A Laser Interference Large-Field-of-View Super-Resolution High-Speed Microscopic Imaging Method

Abstract

The present invention provides a laser interference large-field super-resolution high-speed microscopic imaging method, belonging to the technical field of microscopic imaging. The laser interference large-field super-resolution high-speed microscopic imaging method includes the following steps: making a fluorescent sample by using a fluorescent probe labeling method; constructing a structured light field; collecting a fluorescent image of the fluorescent sample in the structured light field; reconstructing the collected fluorescent image into a super-resolution image; wherein the constructed structured light field includes a two-dimensional grating with fringe projection and an SLM (structured light illumination fluorescence microscopy) for selecting the fringe direction and performing precise phase shift, achieving a large field of view. The present invention breaks through the limitation of the digital projection device on the number of fringes. Compared with the traditional SIM super-resolution imaging method, the main advantage is high-speed and high-precision phase shift; and it avoids the influence of SIM pixelization and dispersion effects.

Description

Technical Field

[0001] The invention belongs to the technical field of microscopic imaging, and in particular relates to a laser interference large-field-of-view super-resolution high-speed microscopic imaging method. Background Art

[0002] With the development of biomedicine and life sciences, microscopic imaging has higher and higher requirements for the exploration of micro-nano biological samples. Micro-nano scientific research is carried out on the basis of higher resolution. However, the lateral resolution limit of ordinary optical microscopes is about 200nm, which makes it difficult to observe biological structures smaller than this characteristic scale. Breaking through the resolution diffraction limit of optical microscopes has become an important technical research direction. In recent years, driven by the demand for nanoscale imaging in biomedicine and life sciences, along with the development of light sources, detectors, new fluorescent probes and new imaging theories, a variety of ultra-high resolution fluorescence imaging technologies that can break through the diffraction limit have been proposed, which has increased the resolution of fluorescence microscopes to below 100nm, bringing us from the era of "micro" microscopes to the era of "nano" microscopes. At present, the field of view of super-resolution microscopes at home and abroad is small, usually around 100-200um, but for the current popular brain science research, a larger field of view is helpful to study the connection between neurons. Therefore, how to achieve super-resolution and a large field of view while having higher accuracy and better convenience is a technical problem that needs to be solved urgently.

[0003] Structured light illumination fluorescence microscopy (SIM) typically uses a digital micromirror device (DMD) or a spatial light modulator to generate and transform structured light patterns in different directions and achieve phase shifts. Using such a projection device can achieve a high frame rate of up to dozens of frames per second. However, the number of fringes that determine the size of the image field of view is limited by the number of pixels of the spatial light modulator (SLM) and DMD. For example, when using a 100X / NA1.4 objective lens and a laser emission wavelength of 532 nm, for a structured light illumination microscope, in order to achieve a 2-fold super-resolution improvement, through diffraction limit calculation, fringes with a structured light period of 190 nm are required, and each fringe can achieve a phase shift and occupy four pixels of the SLM / DMD. Therefore, the SLM / DMD (1920×1080 pixels) can only achieve a field of view of 90×50 um2. Such a small field of view cannot meet many applications, such as super-resolution imaging pictures of tissues and pathological sections. To increase the imaging field of view of SIM, the SIM field of view can be synthesized by mechanically moving the sample, and after taking pictures, the super-resolution image can be synthesized using an image stitching algorithm; furthermore, a wafer or waveguide can also be used to achieve a large field of view, but the implementation is more complex and time-consuming; there is also the use of the projection of a physical grating on the sample to achieve the fringe pattern in a large field of view. However, the change of phase shift and fringe direction is only applied to static samples. The current technical solutions cannot solve the technical problems of high-speed and high-precision phase shift in microscopic imaging and avoiding the influence of SIM pixelization and dispersion effects in biomedical research. Summary of the Invention

[0004] In view of this, the present invention provides a laser interference large-field-of-view super-resolution high-speed microscopic imaging method to solve the technical problems of high-speed and high-precision phase shift in microscopic imaging and avoiding the influence of SIM pixelization and dispersion effects in biomedical research.

[0005] The present invention is implemented as follows:

[0006] The present invention provides a laser interference large-field-of-view super-resolution high-speed microscopic imaging method, which includes the following steps:

[0007] S10: Fabricate a fluorescent sample using the fluorescent probe labeling method;

[0008] S20: Construct a structured light field;

[0009] S30: Collect a fluorescent image of the fluorescent sample in the structured light field;

[0010] S40: Reconstruct the collected fluorescent image into a super-resolution image.

[0011] On the basis of the above technical solution, a laser interference large-field-of-view super-resolution high-speed microscopic imaging method of the present invention can also be improved as follows:

[0012] Among them, the specific steps of the said step S10 include:

[0013] The first step: Open the sterilized packaging of the culture dish in the laminar flow hood, take out the culture dish, add the cell suspension, cover the culture dish lid, place it in the incubator and let it stand, wait until the cells grow to an appropriate density and then take it out for immunofluorescence staining;

[0014] The second step: Aspirate the culture medium, wash the cells with PBS, and then fix the cells with 1 ml of 4% paraformaldehyde PBS solution for 30 min;

[0015] The third step: After 20 minutes, aspirate the fixing solution and add 1 ml of 0.1% Triton X-100 surfactant;

[0016] The fourth step: After 15 minutes, aspirate the Triton, wash the cells and then add BSA for blocking;

[0017] The fifth step: Add the antibody fluorescent stain, aspirate all the reagents, and add the mounting medium for mounting the slide.

[0018] Among them, the said structured light field includes a laser, a polarizer, a first lens, a two-dimensional grating, a second lens, a beam splitter prism, a quarter-wave plate, a spatial light modulator, a third lens, a fourth lens, a pole piece, a fifth lens, a first mirror, a sixth lens, a dichroic mirror, an objective lens, a second mirror, a seventh lens, a camera and a fluorescent sample. The beam emitted by the fiber laser is collimated and emitted after passing through the polarizer and the first lens, and is incident on the surface of the two-dimensional grating. The laser is split by the two-dimensional grating to generate different energy levels. The beams of different energy levels are focused on the liquid crystal panel of the spatial light modulator after passing through the lens and the beam splitter prism. The beam is incident on the 4f system after being reflected by the liquid crystal panel surface of the light modulator and the beam splitter prism. The 4f system is composed of the third lens, the fourth lens and the pole piece coaxially in sequence. The ±1 order beams of the beam passing through the pole piece are parallelly emitted after passing through the fifth lens, are emitted after passing through the first mirror and the sixth lens, and are reflected by the dichroic mirror to the objective lens. A fluorescent sample is arranged behind the objective lens; after being irradiated by the beam passing through the objective lens, the reflected light of the fluorescent sample passes through the objective lens and the dichroic mirror, and is reflected by the second mirror and then focused on the camera through the seventh lens; the said camera is used to observe the fluorescent sample and form a fluorescent sample image.

[0019] Among them, the specific steps of the said S30 are: illuminating the fluorescent sample with three-step phase-shifted structured light respectively, and obtaining 9 low-resolution images based on 3 angles and 3 phases in total.

[0020] Among them, the said S40 specifically includes:

[0021] S41: Perform Fourier transform on the collected fluorescent images to obtain the spectrum of the structured light image;

[0022] S42: Perform spectral translation on the spectrum of the structured light image;

[0023] S43: Normalize the translated spectrum to obtain a normalized spectrum;

[0024] S44: Perform spatial domain OTF stitching on the normalized spectrum to obtain a spatial domain spectrum;

[0025] S45: Perform displacement summation processing on the spatial domain spectrum to obtain a super-resolution image.

[0026] Furthermore, the S41 further includes a pre-step:

[0027] Perform spectral filtering on the collected fluorescence image.

[0028] Furthermore, the S41 further includes:

[0029] Iteratively calculate the spectral translation step so that the correlation of the overlapping region between the 0th and ±1st order spectra is the strongest, and obtain the optimal initial phase.

[0030] Furthermore, the S42 further includes:

[0031] Pad zeros to each order spectrum before spectral translation.

[0032] Furthermore, the S42 further includes:

[0033] Use the generalized Wiener filtering algorithm to superimpose and fuse the high-order spectrum after spectral translation with the 0th order spectrum to obtain an extended spectrum, use a window function to apodize the extended spectrum to eliminate high-frequency interference, and then perform inverse Fourier transform to obtain a super-resolution image.

[0034] Among them, the S40 specifically includes: performing Fourier transform, spectral separation, spectral shift, and spectral stitching on the fluorescence image to obtain a high-resolution image.

[0035] Compared with the prior art, the beneficial effects of a laser interference large-field super-resolution high-speed microscopic imaging method provided by the present invention are: breaking through the limitation of the number of fringes of digital projection equipment, compared with the traditional SIM super-resolution imaging method, the main advantage is high-speed and high-precision phase shift; and avoiding the influence of SIM pixelization and dispersion effects. Brief Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments of the present invention. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0037] Figure 1 is the step flowchart of the present invention;

[0038] Figure 2 is the optical path of super-resolution imaging based on laser interference;

[0039] Figure 3 is the schematic diagram of the light intensity distribution before and after loading the grating;

[0040] Figure 4 is the specific flowchart of the steps to reconstruct the collected fluorescence image into a super-resolution image; Specific Embodiments

[0041] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] It should be noted that: like reference numerals and letters denote like items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0044] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0045] In addition, the terms "first" and "second" are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "a plurality of" means two or more, unless otherwise specifically defined.

[0046] As Figure 1 shown, it is a flowchart of a laser interference large-field super-resolution high-speed microscopic imaging method provided by the present invention. This method includes the following steps:

[0047] S10: Produce a fluorescent sample using the fluorescent probe labeling method;

[0048] S20: Construct a structured light field;

[0049] S30: Collect a fluorescent image of the fluorescent sample in the structured light field;

[0050] S40: Reconstruct the collected fluorescent image into a super-resolution image.

[0051] Among them, in the above technical solution, the specific steps of step S10 include:

[0052] The first step: Open the sterilized packaging of the culture dish in a laminar flow hood, take out the culture dish, add the cell suspension, cover the culture dish lid, place it in an incubator and let it stand. Wait until the cells grow to an appropriate density and then take it out for immunofluorescent staining;

[0053] The second step: Aspirate the culture medium, wash the cells with PBS, and then fix the cells with 1 ml of 4% paraformaldehyde PBS solution for 30 min. Here, PBS is the abbreviation of phosphate buffered saline;

[0054] The third step: After 20 minutes, aspirate the fixing solution, add 1 ml of 0.1% Triton X-100 surfactant. Here, Triton is the abbreviation of polyethylene glycol octyl phenyl ether, also known as a detergent, and X-100 is its model;

[0055] The fourth step: After 15 minutes, aspirate the Triton, wash the cells and then add BSA for blocking. Here, BSA refers to the blocking agent;

[0056] The fifth step: Add the antibody fluorescent staining agent, aspirate all the reagents, and add the blocking agent for sealing the slide.

[0057] Among them, the preparation requirements for the fluorescent sample are as follows:

[0058] (1) Selection of fluorescent dyes: Since super-resolution microscopes use a single-wavelength laser as the light source, when selecting fluorescent dyes, it is necessary to choose according to the wavelength of the laser equipped with the super-resolution microscope. If there are multiple fluorescent dye labels in the same sample, it is also necessary to consider that their emission wavelengths should not overlap as much as possible to avoid crosstalk problems.

[0059] (2) Selection of sample carriers: The high-magnification objective lenses of general super-resolution microscopes are all oil immersion lenses, and their numerical apertures are small, requiring the working distance between the lens and the sample to be no greater than 0.17 mm. Therefore, if the sample to be observed is adherent cells or tissue sections, ordinary glass slides and cover glasses can be used. If it is suspended cells or suspended particles, a special culture dish for super-resolution microscopes can be used to carry the sample for observation.

[0060] (3) Selection of mounting media: If the sample only needs to be observed once and is not a highly quenching fluorescent dye, a glycerol mixture with a certain concentration can be used for mounting. If the sample needs to be placed for a period of time and photographed multiple times, an anti-fluorescence quenching mounting medium should be selected to reduce the loss of fluorescent signals.

[0061] Among them, in the above technical solution, the structured light field includes a laser 1, a polarizer 2, a first lens 3, a two-dimensional grating 4, a second lens 5, a beam splitter prism 6, a quarter-wave plate 7, a spatial light modulator 8, a third lens 9, a fourth lens 10, a mask 11, a fifth lens 12, a first mirror 13, a sixth lens 14, a dichroic mirror 15, an objective lens 16, a second mirror 18, a seventh lens 19, a camera 20, and a fluorescent sample 17. The beam emitted by the fiber laser is collimated and emitted after passing through the polarizer and the first lens, and is incident on the surface of the two-dimensional grating. The laser is split by the two-dimensional grating to generate different energy levels. The beams of different energy levels are focused on the liquid crystal panel of the spatial light modulator after passing through the lens and the beam splitter prism. The beam is incident on the 4f system after being reflected by the liquid crystal panel surface of the light modulator and the beam splitter prism. The 4f system is composed of a third lens, a fourth lens, and a mask coaxially in sequence. The ±1 order beams of the beam passing through the mask are parallelly emitted after passing through the fifth lens, are emitted after passing through the first mirror and the sixth lens, and are reflected by the dichroic mirror to the objective lens. A fluorescent sample is arranged behind the objective lens; after being irradiated by the beam passing through the objective lens, the reflected light of the fluorescent sample passes through the objective lens and the dichroic mirror, and is reflected by the second mirror and then focused on the camera through the seventh lens; the camera is used to observe the fluorescent sample and form a fluorescent sample image.

[0062] Among them, the brand models of the above equipment are as follows:

[0063] Laser: Maiwei Optoelectronics LM405FC-50LM488FC-50MGL-FN-561LM639FC-50

[0064] Polarizer: Thorlabs LPVISE100-A

[0065] Two-dimensional grating: Edmund Optics #49-576

[0066] Dichroic mirror: semrock Di03-R405 / 488 / 561 / 635-t3-25x36

[0067] Objective lens: Olympus UPLXAPO20X

[0068] Camera: Teledyne LT1245RM

[0069] The first to seventh lenses can all adopt conventional lenses;

[0070] As Figure 3 shown, the phase mask on the SLM is divided into 4 quadrants, the ±1 orders cover along the ±45° directions, the ±1 orders can be further filtered through the mask holes, Figures (b) and (c) are the spectral intensity distributions of the mask surface, and the light intensity distributions before and after the grating is loaded onto the SLM can be measured by the camera. Before the grating is loaded onto the quadrant phase mask, the ±1 orders in the orthogonal directions can pass through the four pinholes and generate a two-dimensional lattice pattern ( Figure 3 (b)), after the binary grating is loaded onto the second and fourth quadrants of the SLM, the ±1 orders along the -45° direction will be diffracted by the grating. Except for the zero order, all diffracted orders will be blocked by the pinholes, as Figure 3 (e) shown, the intensities of the remaining energy levels can be ignored because they only account for 4.5% of the total intensity. Figure 3 (f) shows the fringes in one direction. The traditional SIM solution using SLM / DMD can achieve stripe projection within 500, mainly because SLM / DMD only contains a limited number of pixels (phase shift is performed in the way of four pixels per cycle). While the method in this paper only requires a two-dimensional grating and SLM to generate 1760 stripes (22 mm × 80 lines / mm), which is three times that of the traditional SIM, and has a faster rotation and phase shift speed.

[0071] Among them, in the above technical solution, the specific steps of S30 are: illuminating the fluorescent sample with three-step phase-shifted structured light, and obtaining 9 low-resolution images based on 3 angles and 3 phases in total.

[0072] Among them, in the above technical solution, S40 specifically includes:

[0073] S41: Performing Fourier transform on the collected fluorescent images to obtain the spectrum of the structured light image;

[0074] Among them, the collected fluorescent images can be mathematically described as:

[0075]

[0076] In the formula, r is the Cartesian coordinate of the sample space, and S(r) is the intensity of fluorescent molecules in the object to be measured. represents convolution, H(r) is the point spread function (PSF) of the imaging system, and I SI (r) is the intensity distribution of the excitation light I0, m, p, and respectively represent the average intensity of the excitation light, the modulation depth, the spatial frequency, and the initial phase;

[0077] The spatial spectrum of the fluorescence image includes a wide-field spectrum and a mixed spectrum generated by the superposition of high-frequency information beyond the diffraction limit. After performing Fourier transform on the above formula, the spectrum of the structured light image is obtained:

[0078]

[0079] where S(k) is the Fourier transform of S(r), H(k) is the optical transfer function (OTF) of the imaging system, and D1(k), D2(k), and D3(k) are the spectra of the structured light images under three different initial phases;

[0080] S42: Perform spectral translation on the spectrum of the structured light image. The translated spectrum is expressed as:

[0081]

[0082] S43: Assume that the substance is isotropic, that is, the spectral components at each position are uniform. Normalize the translated spectrum to obtain a normalized spectrum. The normalized spectrum obtained by normalizing the spectral components of the image in the above formula is expressed as;

[0083]

[0084] S44: Perform spatial OTF stitching on the normalized spectrum. Add the left components in the three expressions in formula (4). The super-resolution image can be obtained through inverse Fourier transform, and its spatial expression is;

[0085]

[0086] It can be seen from the above formula that for the spatial domain algorithm, the change in the direction of the structured light stripe directly acts on the OTF. That is to say, the equivalent OTF result of the super-resolution system is obtained through the translation and stitching of the left and right of the OTF.

[0087] When the light field illuminated by the structured light generates a displacement (δ j represents the j-th displacement), formula (1) is expressed as

[0088]

[0089] The convolution form of the above formula is expressed as:

[0090] D j (r) = ∫S(r′)I SI (r′ - δ j )H(r - r′)dr′ (7)

[0091] S45: Perform displacement summation processing on the spatial domain spectrum to obtain a super-resolution image. The specific steps are as follows:

[0092] Act on the structured light image of the response displacement with different weight functions B j (r) and perform summation, and we can get

[0093]

[0094] where N is the total number of displacements.

[0095]

[0096] In the formula, D SDR (r) is the super-resolution image, which can be expressed as the convolution of the object S(r) and the function P(r), thus forming a new optical imaging system. P(r) is equivalent to the point spread function PSF of the system. Let N = 3, T(r) be a cosine function, and the light field distribution of the structured light is Substitute them into Equation (9) together to obtain the weight function, and then substitute it into Equation (8) to obtain the super-resolution image.

[0097] Furthermore, in the above technical solution, S41 also includes a pre-step:

[0098] Perform spectral filtering on the collected fluorescence image.

[0099] Specifically: Set a notch filter distributed along the axis to perform spectral filtering on the original fluorescence image and then perform the next structured light parameter calibration, effectively reducing the artifacts in the reconstructed image.

[0100] Furthermore, in the above technical solution, S41 also includes:

[0101] Iteratively calculate the spectral translation step to make the correlation of the overlapping region between the 0th order and the ±1st order spectra the strongest, and obtain the optimal initial phase.

[0102] Furthermore, in the above technical solution, S42 also includes:

[0103] Pad zeros to each order spectrum before spectral translation.

[0104] Further, in the above technical solution, S42 further includes:

[0105] Using the generalized Wiener filtering algorithm, the high-order spectrum after spectrum translation is superimposed and fused with the 0-level spectrum to obtain an extended spectrum. A window function is used to apodize the extended spectrum to eliminate high-frequency interference, and then the inverse Fourier transform is performed to obtain a super-resolution image.

[0106] On the other hand, the steps of S40 specifically include: performing Fourier transform, spectrum separation, spectrum shift, and spectrum splicing on the fluorescence image to obtain a high-resolution image.

[0107] As described above, only the specific embodiments of the present invention are provided, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A laser interference large-field super-resolution high-speed microscopic imaging method, characterized in that, It includes the following steps: S10: Making a fluorescence sample by using the fluorescence probe labeling method; S20: Constructing a structured light field; S30: Collecting a fluorescence image of the fluorescence sample in the structured light field. Specifically, the fluorescence sample is illuminated separately by three-step phase-shifting structured light, and a total of 9 low-resolution images are obtained based on 3 angles and 3 phases; S40: Reconstructing the collected fluorescence image into a super-resolution image, specifically including: S41: Performing a Fourier transform on the collected fluorescence image to obtain the spectrum of the structured light image; S42: Performing spectrum translation on the spectrum of the structured light image; S43: Performing normalization processing on the translated spectrum to obtain a normalized spectrum; S44: Performing spatial domain OTF stitching on the normalized spectrum to obtain a spatial domain spectrum; S45: Performing displacement summation processing on the spatial domain spectrum to obtain a super-resolution image; The structured light field includes a laser, a polarizer, a first lens, a two-dimensional grating, a second lens, a beam splitter prism, a quarter-wave plate, a spatial light modulator, a third lens, a fourth lens, a mask, a fifth lens, a first mirror, a sixth lens, a dichroic mirror, an objective lens, a second mirror, a seventh lens, a camera, and a fluorescence sample. The beam emitted by the fiber laser is collimated and emitted after passing through the polarizer and the first lens, and is incident on the surface of the two-dimensional grating. The laser is split by the two-dimensional grating to generate different energy levels. The beams of different energy levels are focused on the liquid crystal panel of the spatial light modulator after passing through the lens and the beam splitter prism. The beam is incident on the 4f system after being reflected by the liquid crystal panel surface of the light modulator and the beam splitter prism. The 4f system is composed of a third lens, a fourth lens, and a mask coaxially in sequence. The ±1 order beams of the beam passing through the mask are emitted parallel after passing through the fifth lens, pass through the first mirror and the sixth lens, and are reflected by the dichroic mirror to the objective lens, and a fluorescence sample is arranged behind the objective lens; after the fluorescence sample is irradiated by the beam passing through the objective lens, the reflected light of the fluorescence sample passes through the objective lens and the dichroic mirror, and is reflected by the second mirror and then focused on the camera through the seventh lens; the camera is used to observe the fluorescence sample and form a fluorescence sample image.

2. The method for laser interference large-field super-resolution high-speed microscopic imaging according to claim 1, wherein The specific steps of the step S10 include: The first step: Open the sterilization package of the culture dish in a laminar flow hood, take out the culture dish, add cell suspension, cover the culture dish lid, place it in an incubator and let it stand, wait until the cells grow to an appropriate density and then take it out for immunofluorescence staining; The second step: Aspirate the culture medium, wash the cells with PBS, and then fix the cells with 1 ml of 4% paraformaldehyde PBS solution for 30 min; The third step: After 20 minutes, aspirate the fixing solution and add 1 ml of 0.1% Triton X-100 surfactant; The fourth step: After 15 minutes, aspirate the Triton, wash the cells and then add BSA for blocking; The fifth step: Add an antibody fluorescence staining agent, aspirate all the reagents, and add a mounting medium for mounting the slide.

3. The method for laser interference large-field super-resolution high-speed microscopic imaging according to claim 2, wherein, The S41 further includes a pre-step: performing spectrum filtering on the collected fluorescence image.

4. The method for laser interference large-field super-resolution high-speed microscopic imaging according to claim 3, characterized in that, The S41 further includes: repeatedly performing iterative calculations on the spectrum translation step so that the correlation of the overlapping region between the 0th order and the ±1st order spectra is the strongest, and obtaining an optimal initial phase.

5. The method for laser interference large-field super-resolution high-speed microscopic imaging according to claim 4, wherein The S42 further includes: padding zeros to each order of spectrum before spectrum translation.

6. The method for laser interference large-field super-resolution high-speed microscopic imaging according to claim 5, wherein, The S42 further includes: using the generalized Wiener filtering algorithm to superimpose and fuse the high-order spectrum after spectrum translation with the 0-level spectrum to obtain an extended spectrum, using a window function to apodize the extended spectrum to eliminate high-frequency interference, and then performing an inverse Fourier transform to obtain a super-resolution image.

Images