CN104515759B - Nonlinear organization optical illumination micro imaging method and system - Google Patents

Abstract

The invention discloses a non-linear structured illumination microscopic imaging method, which comprises the following steps: 1) loading a computational hologram on the digital micromirror array; A spatially structured light field, the first spatially structured light field irradiates the surface of the sample, causing some proteins to switch from a dark state to a bright state; 3) the second spatially structured light field irradiates the sample, causing the fluorescent proteins in the bright state to fluoresce, and Collect fluorescence and image it in the photodetector; 4) Repeat steps 2) and 3) to collect multiple spatial frequencies, and collect multiple initial phases in each direction to obtain multiple original images, and reconstruct super Resolve images. At the same time, the invention also discloses a non-linear structured illumination microscopic imaging system. The invention has the advantages of high system imaging resolution, high resistance to fluorescent drift, low phototoxicity and fast imaging speed.

Description

Nonlinear Structured Illumination Microscopic Imaging Method and System

technical field

The invention relates to the field of biological imaging, in particular to a non-linear structured illumination microscopic imaging method and system.

Background technique

Bioimaging is a frontier field of life science involving multiple disciplines. It is a cutting-edge imaging technology for studying biological functions by "seeing" the interaction of living cells in real time and dynamically at different levels such as molecules, cells, tissues, and living bodies. It has become a breakthrough point for major achievements in life sciences today.

The traditional optical microscope is limited by the optical diffraction limit. The size of the diffraction spot (Airy disk) is about half of the ratio of the wavelength of the light source to the numerical aperture of the microscope objective lens. Generally, the highest imaging resolution is around 200-300nm, and it is impossible to directly observe some How important molecules such as genes, structural proteins, immune proteins, RNA and their complexes, and active factors are expressed in cells, how to form the basic structural system of cells, and how to regulate the main life activities of cells, such as cell proliferation and cell differentiation , apoptosis and cell signaling. The characteristic scales reflecting the properties of these systems are all in the order of nanometers. Electron microscopy (EM) can achieve nanometer-level resolution and can locate organelles such as vesicles and mitochondria inside cells, but it is not suitable for locating individual protein molecules due to the lack of corresponding probe labels. Electron microscope samples need to be fixed, and the irradiation of electron beams will also damage biological samples, so it is not suitable for the observation of dynamic changes in living cells.

Super-resolution optical microscopy with submicron or even nanometer resolution can overcome the defects of traditional optical microscopes and electron microscopes, break through the diffraction limit, and achieve super-resolution imaging capabilities at the cellular level.

Super-resolution optical microscopy mainly includes three categories: 1. Photo-activated localization microscopy (PALM for short) based on fluorescence single-molecule localization and stochastic optical reconstruction microscopy (STORM for short). ); 2. Stimulated emission depletion (STED) by changing the point spread function; 3. Structured illumination microscopy (SIM) using structured light illumination to excite fluorescence.

The imaging principles of PALM/STORM are the same, they both use the random excitation of fluorescent molecules to emit fluorescent photons one by one, and obtain their central positions through point spread function digitization, thus breaking through the limitation of imaging resolution by light wave diffraction. Although PALM/STORM uses surface detection imaging, only a small number of fluorescent molecules are imaged at a time, and it is necessary to repeatedly activate-quench fluorescent molecules for imaging. To obtain a complete cell super-resolution image, generally 10,000 images need to be collected. The amount of imaging is large and the imaging time is very long, which makes most experiments done on fixed cells and cannot be used for live cell research.

In the process of STED imaging, a beam of excitation light is used to make the fluorescent material emit light, and at the same time, another high-energy pulse laser is used to emit a beam of overlapping, ring-shaped, and longer-wavelength laser to pass most of the fluorescent material in the first beam spot. The stimulated emission depletion process is quenched, thereby reducing the diffraction area of the fluorescent spot and significantly improving the resolution of the microscope. However, since STED needs to use high-intensity lasers (the required peak power is 400-800 MW/cm2 at the resolution of 50-70 nanometers, and the required laser power density reaches the order of GW/cm2 at the resolution of 5.8 nanometers), high-power lasers are easy to cause Biological tissue damage greatly restricts the application range of STED.

SIM uses a modulated light source to illuminate the sample, and the illumination light and fluorescence undergo frequency mixing and frequency shifting in the frequency domain, encoding the originally indistinguishable high-resolution information into the fluorescence image, and combining calculation and decoding fast Fourier transform to obtain high-resolution information. However, since the illumination light field itself is also limited by the diffraction limit of the system, theoretically SIM can increase the resolution by about one time at most, and the lateral resolution can reach about 100-150nm. In 2005, Gustafsson.etal. [PNAS, 102: 13801 (2005)] proposed a nonlinear structured light illumination super-resolution imaging method (NL-SIM), which can obtain high-frequency information of samples by using the saturated nonlinear characteristics of fluorescent molecules , in theory, any high-resolution fluorescence imaging can be achieved. However, in order to achieve saturated nonlinear fluorescence emission, a high excitation light power density is required, which is easy to cause damage to biological tissues. At the same time, NL-SIM needs to collect data that is several times that of SIM. Only by reconstructing a super-resolution image, the imaging speed is slow, these problems limit the practical application of NL-SIM in the field of biomedicine.

Contents of the invention

In order to solve the above technical problems, the present invention provides a non-linear structured light microscopy imaging method and system, which activates fluorescently labeled samples through a spatially structured light field of low power density, and then uses spatially structured light that is in phase with the activated light field Field excitation of fluorescence and image acquisition can achieve 3 times the optical cut-off frequency imaging, the imaging lateral resolution can reach 60 nanometers, the system has good mechanical stability, and the Z-direction drift is less than 5 nanometers per hour, and the image acquisition speed can reach 100Hz. Realize super-resolution imaging of living cells.

In order to achieve the above object, technical scheme of the present invention is as follows:

A non-linear structured illumination microscopy imaging method, comprising the following steps:

1) Let the digital micromirror array work in the locked mode, load the computational hologram on the digital micromirror array, and the transmittance function of the computational hologram is: where γ represents the degree of modulation, k ₀ represents the spatial frequency, Indicates the initial phase;

2) Let the first laser light with uniform spatial distribution shine on the digital micromirror array, the first laser light is diffracted by the holographic grating on the digital microscope array to each order, and the light of each order is filtered by the Fourier surface of the system composed of lenses , select a pair of positive and negative first-order diffracted lights, and a pair of positive and negative first-order diffracted lights sequentially pass through the lens and the microscope objective, and irradiate the surface of the sample, so that the positive and negative first-order diffracted lights interfere on the protein surface of the sample, resulting in The first spatially structured light field satisfying the sinusoidal distribution for activating the fluorescent protein, the first spatially structured light field irradiates the surface of the sample, so that part of the protein switches from a dark state to a bright state;

3) Repeat step 2) using a second laser with a wavelength greater than that of the first laser in step 2), to obtain a second spatially structured light field that satisfies a sinusoidal distribution for exciting the fluorescent protein, and use the second spatially structured light field to irradiate the sample, causing the fluorescent protein in its bright state to fluoresce, and the fluorescence is collected and imaged in a photodetector;

4) Change the computational hologram loaded on the digital microarray, repeat steps 2) and 3), collect multiple spatial frequencies, collect multiple initial phases in each direction, and obtain multiple original images, and finally use the GPU acceleration algorithm, according to Super-resolution images are reconstructed from multiple original images.

Preferably, in step 4), before adopting the GPU acceleration algorithm to reconstruct the super-resolution image according to multiple original images, a step is also included: judging whether all gratings have been loaded and imaged: if not, return to step 2); if so, judge Whether multiple wavelength windows have been imaged: if not, still return to step 2); if yes, turn off the digital microscope array, stop collecting images, and GPU accelerates image reconstruction.

Preferably, in the above step 2), the system formed by the lens is a 4F system, and the light of each order is filtered on the Fourier plane of the 4F system.

Preferably, the aforementioned first spatially structured light field and the second spatially structured light field have the same phase.

Preferably, the aforementioned pair of positive and negative first-order diffracted lights is a pair of coherent light beams.

Non-linear structured illumination microscope imaging system, which includes:

A light source system (1), comprising at least two lasers (11, 12) with different wavelengths, at least two neutral filters (15, 16), at least two half-wave plates (19, 110), at least two two Chromatic mirrors (113, 114), acousto-optic tunable filters (117), fiber couplers (118) and high-frequency vibrating tables (119), at least one of the lasers (11, 12) is used as an active light source, and at least The other is used as an excitation light source, and the laser light emitted by the laser as the excitation light source and the laser light emitted from the laser as the excitation light source pass through the neutral filter (15, 16), the half-wave plate (19, 110), the dichroic filter in sequence, respectively. Mirrors (113, 114), acousto-optic tunable filters (117), fiber couplers (118) and high-frequency vibrating tables (119), finally obtain at least one active beam and at least one beam propagating in the same optical fiber excitation beam;

Diffraction system (2), at least one active light beam and at least one excitation light beam pass through the fiber coupler (21), lens (22), reflector (23), digital micromirror array sequentially placed along the optical path in the diffraction system respectively (24), lens (25), intensity mask (26), lens (27), reflector (28) and lens (29), each bundle of light all obtains a pair of corresponding positive and negative first-order diffracted light;

The imaging system (3), the first positive and negative first-order diffracted light corresponding to the activation beam generated by the diffraction system (2) and the second positive and negative first-order diffracted light corresponding to the excitation beam pass through the dichroic light in the imaging system (3) in sequence mirror (31) and microscope objective lens (32), and irradiate the surface of the sample, so that the first positive and negative first-order diffracted light and the second positive and negative first-order diffracted light interfere on the surface of the sample protein respectively, generating a spatially structured light field, Wherein the first positive and negative first-order diffracted light generates at least one first spatially structured light field satisfying a sinusoidal distribution for activating the fluorescent protein, and the second positive and negative first-order diffracted light generates at least one first spatially structured light field satisfying a sinusoidal distribution for exciting the fluorescent protein The second spatially structured light field, the first spatially structured light field and the second spatially structured light field cause the sample to emit fluorescence, and the emitted fluorescence is collected by the objective lens (32), passed through the dichroic mirror (31), dichroic mirror ( 34), the interference filter (35), multiple imaging on the detection surface of the photodetector through the tube lens (36), finally adopts the GPU acceleration algorithm by the GPU acceleration data processing subsystem in the imaging system (3), according to multiple Super-resolution images of fluorescent samples are reconstructed from raw images collected by Zhang photodetectors.

Preferably, the above-mentioned GPU accelerated data processing subsystem includes a data acquisition card, a graphics workstation and a graphics processing graphics card. The GPU accelerated data processing subsystem reconstructs the super-resolution of the sample from the raw data collected by the photodetector through the GPU accelerated algorithm. microscopic image.

Preferably, the above-mentioned nonlinear structured illumination microscopic imaging system further includes an electronic viewing control system for realizing synchronous triggering of the acousto-optic tunable filter (117), the digital micromirror array (24) and the photodetector.

Preferably, the above-mentioned light source system (1) is placed on an air flotation shockproof experimental platform, the diffraction system (2) and the imaging system (3) are placed on another air flotation shockproof experimental platform, and the two air flotation shockproof experimental platforms pass through A multimode fiber jumper with a standard FC interface is connected, and a part of the multimode fiber jumper is fixed on the vibration table, which continues to vibrate randomly during the imaging process.

Preferably, the above-mentioned first positive and negative first-order diffracted light and second positive and negative first-order diffracted light are a pair of coherent light beams.

The beneficial effects of the present invention are:

1) The imaging resolution of the system is significantly improved compared with traditional SIM; the present invention uses structured light to activate fluorescent samples in the first step, and uses a structured light field in phase with the activation light field to excite fluorescence in the second step. This two-step structured light activation - The excitation imaging method makes the maximum effective spatial frequency of the system three times that of the traditional optical cutoff frequency, and theoretically the imaging resolution can be increased by up to three times, while the traditional SIM imaging method uses one-step structured light excitation for direct imaging, and the theoretical maximum effective space The frequency can only reach twice the cutoff frequency, and the imaging resolution can only be doubled;

2) High anti-fluorescence drift, low phototoxicity, the activation light field and the excitation light field used in the present invention are all low power density light fields (typical value W/cm2), far lower than traditional NL-SIM and STED (typical The value is MW/cm2), the lower power density weakens the bleaching effect of the laser on the fluorescent molecules, which is beneficial to prolong the fluorescence emission time, and at the same time avoids damage to cells or biological tissues and reduces phototoxicity, which is of great significance for biomedical microscopy imaging important meaning;

3) The imaging speed is fast, and super-resolution imaging of living cells can be realized; the present invention constructs an optical path based on a structured light field of a digital micro-mirror device (DMD for short), and displays a computational hologram on the DMD. The transmittance function can be expressed as: Among them, γ represents the degree of modulation, k ₀ represents the spatial frequency (determines the direction of diffraction and the size of the diffraction angle), Represents the initial phase (determines the intensity distribution of the structured light field) t, and a set of computational holograms with different spatial frequencies and initial phases can be designed to obtain a set of diffracted light fields with different relative phase differences. The spatially evenly distributed laser light is irradiated on the DMD, and is diffracted to various orders by the holographic grating. After passing through the 4f system and filtering on the Fourier surface, the positive and negative first-order diffracted light is selected, and through the combination of the ITRF lens and the microscope objective lens, the The positive and negative first-order diffracted light interferes on the surface of the sample to produce a spatially structured light field that satisfies a sinusoidal distribution; it is worth pointing out that when the DMD is used to generate the structured light field in the patent CN102540446A and other related patents, most of the DMD is used by the imaging system. Direct imaging to the sample surface theoretically produces fringe distribution that does not strictly satisfy the sinusoidal distribution, and the effective spatial frequency is low. However, the structured light field produced by the DMD holographic grating diffraction light interference method set by the present invention strictly satisfies the sinusoidal distribution, and can achieve higher spatial frequencies. In the pixel locking mode, the switching speed is higher than 1KHz. At the same time, a high-speed scientific research-grade SCOMS camera is used, and its highest frame rate also reaches 1KHz, so that the overall image acquisition speed of the system of the present invention is higher than 100Hz, which can meet the needs of live cell imaging.

Description of drawings

FIG. 1 is a schematic flow chart of the non-linear structured illumination microscopy imaging method of the present invention.

Fig. 2 is a structural schematic diagram of the nonlinear structured illumination microscopy imaging system of the present invention.

Fig. 3 is a schematic diagram of the image reconstruction algorithm involved in the nonlinear structured illumination microscopic imaging method and system of the present invention.

detailed description

Preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings.

Fig. 1 is a flowchart of a super-resolution fluorescence imaging method with nonlinear structured light illumination. For the convenience of description, in this embodiment, the activation light specifically refers to a continuous solid-state laser with a wavelength of 405 nm, and the excitation light specifically refers to a continuous solid-state laser with a wavelength of 488 nm. The output power of the two laser beams is adjustable between 0 and 100 mW.

In order to achieve the purpose of the present invention, as shown in Figures 1 and 3, in some embodiments of the non-linear structured illumination microscopic imaging method of the present invention, it includes steps:

Step S101: Uniformly illuminate the sample with excitation light, collect traditional wide-field illumination imaging images through SCOMS, and select the area requiring super-resolution imaging by moving the stage;

Step S102: the computer automatically locks the focus;

Step S103: increasing the power of the excitation light so that all the fluorescent protein molecules are in a dark state and do not emit fluorescence;

Step S104: The control system modifies the working frequency of the AOTF, only allowing the activation light to pass through the AOTF, and then irradiate the DMD, and load a computational hologram on the DMD. The intensity transmittance function of the computational hologram is: Among them, γ represents the degree of modulation, k ₀ represents the spatial frequency (determines the direction of diffraction and the size of the diffraction angle), Represents the initial phase (determines the intensity distribution of the structured light field); the activation light is first diffracted by the calculated hologram to various orders, and then collected by a 4f system consisting of two lenses placed after the DMD, in the Fourier of the 4f system On the leaf surface, an intensity mask is used to select the frequency, and only the positive and negative first-order diffracted light is allowed to pass through. After exiting this 4f system, it passes through a 4f system composed of a TIRF lens and a microscope objective lens. The surface of the sample interferes and produces Periodic illumination light field with cosine distribution, after the activation light is irradiated for 2ms, the AOTF is cut off the activation light;

In step S105, the control system adjusts the working frequency of the AOTF, allowing only the excitation light to pass through the AOTF and irradiate the DMD. After the excitation light is diffracted by the computational hologram, a periodic illumination light field with a cosine distribution is generated on the sample surface, so that in step S104 The activated fluorescent protein molecules emit fluorescence, and the fluorescence intensity distribution at this time is expressed as:

Among them, S(r) represents the spatial distribution function of sample fluorescent protein molecules, represents the spatial frequency of the activating light, Indicates the spatial frequency of the excitation light, it can be seen that the spatial frequency contained in I(r) is components, in the same microscopic imaging system Where k _C represents the optical cut-off frequency of the system, so the imaging system can achieve super-resolution imaging with a maximum of 3 times the optical cut-off frequency. At the same time, trigger SCOMS to collect data and store the data in the memory of the graphics workstation. The data collection ends after the excitation light is irradiated for 2ms, and the AOTF cuts off the excitation light;

Step S106: Determine whether the imaging of the excitation light in the 9 stripe directions is completed, the 9 stripe directions correspond to ₉ different k0 values, and the angles between the azimuth angles of the excitation light in the 9 stripe directions and the x-axis direction are respectively 2Nπ /9, where N=0, 1, 2, .., 8, the initial bit of excitation light in each stripe direction corresponds to 7 different The values are respectively Mπ/7, where M=0, 1, 2, ..., 6, and a total of 63 original images are collected; if 63 original images are not collected, then return to step S103; if 63 are collected original image, proceed to step S107;

Step S107: On the graphics workstation, accelerate the non-linear structured light illumination image processing algorithm through the GPU, and reconstruct the super-resolution fluorescence image of the sample through the 63 original images, the algorithm structure is shown in Figure 3; the activation light and excitation The wavelength of the light can be changed according to the absorption-emission spectral characteristics of the switch protein, or two beams of excitation light with different wavelengths can be set to perform dual-color imaging on biological samples labeled with two switch proteins.

Wherein, in step S103, part of the protein on the surface of the sample is converted from a dark state to a bright state, the dark state is a spatial configuration that cannot be excited by laser light and emits fluorescence, and the bright state is a spatial configuration that can be excited by laser light and emit fluorescence. Among the lenses through which the positive and negative relic diffracted light passes sequentially and the microscope objective lens, the lens can be an ITRF lens.

In order to achieve the object of the present invention, as shown in Figures 2 and 3, in some embodiments of the nonlinear structured illumination microscopy imaging system of the present invention, it includes:

A light source system 1 comprising two or more lasers with different wavelengths 11, 12, two or more neutral filters 15, 16, two or more half-wave plates 19, 110, two or more More dichroic mirrors 113, 114, acousto-optic tunable filter (AOTF) 117, fiber coupler 118 and high-frequency vibrating table 119, high-frequency vibrating table 119 continuously vibrates at a frequency of 5KHz during the imaging process to eliminate laser The spatial coherence makes the illumination light field spatially uniform. One of the lasers 11 and 12 with different wavelengths is used as an active light source, and the other is used as an exciting light source. The laser light emitted from the laser used as the active light source and the laser light emitted from the laser used as the excited light source pass through the neutral filters 15 and 16 respectively. , half-wave plates 19, 110, dichroic mirrors 113, 114, acousto-optic tunable filter (AOTF) 117, fiber coupler 118 and high-frequency vibrating table 119, finally obtain a bundle of activation propagating in the same optical fiber beam and an excitation beam;

Diffraction system 2, an activation beam and an excitation beam generated by light source system 1 respectively pass through the fiber coupler 21, lens 22, mirror 23, digital micromirror array DMD 24, and lens placed sequentially along the optical path in the diffraction system 2 25. Intensity mask 26, lens 27, mirror 28 and TIRF lens 29, each beam of light obtains a pair of corresponding positive and negative first-order diffracted lights, wherein the positive and negative first-order diffracted lights are a pair of coherent beams;

The imaging system 3, the positive and negative first-order diffracted light corresponding to the activation beam generated by the diffraction system 2, and the positive and negative first-order diffracted light corresponding to the excitation beam pass through the dichroic mirror 31 and the microscopic objective lens 32 (100× , NA1.49, oil), irradiate the surface of the sample, and the sample is fixed on the three-dimensional precision motorized stage 33, so that the positive and negative first-order diffracted lights interfere on the surface of the sample protein respectively to generate a spatially structured light field, wherein one beam activates The positive and negative first-order diffracted light corresponding to the beam produces a spatially structured light field satisfying the sinusoidal distribution for activating the fluorescent protein, and the positive and negative first-order diffracted light corresponding to an excitation beam produces a sinusoidal distribution for exciting the fluorescent protein. Spatial structured light field, the structured light field causes the sample to emit fluorescence, and the emitted fluorescence is collected by the objective lens 32, passes through the dichroic mirror 31, the dichroic mirror 34, the interference filter 35, and passes through the tube lens 36. The photodetector detection surface is imaged multiple times, and finally the GPU-accelerated data processing subsystem in the imaging system 3 uses a GPU-accelerated algorithm to reconstruct the super-resolution image of the fluorescent sample based on the original images collected by multiple photodetectors.

Among them, the above-mentioned GPU-accelerated data processing subsystem includes a data acquisition card, a graphics workstation and a graphics processing graphics card. The GPU-accelerated data-processing subsystem reconstructs the super-resolution display of the sample from the original data collected by the photodetector through the GPU-accelerated algorithm. micro image.

Preferably, the above-mentioned non-linear structured illumination microscopic imaging system also includes an electronic viewing control system, which can include a single-chip microcomputer and a switching power supply for realizing an acousto-optic tunable filter (117), a digital micromirror array ( 24) Synchronous triggering with photodetectors.

The above-mentioned light source system (1) is placed on an air flotation shockproof experimental platform, the diffraction system (2) and imaging system (3) are placed on another air flotation shockproof experimental platform, and the two air flotation shockproof experimental platforms pass through a standard FC The multi-mode fiber jumper of the adapter is connected, and a part of the multi-mode fiber jumper is fixed on a 5KHz vibration table, which continues to vibrate randomly during the imaging process.

For the light source subsystem of the light source system, the light source used to activate the fluorescent protein can be a continuous solid-state laser with a wavelength of 405nm, or a laser with other wavelengths or an LED light source (the wavelength selection depends on the type of fluorescent protein used in the experiment) .

The excitation light source used in the system of the present invention to excite fluorescent proteins to emit fluorescence can be 488nm, 514nm, 561nm, 647nm continuous output solid-state lasers, or lasers of other wavelengths. As shown in Figure 2, up to 4 channels can be set in the system at the same time Lasers 11 , 12 , 13 , 14 , for each additional laser path, such as 13 , 14 , neutral filters 17 , 18 , half-wave plates 111 , 112 , and dichroic mirrors 115 , 116 need to be added accordingly.

As shown in Figure 2, the imaging system includes at least one SCMOS 1 photodetector, where the photodetector can also be an EMCCD, and up to four SCMOS photodetectors can be set according to the different fluorescently labeled proteins, and each additional SCMOS For the detection optical path, such as SCMOS2, a dichroic mirror 38 , an interference filter 39 , and a tube lens (Tube Lens) 310 need to be added accordingly.

The light source used to activate the fluorescent protein in the light source system 1 can be a continuous solid-state laser with a wavelength of 405 nm, and lasers with other wavelengths can also be selected according to the type of fluorescent protein used in the experiment.

The above is only the preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the inventive concept of the present invention, some modifications and improvements can also be made, and these all belong to the present invention. protection scope of the invention.

Claims (9)

1. The non-linear structured illumination microscopic imaging method, is characterized in that, comprises the following steps: 1) Make the digital micromirror array work in the locked mode, load the calculation hologram on the digital micromirror array, and the transmittance function of the calculation hologram is: where γ represents the degree of modulation, k ₀ represents the spatial frequency, Indicates the initial phase, r indicates the transmittance; 2) irradiating the first laser light uniformly distributed in space onto the digital micromirror array, the first laser light is diffracted to various orders by the holographic grating on the digital micromirror array, and the light of each order is After the Fourier surface filtering of the system composed of lenses, a pair of positive and negative first-order diffracted lights is selected, and the pair of positive and negative first-order diffracted lights pass through the lens and the microscope objective lens in turn, and irradiate the sample surface, so that the positive and negative first-order diffracted lights The first-order diffracted light interferes on the protein surface of the sample to generate a first spatially structured light field that satisfies the sinusoidal distribution for activating the fluorescent protein, and the first spatially structured light field irradiates the sample surface, causing some proteins to switch from the dark state to bright state; 3) Repeat step 2) using a second laser with a wavelength greater than that of the first laser in step 2), to obtain a second spatially structured light field satisfying a sinusoidal distribution for exciting the fluorescent protein, using the second spatial The structured light field irradiates the sample, causing the fluorescent protein in the bright state to fluoresce, and the fluorescence is collected and imaged in the photodetector; 4) Change the calculation hologram loaded on the digital micromirror array, repeat the 2) and 3) steps, collect multiple spatial frequencies, collect multiple initial phases in each fringe direction, and obtain multiple original images, and finally Using a GPU accelerated algorithm to reconstruct a super-resolution image based on the multiple original images. 2. The non-linear structured illumination microscopic imaging method according to claim 1, characterized in that, in the step 4), the super-resolution image is reconstructed according to the plurality of original images using the GPU acceleration algorithm It also includes steps before: judging whether all gratings have been loaded and imaged: if no, return to step 2); if yes, judge whether multiple wavelength windows have been imaged: if no, still return to step 2); if yes, close the digital The micromirror array stops collecting images, and the GPU accelerates the image reconstruction. 3. The non-linear structured illumination microscopic imaging method according to claim 1, characterized in that, in the step 2), the system formed by the lens is a 4F system, and the light of each order is in the The Fourier surface of the 4F system described above is filtered. 4 . The nonlinear structured illumination microscopy imaging method according to claim 1 , wherein the first spatially structured light field and the second spatially structured light field have the same phase. 5 . The nonlinear structured illumination microscopic imaging method according to claim 1 , wherein the pair of positive and negative first-order diffracted lights is a pair of coherent light beams. 6. A non-linear structured light microscopy imaging system, characterized in that it includes: A light source system (1), comprising at least two lasers (11, 12) with different wavelengths, at least two neutral filters (15, 16), at least two half-wave plates (19, 110), at least two two Chromatic mirrors (113, 114), acousto-optic tunable filters (117), fiber couplers (118) and high-frequency vibration tables (119), at least one of the lasers (11, 12) is used as an active light source, At least one other is used as the excitation light source, and the laser light emitted by the laser as the excitation light source and the laser light emitted from the laser as the excitation light source respectively pass through the neutral filter (15, 16), the half-wave plate (19, 110), the dichroic mirror (113, 114), the acousto-optic tunable filter (117), the fiber coupler (118) and the high-frequency vibrating table (119), finally obtain the same at least one activation beam and at least one excitation beam propagating in an optical fiber; Diffraction system (2), said at least one beam of activation light beam and said at least one beam of excitation beam respectively successively pass through fiber coupler (21), first lens (22), digital micromirror array placed in sequence along the optical path in the diffraction system (24), first mirror (23), second lens (25), intensity mask (26), third lens (27), second mirror (28) and fourth lens (29), each beam The light gets a pair of corresponding positive and negative first-order diffracted light; Imaging system (3), the first positive and negative first-order diffracted light corresponding to the activation beam generated by the diffraction system (2) and the second positive and negative first-order diffracted light corresponding to the excitation beam pass through the first positive and negative first-order diffracted light in the imaging system (3) in sequence a dichroic mirror (31) and a microscope objective lens (32), and irradiate the sample surface, so that the first positive and negative first-order diffracted light and the second positive and negative first-order diffracted light interfere on the sample protein surface respectively, Generate a spatially structured light field, wherein the first positive and negative first-order diffracted light generates at least one first spatial structured light field satisfying a sinusoidal distribution for activating the fluorescent protein, and the second positive and negative first-order diffracted light generates at least one The second spatially structured light field satisfying the sinusoidal distribution for exciting the fluorescent protein, the first spatially structured light field and the second spatially structured light field cause the sample to emit fluorescence, and the emitted fluorescence is collected by the objective lens (32), passed through the second A dichroic mirror (31), a second dichroic mirror (34), an interference filter (35), multiple images on the detection surface of the photodetector through the tube lens (36), and finally pass through the imaging system (3 The GPU-accelerated data processing subsystem in ) uses GPU-accelerated algorithms to reconstruct the super-resolution images of fluorescent samples from the original images collected by multiple photodetectors. 7. The non-linear structured illumination microscopic imaging system according to claim 6, wherein the GPU accelerated data processing subsystem comprises a data acquisition card, a graphics workstation and a graphics processing graphics card, and the GPU accelerated data processing subsystem The system reconstructs the super-resolution microscopic image of the sample from the original data collected by the photodetector through the GPU acceleration algorithm. 8. The non-linear structured illumination microscopy imaging system according to claim 6, is characterized in that, also comprises electronic control system, is used to realize described acousto-optic tunable filter (117), digital micromirror array (24) ) and the synchronous triggering of the photodetector. 9. The non-linear structured illumination micro-imaging system according to claim 6, characterized in that, the light source system (1) is placed on an air flotation shockproof experimental platform, and the diffraction system (2) and the imaging The system (3) is placed on another air flotation anti-vibration experimental platform, and the two air flotation anti-vibration experimental platforms are connected by a multi-mode optical fiber jumper with a standard FC interface, and a part of the multi-mode optical fiber jumper is fixed on the On the vibration table, the random vibration is continued during the imaging process.