradually decreases; wherein, the axial half-width at half maximum (HWHM) of the main lobe of the interference light field is significantly compressed, and its HWHM is 4-7 times higher than that of the spot after the laser of the excitation wavelength is focused by the objective lens; the intensity of the first-order side lobe is half that of the main lobe of the interference light field, and the intensity of the side lobe decreases as the order increases.

[0044] Furthermore, the present invention achieves layer-by-layer scanning in the Z direction through high / super-compressed full width at half maximum (FWHM) of the main lobe, thereby realizing continuous axial high / super-resolution imaging at different distances from the mirror surface.

[0045] Furthermore, the fluorescent probe of the present invention comprises rare-earth-doped upconversion nanoparticles with a nonlinear order n≥4, which can be matched with its activating ion Tm by continuous / femtosecond near-infrared light. 3+ The excited state absorbs energy, Nd 3+ or Yb 3+ As a sensitizer co-doped upconversion nanoparticle, it can be excited by lasers with wavelengths of 980nm, 795nm, and 730nm to generate a four-photon fluorescence radiation process, eliminating sidelobes of the interference light field and improving lateral resolution, breaking through the diffraction limit, and realizing three-dimensional sidelobe-free high-resolution imaging.

[0046] Furthermore, the fluorescent probe of the present invention may also include one of quantum dots or organic dyes.

[0047] Furthermore, the fluorescent probe of the present invention can also be a photon avalanche upconversion fluorescence generated by an avalanche fluorescent nanoprobe, producing a higher-order nonlinear effect n≥28, in order to obtain a higher-order nonlinear effect, eliminate sidelobes of the interference light field and improve lateral resolution, break through the diffraction limit, and realize three-dimensional sidelobe-free isotropic ultra-high resolution imaging.

[0048] This invention also provides a device for three-dimensional isotropic single-lens super-resolution imaging based on high-order nonlinear fluorescence. The device includes an excitation light generation module, a dual-focus modulation module, a multiphoton microscopy scanning module, a mirror sample module, and a photoelectric detection module. The laser generation module generates continuous near-infrared light, femtosecond near-infrared light, or continuous visible light as excitation light. After shaping, the excitation light enters the dual-focus modulation module for wavefront modulation to obtain two focal points distributed along the optical axis. These focal points are then focused onto the mirror sample module via the multiphoton microscopy scanning module to generate dual-focus interference. The nonlinearity of the high-order fluorescence probe is used to obtain a sidelobe-free isotropic central spot, which scans different regions of the sample. The relevant fluorescence signals are collected by the photoelectric detection module.

[0049] Furthermore, the present invention also includes a filter, a collimating and expanding system, a half-wave plate, and a polarizer, arranged sequentially along the optical axis of the incident beam emitted by the laser in the laser generating module. The laser generates a stable, single-peak laser beam, which, after being filtered by the filter to remove incident stray light, enters the collimating and expanding system to enlarge the excitation spot size and improve the excitation light power utilization. The incident laser power is adjusted by the half-wave plate and polarizer to achieve matching of the absorption of the corresponding activated ion excited state in the sample.

[0050] Furthermore, the dual-focus modulation module of the present invention includes a mirror and a spatial light modulator placed along the laser beam propagation direction; the mirror reflects the collimated beam incident through the laser generation module to the spatial light modulator, and its modulation focal plane is optically conjugate to the rear focal plane of the objective lens. By inputting different phase modulations, two focal points with different distances along the optical axis are obtained, which are used to modulate the subsequent single-objective lens interference imaging light field.

[0051] Furthermore, the multiphoton microscopy scanning module of the present invention includes a scanning galvanometer, a scanning lens, a tube mirror, a high-reflection, low-transmission dichroic mirror, and an objective lens arranged sequentially along the laser beam propagation direction; the scanning galvanometer controls the optical path deflection of the laser beam to perform two-dimensional scanning of the sample; the scanning lens and the field lens focus and collimate the laser beam emitted from the scanning galvanometer; the high-reflection, low-transmission dichroic mirror can reflect near-infrared excitation light and transmit sample fluorescence, and is used to separate excitation light and fluorescence; the incident light field from the dual-focus modulation module is focused onto the reflector sample module by the objective lens.

[0052] Furthermore, the reflector sample module of the present invention comprises, from top to bottom, a glass / silicon substrate, a silver / gold / aluminum plating layer, a silicon dioxide protective layer, a sample labeled with a fluorescent probe, and a cover glass. The two focal points, distributed along the optical axis and obtained through wavefront modulation, converge at the objective lens exit end due to the action of the reflector. This causes the incident light field at the front focal point and the reflected light field at the rear focal point to converge at the front focal point, forming an alternating bright and dark interference light field on the mirror surface. This eliminates the side lobes of the interference light field through a highly nonlinear effect and improves the lateral resolution of the central main lobe, ultimately resulting in a three-dimensional, side-lobe-free, isotropic interference light field.

[0053] Furthermore, the photoelectric detection module of the present invention includes a focusing lens and a photodetector placed coaxially in sequence; wherein, the focusing lens and the photodetector are positioned in the forward direction of the fluorescence collected by the objective lens, the sample emits fluorescence in various directions under the excitation of the incident laser, the objective lens collects a portion of the fluorescence signal, the signal passes through a high-reflection, low-transmission dichroic mirror and the focusing lens, and is received by the photodetector, after the photodetector receives a detection signal each time, the photodetector sends the signal to the computer, the computer controls the rotation of the scanning galvanometer through a rotating device, moves the focused spot to scan the next pixel, thereby obtaining a two-dimensional laser scanning super-resolution fluorescence image, a motor is provided on one side of the stage for driving the stage to move along the Z-axis direction, and through computer control, Z-axis scanning can be performed on the basis of the above, and finally a three-dimensional laser scanning super-resolution fluorescence image is obtained.

[0054] Furthermore, in this invention, in addition to using an XY scanning galvanometer paired with a Z-axis displacement stage to achieve laser point scanning, an XYZ three-axis displacement stage can also be used to achieve three-dimensional imaging. The XYZ displacement stage is linked with the input and output signals through a data acquisition card. By moving the sample in three-dimensional space, three-dimensional scanning of the sample is achieved. The signals collected in each scan are received by a photodetector. After receiving each detection signal, the photodetector sends the signal to a computer, ultimately obtaining a three-dimensional laser scanning super-resolution fluorescence image.

[0055] Example 1

[0056] The S1 laser emits a stable laser beam. This beam is filtered to remove other wavelengths, then collimated and expanded by a biconvex lens, filtered and shaped by a pinhole filter, and finally enters the dual-focus modulation system. Input is as follows: Figure 1 The phase modulation plate shown is wavefront modulated by a spatial light modulator to obtain two focal points distributed along the optical axis. For example... Figure 2 As shown in (a), the distance between the two focal points can be controlled by adjusting the phase plate parameters;

[0057] The two focal points, distributed along the optical axis and obtained by S2 wavefront modulation, converge at the objective lens exit end due to the effect of the mirror. The incident light field at the front focal point and the reflected light field at the rear focal point converge at the front focal point, forming an alternating bright and dark interference field on the mirror surface. The axial movement of the interference field can be achieved by adjusting the positions of the two focal points. Figure 2 As shown in (b);

[0058] S3 demonstrated, through energy level analysis and rate equation calculations of Yb³⁺ and Pr³⁺ ions, that the doped system can achieve an ultra-high-order nonlinear (n=28) photon avalanche effect. Under the excitation of an 852 nm laser, by adjusting the laser power, when the power is near the photon avalanche threshold, the photon avalanche fluorescence emitted by the nanoprobe exhibits an ultra-high-order nonlinear response, and the fluorescence dot spread function is significantly compressed, achieving a resolution that breaks through the diffraction limit. Simulated light field diagrams of confocal microscopy and bifocal interferometry based on high-order fluorescence nonlinearity at different nonlinear orders are shown below. Figure 3 As shown.

[0059] Example 2

[0060] A schematic diagram of the device of the present invention is shown below. Figure 4 The system includes an excitation light generation module, a dual-focus modulation module, a multiphoton microscopy scanning module, a mirror sample module, and a photoelectric detection module. The excitation light generation module comprises a continuous-wave near-infrared (700nm-1100nm) / femtosecond near-infrared (700nm-1100nm) / continuous-wave visible (400nm-700nm) laser 1, a filter 2, a collimating beam expander 3 (including a pinhole filter), a half-wave plate 4, and a polarizer 5. The near-infrared laser generates Gaussian laser output. The filter removes stray light from other wavelengths in the laser beam. The collimating beam expander enlarges the excitation spot size, improving the excitation light power utilization. Simultaneously, a pinhole filter is placed at the focal point to filter out high-frequency stray light. The half-wave plate 4 is mounted on a rotatable mount and used with a linear polarizer 5 to adjust the laser beam power.

[0061] The dual-focus modulation module includes a mirror and a spatial light modulator 6. The mirror reflects the collimated beam incident from the laser generation module to the spatial light modulator, whose modulation focal plane is optically conjugate to the rear focal plane of the objective lens. By inputting different phase modulations, two focal points with different distances along the optical axis are obtained, which are used to modulate the subsequent single-objective interference imaging light field.

[0062] The multiphoton microscopy scanning module includes a scanning galvanometer 7, a scanning lens 8, a tube mirror 9, a high-reflection, low-transmission dichroic mirror 10, and an objective lens 11. The scanning galvanometer controls the deflection of the laser beam to achieve two-dimensional scanning of the sample. The high-reflection, low-transmission dichroic mirror reflects near-infrared excitation light and transmits sample fluorescence, used to separate the excitation light from the fluorescence. The scanning lens and tube mirror focus and collimate the beam emitted from the scanning galvanometer, ensuring that the laser beam remains matched to the entrance pupil size of the microscope objective lens during scanning. Finally, the incident light field from the dual-focus modulation module is focused by the objective lens onto the reflector sample module 12.

[0063] Mirror sample module, such as Figure 5 As shown, from top to bottom, the structure consists of a glass / silicon substrate, a silver / gold / aluminum plating layer, a silica protective layer, a high-order upconversion fluorescent probe, a quantum dot or organic dye-labeled sample, and a coverslip. The two focal points, distributed along the optical axis and obtained through wavefront modulation, converge at the objective lens exit end due to the action of the mirror. This convergence of the incident light field at the front focal point and the reflected light field at the rear focal point creates an alternating bright and dark interference field on the mirror surface. The high nonlinearity of the rare-earth-doped upconversion fluorescent probe eliminates the sidelobes of the interference field and improves the lateral resolution of the central main lobe, ultimately resulting in a three-dimensional, sidelobe-free, isotropic interference field.

[0064] The photoelectric detection module includes a focusing lens 13 and a photodetector 14. The focusing lens and photodetector are positioned along the direction of fluorescence collected by the objective lens. Under the excitation of the incident laser, the sample emits fluorescence in various directions. The objective lens collects a portion of the fluorescence signal, which passes through a high-reflection, low-transmission dichroic mirror and the focusing lens before being received by the photodetector. After receiving each detection signal, the photodetector sends the signal to the computer. The computer controls the rotation of the scanning galvanometer via a rotating device, moving the focused spot to scan the next pixel, thereby obtaining a two-dimensional laser scanning super-resolution fluorescence image. A motor is located on one side of the stage to drive the stage to move along the Z-axis. Through computer control, Z-axis scanning can be performed based on the above, ultimately obtaining a three-dimensional laser scanning super-resolution fluorescence image. Besides using an XY scanning galvanometer with a Z-axis displacement stage to achieve laser point scanning, a three-dimensional imaging system can be achieved using an XYZ three-axis displacement stage. The XYZ displacement stage uses a data acquisition card to link with the input and output signals. It achieves three-dimensional scanning of the sample by moving the sample in three-dimensional space. The signals collected in each scan are received by a photodetector. After receiving the signal from each scan, the photodetector sends the signal to the computer, ultimately obtaining a three-dimensional laser scanning super-resolution fluorescence image.

[0065] For those skilled in the art, various corresponding changes and modifications can be made based on the above technical solutions and concepts, and all such changes and modifications should be included within the protection scope of the claims of this invention.

Claims

1. A three-dimensional isotropic single-objective lens super-resolution imaging method based on high-order nonlinear fluorescence, characterized in that: The method comprises the following steps: The S1 laser emits a stable laser beam, which is filtered out by a filter to remove other wavelengths of laser light, then collimated and expanded by a double convex lens, filtered and shaped by a pinhole filter, and then enters a dual-focus modulation system. The spatial light modulator performs wavefront modulation to obtain two front and rear focuses of the axial distribution. S2 The front and rear focal points are emitted at the end of the objective lens. Due to the effect of the reflector, the incident light field of the front focal point and the reflected light field of the rear focal point converge at the front focal point position, forming an interference light field of alternating light and dark on the mirror surface; wherein the reflector is placed at the midpoint of the front and rear focal points distributed along the optical axis, and the incident light field of the front focal point emitted by the objective lens and the reflected light field of the rear focal point emitted by the objective lens through the reflector converge at the front focal point position; The S3 upconversion fluorescence emission has a high-order nonlinear response relationship with the excitation light intensity. Through the multi-photon nonlinear fluorescence effect of the designed fluorescent probe, the fluorescence point spread function is greatly compressed in the X, Y, and Z directions, and the resolution is greatly improved, breaking through the diffraction limit. S4: Place the sample labeled with the fluorescent probe on the surface of the reflector, scan it point by point in the X, Y, and Z directions using the interference light field, and then use a photodetector to detect the fluorescence signal corresponding to each point. Combined with the nonlinear effect, the three-dimensional isotropic high / super-resolution fluorescence image without side lobes can be obtained.

2. The three-dimensional isotropic single-objective lens super-resolution imaging method based on high-order nonlinear fluorescence according to claim 1, characterized in that: The position of the interference light field distribution changes with the relative position of the front focus and the reflector. By changing the distance between the front and rear focuses, the axial movement of the interference light field is ensured to achieve axial high / super resolution imaging.

3. The three-dimensional isotropic single-objective lens super-resolution imaging method based on high-order nonlinear fluorescence according to claim 1 or 2, characterized in that: The interference light field is symmetrically distributed along the central spot, and the longitudinal spot intensity perpendicular to the optical axis gradually weakens; among them, the axial half-width of the main lobe of the interference light field is greatly compressed, and its axial half-width is 4-7 times higher than the axial half-width of the spot after the excitation wavelength laser is focused by the objective lens; the intensity of the first-order side lobe is half of the main lobe of the interference light field, and the side lobe intensity decreases with the increase of the order.

4. The three-dimensional isotropic single-objective lens super-resolution imaging method based on high-order nonlinear fluorescence according to claim 3, characterized in that: Through the half-width of the main lobe high / super compression, layer-by-layer scanning in the Z direction is achieved, realizing continuous axial high / super-resolution imaging at different distances from the mirror.

5. The three-dimensional isotropic single-objective lens super-resolution imaging method based on high-order nonlinear fluorescence according to claim 3, characterized in that: The fluorescent probe comprises rare earth doped up-conversion nanoparticles, whose nonlinear order n≥4, and whose activation ion Tm is matched by continuous / femtosecond near-infrared light. 3+ The excited state absorption energy of Nd 3+ or Yb 3+ As a sensitizer, co-doped upconversion nanoparticles can be excited by lasers with wavelengths of 980 nm, 795 nm, and 730 nm, respectively, to produce a four-photon fluorescence radiation process, eliminating the side lobes of the interference light field and improving the lateral resolution, breaking through the diffraction limit, and achieving three-dimensional sidelobe-free high-resolution imaging.

6. The three-dimensional isotropic single-objective lens super-resolution imaging method based on high-order nonlinear fluorescence according to claim 1, characterized in that: The fluorescent probe uses an avalanche fluorescent nanoprobe to generate photon avalanche up-conversion fluorescence, generating a high-order nonlinear effect n≥28, so as to obtain a higher-order nonlinear effect, eliminate the side lobes of the interference light field and improve the lateral resolution, break through the diffraction limit, and realize three-dimensional sidelobe-free isotropic ultra-high resolution imaging.

7. A three-dimensional isotropic single-objective lens super-resolution imaging device based on high-order nonlinear fluorescence according to any one of claims 1 to 6, characterized in that: The device includes an excitation light generation module, a dual-focus modulation module, a multi-photon microscopic scanning module, a reflector sample module and a photoelectric detection module; wherein the laser generation module is used to generate continuous near-infrared light, femtosecond near-infrared light or continuous visible light as excitation light, the excitation light enters the dual-focus modulation module for wavefront modulation after shaping to obtain two front and rear focuses distributed along the optical axis, and is focused to the reflector sample module via the multi-photon microscopic scanning module to generate dual-focus interference, and the nonlinearity of the high-order fluorescent probe is used to obtain a sidelobe-free isotropic central light spot, and different areas of the sample are scanned, and the relevant fluorescence signals are collected by the photoelectric detection module.

8. The three-dimensional isotropic single-objective lens super-resolution imaging device based on high-order nonlinear fluorescence according to claim 7, characterized in that: It also includes a filter, a collimating beam expansion system, a half-wave plate and a polarizer placed in sequence along the optical axis direction of the incident light beam emitted by the laser in the laser generating module; the laser generates a stable, single-peak laser, which is filtered out by the filter to remove incident lightless stray light and enters the collimating beam expansion system to expand the size of the excitation light spot and improve the utilization rate of the excitation light power; the incident laser is adjusted in power through the half-wave plate and the polarizer to achieve matching of the excited state absorption of the corresponding activated ions of the sample.

9. The three-dimensional isotropic single-objective lens super-resolution imaging device based on high-order nonlinear fluorescence according to claim 7, characterized in that: The dual-focus modulation module includes a reflector and a spatial light modulator placed along the direction of laser beam advance; the reflector reflects the collimated light beam incident through the laser generating module to the spatial light modulator, and its modulation focal plane is optically conjugate with the rear focal plane of the objective lens; by inputting different phase modulations, two front and rear focal points with different distribution distances along the optical axis are obtained, which are used to modulate the subsequent single objective lens interference imaging light field.

10. The three-dimensional isotropic single-objective lens super-resolution imaging device based on high-order nonlinear fluorescence according to claim 7, characterized in that: The multiphoton microscopy scanning module includes a scanning galvanometer, a scanning lens, a tube lens, a high-reflection and low-transmittance dichroic mirror and an objective lens which are sequentially placed along the advancing direction of the laser beam; the scanning galvanometer controls the optical path deflection of the laser beam to perform two-dimensional scanning of the sample, the scanning lens and the field lens focus and collimate the laser beam emitted through the scanning galvanometer, the high-reflection and low-transmittance dichroic mirror can reflect near-infrared excitation light and transmit sample fluorescence, and is used to separate the excitation light and the fluorescence, and the incident light field of the dual-focus modulation module is focused to the reflector sample module through the objective lens.

11. The three-dimensional isotropic single-objective lens super-resolution imaging device based on high-order nonlinear fluorescence according to claim 7, characterized in that: The reflector sample module comprises, from top to bottom, a glass / silicon substrate, a silver / gold / aluminum layer, a silicon dioxide protective layer, a sample marked with a fluorescent probe, and a cover glass; The front and rear focal points along the optical axis obtained by wavefront modulation converge at the front focus position due to the effect of the reflector at the output end of the objective lens. Alternating light and dark interference light fields are formed on the mirror surface. The side lobes of the interference light field are eliminated and the lateral resolution of the central main lobe is improved through the high nonlinear effect, and finally a three-dimensional sidelobe-free isotropic interference light field is obtained.

12. The three-dimensional isotropic single-objective lens super-resolution imaging device based on high-order nonlinear fluorescence according to claim 7, characterized in that: The photoelectric detection module includes a focusing lens and a photoelectric detector which are coaxially placed in sequence; wherein the focusing lens and the photoelectric detector are arranged in the forward direction of the fluorescence collected by the objective lens, the sample emits fluorescence in all directions under the excitation of the incident laser, the objective lens collects a part of the fluorescence signal, the signal passes through the high-reflection and low-transmittance dichroic mirror and the focusing lens, and is received by the photoelectric detector. After receiving each detected signal, the photoelectric detector sends the signal to the computer, the computer controls the rotation of the scanning galvanometer through the rotating device, moves the focusing spot to scan the next pixel, thereby obtaining a two-dimensional laser scanning super-resolution fluorescence image, a motor for driving the stage to move along the Z-axis direction is provided on one side of the stage, and the Z-axis scanning is performed through computer control, and finally a three-dimensional laser scanning super-resolution fluorescence image is obtained.

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