A kind of method and device based on mirror interference enhancement multi-focal wide-field super-resolution microscopic imaging

Abstract

The application discloses a kind of multi-focal wide-field super-resolution microscopic imaging method and device based on mirror interference enhancement, specifically, sparse multi-focal excitation point array is formed by digital micromirror modulation or spatial light modulator using excitation light, due to the effect of mirror, the axial size of focused Gaussian spot is compressed and light intensity is significantly increased, the imaging tomography ability and image signal-to-noise ratio are greatly improved, and the time resolution of imaging and fluorescence collection efficiency are also improved.Switching point array position can realize multi-focal scanning of sample and acquire signal through camera, and high signal-to-noise ratio super-resolution image can be obtained in a short time by using computer for a series of post-processing.Based on the above method, a multi-focal wide-field super-resolution microscopic imaging device composed of multi-focal excitation point array generation module, wide-field microscopic imaging module, mirror sample module and signal acquisition module is built, to realize high signal-to-noise ratio, simple operation super-resolution microscopic imaging technology under high time resolution.

Description

Technical Field

[0001] This invention relates to the technical field of optical microscopy imaging, and in particular to a multifocal wide-field super-resolution microscopy imaging method and apparatus based on mirror interference enhancement. Background Technology

[0002] Optical super-resolution microscopy is an important tool in biomedical research. Multifocal structured illumination microscopy, in particular, has great application potential due to its larger imaging range and higher imaging speed. However, there is still significant room for improvement in imaging speed. In the process of generating sparse excitation lattices, the sparser the lattice spacing, the more scanning frames are required, resulting in a higher signal-to-noise ratio for the final reconstructed image. However, this also means longer imaging time, which is detrimental to real-time sample observation and can lead to photobleaching, causing distortion in the reconstructed image.

[0003] Therefore, given the problems with this technology, there is an urgent need to develop a method that can improve the imaging signal-to-noise ratio while maintaining temporal resolution. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide a multifocal wide-field super-resolution microscopy imaging method and apparatus based on mirror interference enhancement, which improves the signal-to-noise ratio of sample images while maintaining a high level of temporal resolution.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement, the method comprising the following steps: The S1 laser emits excitation light, which is expanded twice by a double convex lens and then illuminates a digital micromirror device or a spatial light modulator. The reflected beam modulated by the digital micromirror device or spatial light modulator converges on the back focal plane of the 4f filter system to form a sparse excitation lattice. Its position coincides with the front focal plane of the microscope excitation optical path tube, ensuring that the optical reflection surface of the digital micromirror device is conjugate with the front focal plane of the excitation optical path tube. The excitation lattice then passes through a microscopic system consisting of the excitation optical path tube and the objective lens, and finally obtains a sparse multifocal excitation lattice conjugate with the optical reflection surface of the digital micromirror device on the back focal plane of the objective lens. After the Gaussian spot focused by S2 exits from the objective lens, due to the effect of the mirror, the part of the focused spot that is not incident on the mirror surface and the part reflected from the mirror surface interfere with each other, forming an interference enhancement point on the surface of the mirror with an axial size compression of 4 times and a light intensity increase of about 3.5 times. The S3 uses fluorescent probes to label samples and places them on the surface of a mirror. By controlling a digital micromirror device or a spatial light modulator to switch the position of the excitation array, the camera simultaneously records the fluorescence signal at each position, achieving the effect of multi-focal parallel scanning. Due to the enhanced effect of mirror interference, the image signal-to-noise ratio is improved, which can effectively reduce the number of scanning steps and improve the temporal resolution by 2-3 times. Digital pinhole filtering, pixel relocation, image superposition, and deconvolution processing are performed on a series of multi-focal excited sample images to reconstruct a high signal-to-noise ratio super-resolution image.

[0006] It should be noted that, due to the effect of the mirror, the portion of the focused spot that is not incident on the mirror surface and the portion reflected from the mirror surface in the sparse multifocal excitation lattice self-interfere on the sample surface, forming an interference enhancement point with an axial size compressed by 4 times on the mirror surface. The smaller axial size of the spot greatly reduces the defocus background signal and effectively improves the optical tomography capability of the multifocal wide-field microscope.

[0007] It should be noted that, due to the effect of the mirrors, the portion of the focused spot that is not incident on the mirror surface and the portion reflected from the mirror surface in the sparse multifocal excitation array undergo self-interference on the sample surface, forming an interference enhancement point with an increase in light intensity of about 3.5 times on the mirror surface. At the same time, the addition of the mirrors can further improve the fluorescence collection efficiency. Therefore, the signal-to-noise ratio of the image after fluorescence signal enhancement will be significantly improved. The high signal-to-noise ratio image can effectively reduce the impact of fluorescence crosstalk on multifocal reconstruction, thereby allowing for a smaller focal spacing to be set for multifocal parallel scanning and improving the temporal resolution of imaging.

[0008] It should be noted that the reconstruction of the multifocal excited sample image in step S3 includes: ① Locate the positions of all excitation points and add digital pinholes for spatial filtering; ② Perform pixel relocation on the image after pinhole filtering, i.e., interpolate to obtain an image with twice the number of original pixels; ③ Overlay all the images processed in steps ① and ② to obtain a reconstructed image with 1.5 times higher resolution than a wide-field microscope; ④ The superimposed images are deconvolved to obtain a super-resolution image with a resolution improvement of 2 times compared to a wide-field microscope.

[0009] This invention also provides a multifocal wide-field super-resolution microscopy imaging device based on mirror interference enhancement. The device includes a multifocal excitation array generation module, a wide-field microscopy imaging module, a mirror sample module, and a signal acquisition module. The excitation light generation module generates a sparse multifocal excitation array and switches between different excitation array positions. After passing through the mirror interference module, the excitation array undergoes interference enhancement. The signal acquisition module then acquires the fluorescence signal generated by the scanned excitation of the sample.

[0010] It should be noted that the multifocal excitation lattice generation module includes a continuous laser, a biconvex lens beam expander system, a reflector, a digital micromirror device (DMM) or a spatial light modulator, and a 4f filtering system. The excitation beam emitted from the continuous laser is expanded to twice its original diameter by the beam expander system, and then the reflector changes the beam direction, causing it to illuminate the DMM at a 24° incident angle. The DMM consists of several miniature square reflectors, which can be rapidly switched between ±12° via hinges below the reflectors. When the incident light forms a 24° angle with the normal to the DMM surface, the micro-reflectors reflect the illumination light into the subsequent 4f filtering system, filtering out excess diffraction-level reflected light to prevent stray light interference. The controlled reflected beam converges on the back focal plane of the 4f filtering system lens to form a sparse excitation lattice, and its position coincides with the front focal plane of the excitation optical path tube of the wide-field microscopy imaging module, ensuring that the optical reflecting surface of the DMM is conjugate to the front focal plane of the excitation optical path tube.

[0011] It should be noted that the wide-field microscopy imaging module includes an excitation optical path tube, a dichroic mirror, and an objective lens; the sparse lattice generated by the multi-focal excitation lattice generation module is focused by the excitation optical path tube and then by the dichroic mirror, which can be used to separate the excitation light and fluorescence, before being focused by the objective lens onto the fluorescent probe-labeled reflector sample module; the sparse lattice generated by the focal excitation lattice generation module is conjugate with the sample surface, and after passing through the microscopy system composed of the excitation optical path tube and the objective lens, its size is reduced to 1 / 167 of the original before being imaged onto the sample surface.

[0012] It should be noted that the reflector sample module consists of, from top to bottom, a glass / silicon substrate, a silver / gold plating layer, a silicon dioxide protective layer, a sample labeled with a fluorescent probe, and a cover glass.

[0013] It should be noted that the signal acquisition module includes a coaxially placed fluorescence optical path tube, a filter, and a camera. The fluorescence signal generated after the sample is excited by the enhanced excitation mode is filtered by a two-way mirror and a filter, then focused onto the detection surface by the fluorescence optical path tube. The camera acquires the signal and sends it to the computer. By switching the position of the excitation point array to scan the sample, a series of multifocal excited sample images are collected. Finally, the computer performs digital pinhole filtering, pixel relocation, image overlay, and deconvolution processing on the sample images to reconstruct a high signal-to-noise ratio super-resolution image.

[0014] The beneficial effects of this invention are as follows: It provides a multifocal wide-field super-resolution microscopy imaging method and apparatus enhanced by mirror interference. By using mirror interference of a sparse multifocal excitation array, an excitation mode with a 4-fold compression of axial size and a 3.5-fold increase in light intensity is obtained. The reduction in axial size significantly reduces defocus background signal and effectively improves the optical tomography capability (~110 nm) of the multifocal wide-field microscope. After fluorescence signal enhancement, the image signal-to-noise ratio is significantly improved through computer reconstruction. The high signal-to-noise ratio image effectively reduces the impact of fluorescence crosstalk on multifocal reconstruction, allowing for smaller focal spacing for parallel multifocal scanning and improving the temporal resolution of imaging by approximately 2-3 times. Attached Figure Description

[0015] Figure 1 This is the optical path diagram of the multifocal wide-field super-resolution microscopy imaging device based on mirror interference enhancement provided by the present invention; Figure 2 a and Figure 2 b is the diffusion function of the axial illumination point of the microscope under the conditions of no mirror and with mirror; Figure 2 c represents the normalized intensity distribution of the diffusion function of the axial illumination point under conditions with and without a mirror. Figure 3 a is a schematic diagram of the scanning mode of the sparse multifocal excitation array in the multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement provided by the present invention; Figure 3 b is an experimental diagram of the sparse multifocal excitation lattice in the multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement provided by the present invention; Figure 4 a and Figure 4 b is the super-resolution star pattern image with and without a mirror when the number of multifocal scanning frames is 80 frames simulated by computer in Example 1. Figure 4 c is the super-resolution star image in Example 2, where the number of scanning frames is increased to 224 by computer simulation and no mirror is added; Figure 5 a and Figure 5 b represents the single-particle super-resolution images in Example 2, with and without a mirror, when the number of multifocal scanning frames is 80 frames simulated by computer. Figure 5 c is a single-particle super-resolution image in Example 3, obtained by increasing the focal distance through computer simulation to achieve 224 scan frames without adding a mirror. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings, but this is not intended to limit the scope of the invention.

[0017] This invention relates to a multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement, the method comprising the following steps: The S1 laser emits excitation light, which is expanded twice by a double convex lens and then illuminates a digital micromirror device or a spatial light modulator. The reflected beam modulated by the digital micromirror device or spatial light modulator converges on the back focal plane of the 4f filter system to form a sparse excitation lattice. Its position coincides with the front focal plane of the microscope excitation optical path tube, ensuring that the optical reflection surface of the digital micromirror device is conjugate with the front focal plane of the excitation optical path tube. The excitation lattice then passes through a microscopic system consisting of the excitation optical path tube and the objective lens, and finally obtains a sparse multifocal excitation lattice conjugate with the optical reflection surface of the digital micromirror device on the back focal plane of the objective lens. After the Gaussian spot focused by S2 exits from the objective lens, due to the effect of the mirror, the part of the focused spot that is not incident on the mirror surface and the part reflected from the mirror surface interfere with each other, forming an interference enhancement point on the surface of the mirror with an axial size compression of 4 times and a light intensity increase of about 3.5 times. The S3 uses fluorescent probes to label samples and places them on the surface of a mirror. By controlling a digital micromirror device or a spatial light modulator to switch the position of the excitation array, the camera simultaneously records the fluorescence signal at each position, achieving the effect of multi-focal parallel scanning. Due to the enhanced effect of mirror interference, the image signal-to-noise ratio is improved, which can effectively reduce the number of scanning steps and improve the temporal resolution by 2-3 times. Digital pinhole filtering, pixel relocation, image superposition, and deconvolution processing are performed on a series of multi-focal excited sample images to reconstruct a high signal-to-noise ratio super-resolution image.

[0018] It should be noted that, due to the effect of the mirror, the portion of the focused spot that is not incident on the mirror surface and the portion reflected from the mirror surface in the sparse multifocal excitation array self-interfere on the sample surface, forming an interference enhancement point with an axial size compressed by 4 times on the mirror surface. The smaller axial size of the spot greatly reduces the defocus background signal and effectively improves the optical tomography capability of the multifocal wide-field microscope, while achieving an axial resolution of approximately 110 nm.

[0019] It should be noted that, due to the effect of the mirrors, the portion of the focused spot that is not incident on the mirror surface and the portion reflected from the mirror surface in the sparse multifocal excitation array undergo self-interference on the sample surface, forming an interference enhancement point with an increase in light intensity of about 3.5 times on the mirror surface. At the same time, the addition of the mirrors can further improve the fluorescence collection efficiency. Therefore, the signal-to-noise ratio of the image after fluorescence signal enhancement will be significantly improved. The high signal-to-noise ratio image can effectively reduce the impact of fluorescence crosstalk on multifocal reconstruction, thereby allowing for a smaller focal spacing to be set for multifocal parallel scanning and improving the temporal resolution of imaging.

[0020] It should be noted that the reconstruction of the multifocal excited sample image in step S3 includes: ① Locate the positions of all excitation points and add digital pinholes for spatial filtering; ② Perform pixel relocation on the image after pinhole filtering, i.e., interpolate to obtain an image with twice the number of original pixels; ③ Overlay all the images processed in steps ① and ② to obtain a reconstructed image with 1.5 times higher resolution than a wide-field microscope; ④ The superimposed images are deconvolved to obtain a super-resolution image with a resolution improvement of 2 times compared to a wide-field microscope.

[0021] This invention also provides a multifocal wide-field super-resolution microscopy imaging device based on mirror interference enhancement. The device includes a multifocal excitation array generation module, a wide-field microscopy imaging module, a mirror sample module, and a signal acquisition module. The excitation light generation module generates a sparse multifocal excitation array and switches between different excitation array positions. After passing through the mirror interference module, the excitation array undergoes interference enhancement. The signal acquisition module then acquires the fluorescence signal generated by the scanned excitation of the sample.

[0022] It should be noted that the multifocal excitation lattice generation module includes a continuous laser, a biconvex lens beam expander system, a reflector, a digital micromirror device (DMM) or a spatial light modulator, and a 4f filtering system. The excitation beam emitted from the continuous laser is expanded to twice its original diameter by the beam expander system, and then the reflector changes the beam direction, causing it to illuminate the DMM at a 24° incident angle. The DMM consists of several miniature square reflectors, which can be rapidly switched between ±12° via hinges below the reflectors. When the incident light forms a 24° angle with the normal to the DMM surface, the micro-reflectors reflect the illumination light into the subsequent 4f filtering system, filtering out excess diffraction-level reflected light to prevent stray light interference. The controlled reflected beam converges on the back focal plane of the 4f filtering system lens to form a sparse excitation lattice, and its position coincides with the front focal plane of the excitation optical path tube of the wide-field microscopy imaging module, ensuring that the optical reflecting surface of the DMM is conjugate to the front focal plane of the excitation optical path tube.

[0023] It should be noted that the wide-field microscopy imaging module includes an excitation optical path tube, a dichroic mirror, and an objective lens; the sparse lattice generated by the multi-focal excitation lattice generation module is focused by the excitation optical path tube and then by the dichroic mirror, which can be used to separate the excitation light and fluorescence, before being focused by the objective lens onto the fluorescent probe-labeled reflector sample module; the sparse lattice generated by the focal excitation lattice generation module is conjugate with the sample surface, and after passing through the microscopy system composed of the excitation optical path tube and the objective lens, its size is reduced to 1 / 167 of the original before being imaged onto the sample surface.

[0024] It should be noted that the reflector sample module consists of, from top to bottom, a glass / silicon substrate, a silver / gold plating layer, a silicon dioxide protective layer, a sample labeled with a fluorescent probe, and a cover glass.

[0025] It should be noted that the signal acquisition module includes a coaxially placed fluorescence optical path tube, a filter, and a camera. The fluorescence signal generated after the sample is excited by the enhanced excitation mode is filtered by a two-way mirror and a filter, then focused onto the detection surface by the fluorescence optical path tube. The camera acquires the signal and sends it to the computer. By switching the position of the excitation point array to scan the sample, a series of multifocal excited sample images are collected. Finally, the computer performs digital pinhole filtering, pixel relocation, image overlay, and deconvolution processing on the sample images to reconstruct a high signal-to-noise ratio super-resolution image.

[0026] Example 1 The device of the present invention, such as Figure 1 As shown, it includes a multifocal excitation array generation module, a wide-field microscopic imaging module, a mirror sample module, and a signal acquisition module.

[0027] The multifocal excitation lattice generation module includes a continuous laser 1, a biconvex lens beam expander 2, a reflector 3, a digital micromirror device (DMD) or spatial light modulator 4, and a 4f filtering system 5. The excitation light emitted from the continuous laser is expanded in diameter by the beam expander and then its direction is changed by the reflector, causing it to illuminate the DMD at a 24° incident angle. The DMD is composed of millions of miniature square reflectors, which can be rapidly switched between ±12° via hinges below the reflectors, corresponding to 'on' and 'off' in the display mode. When the incident light makes a 24° angle with the normal to the DMD surface, the micromirrors in the 'on' state reflect the illumination light into the subsequent 4f filtering system, filtering out excess diffraction-level reflected light. The controlled reflected beam converges on the back focal plane of the 4f filter system lens to form a sparse excitation lattice, and its position coincides with the front focal plane of the excitation optical path tube of the wide field microscopy imaging module, so as to ensure that the optical reflecting surface of the digital micromirror device is conjugate with the front focal plane of the excitation optical path tube.

[0028] The wide-field microscopy imaging module includes an excitation optical path tube (6), a dichroic mirror (7), and an objective lens (8). The sparse lattice generated by the multifocal excitation lattice generation module is focused by the excitation optical path tube and, after passing through a dichroic mirror used to separate the excitation light from the fluorescence, is focused by the objective lens onto the fluorescent probe-labeled reflector sample module. The sparse lattice generated by the focal excitation lattice generation module is conjugate with the sample surface. After passing through the microscopy system composed of the excitation optical path tube and the objective lens, its size is reduced to 1 / 167th of its original size before being imaged onto the sample surface. Figure 3 b illustrates the multifocal excitation modes of the system.

[0029] The reflector sample module 9, from top to bottom, consists of a glass / silicon substrate, a silver / gold plating layer, a silicon dioxide protective layer, a sample labeled with a fluorescent probe, and a coverslip. Figure 2 The effect of mirror interference on the focused spot is demonstrated. The signal acquisition module includes a coaxially placed fluorescence optical path tube 10, a filter 11, and a camera 12. The fluorescence signal generated after the sample is excited by the enhanced excitation mode is filtered by the dichroic mirror and the filter, and then focused onto the detector surface by the fluorescence optical path tube. The camera acquires the signal and sends it to the computer. By switching the position of the excitation point array to scan the sample, a series of multifocal excited sample images are collected. Finally, the computer performs digital pinhole filtering, pixel relocation, image overlay, and deconvolution processing on the sample images to reconstruct a high signal-to-noise ratio super-resolution image.

[0030] Example 2 This embodiment demonstrates the effect of super-resolution reconstruction of star-shaped sample images based on a multifocal wide-field super-resolution microscopy method enhanced by mirror interference.

[0031] Using computer programming software, a Gaussian spot with diffraction-limited single excitation wavelength is made... Figure 3 The pixels shown in image 'a' are convolved to obtain a simulated sparse multifocal excitation matrix. This excitation matrix is ​​then multiplied by the star-shaped image to simulate the sample excitation process in the experiment. Additionally, a single Gaussian spot with diffraction-limited radiation wavelength is generated and convolved with the multiplied sample image to simulate the sample image obtained by microscopic detection. Finally, the 'imnoise' function is used to add Gaussian noise to the sample image to simulate background noise in a real-world environment. Figure 3 The scanning pattern shown in a is used to scan the star pattern, resulting in a series of sample images.

[0032] The series of sample images generated by the simulated multifocal excitation need to be post-processed before they can be reconstructed to obtain super-resolution images. The steps include: (1) Locate the positions of all excitation points and add digital pinholes for spatial filtering; (2) Perform pixel relocation on the image after pinhole filtering, that is, interpolate to obtain an image with twice the number of original pixels; (3) Overlay all the images processed by steps (1) and (2) to obtain a reconstructed image with a resolution improvement of 1.5 times for a wider field microscope; (4) Perform deconvolution processing on the superimposed image to obtain a super-resolution image with a resolution improvement of 2 times that of a wide-field microscope.

[0033] In this embodiment, the signal-to-noise ratio (SNR) of the reconstructed image is used to measure the reconstruction quality of the star pattern. The SNR is defined as: (maximum value of the sample image - average value of Gaussian noise) / standard deviation of Gaussian noise. To simulate the effect of mirror interference, the value of the Gaussian spot is set to 3.5 times its original value, simulating an interference enhancement lattice with 3.5 times the light intensity enhancement under the action of a mirror. The subsequent post-processing steps are the same as those described above. Based on the definition of SNR, the SNR of the simulated super-resolution reconstructed image is improved by about 10-20 after mirror interference. The number of scanning frames is 80 frames. The super-resolution star pattern images without and with mirror interference are shown below. Figure 4 a and Figure 4 As shown in b. High signal-to-noise ratio images can effectively reduce the impact of fluorescence crosstalk on multifocal reconstruction, thus allowing for smaller focal spacing to be set for parallel multifocal scanning, improving the temporal resolution of imaging. For comparison, Figure 4 c represents the reconstruction effect of the star-shaped sample image at 224 frames (with a larger focal spacing). Therefore, when achieving a similar signal-to-noise ratio, the mirror effect can improve the temporal resolution by approximately 2 to 3 times.

[0034] Example 3 This embodiment demonstrates the effect of super-resolution reconstruction of single-particle sample images based on a multifocal wide-field super-resolution microscopy method enhanced by mirror interference.

[0035] Using computer programming software, a Gaussian spot with diffraction-limited single excitation wavelength is made... Figure 3 The pixels shown in image 'a' are convolved to obtain a simulated sparse multifocal excitation matrix. This excitation matrix is ​​then multiplied by the star-shaped image to simulate the sample excitation process in the experiment. Additionally, a single Gaussian spot with diffraction-limited radiation wavelength is generated and convolved with the multiplied sample image to simulate the sample image obtained by microscopic detection. Finally, the 'imnoise' function is used to add Gaussian noise to the sample image to simulate background noise in a real-world environment. Figure 3 The scanning pattern shown in a is used to scan the star pattern, resulting in a series of sample images.

[0036] The series of sample images generated by the simulated multifocal excitation need to be post-processed before they can be reconstructed to obtain super-resolution images. The steps include: (1) Locate the positions of all excitation points and add digital pinholes for spatial filtering; (2) Perform pixel relocation on the image after pinhole filtering, that is, interpolate to obtain an image with twice the number of original pixels; (3) Overlay all the images processed by steps (1) and (2) to obtain a reconstructed image with a resolution improvement of 1.5 times for a wider field microscope; (4) Perform deconvolution processing on the superimposed image to obtain a super-resolution image with a resolution improvement of 2 times that of a wide-field microscope.

[0037] In this embodiment, the reconstruction quality of a single-particle image is measured by the signal-to-noise ratio (SNR) of the reconstructed image. The SNR is defined as: (maximum value of the sample image - average value of Gaussian noise) / standard deviation of Gaussian noise. To simulate the effect of mirror interference, the value of the Gaussian spot is set to 3.5 times its original value, simulating an interference enhancement lattice with 3.5 times increased light intensity under the action of a mirror. The subsequent post-processing steps are the same as those described above. Based on the definition of SNR, the SNR of the simulated super-resolution reconstructed image is improved by about 10-20 after mirror interference. The number of scanning frames is 80 frames. The single-particle super-resolution images without and with mirror interference are shown below. Figure 5 a and Figure 5 As shown in b. High signal-to-noise ratio images can effectively reduce the impact of fluorescence crosstalk on multifocal reconstruction, thus allowing for smaller focal spacing to be set for parallel multifocal scanning, improving the temporal resolution of imaging. For comparison, Figure 5 c shows the reconstruction effect of the single-particle image when the frame rate is 224 frames (with a larger focal spacing). Therefore, when achieving a similar signal-to-noise ratio, the mirror effect can improve the temporal resolution by about 2 to 3 times, while achieving an axial resolution of about 110 nm.

[0038] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations to these embodiments without departing from the principles and spirit of the present invention still fall within the protection scope of the present invention.

Claims

1. A multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement, characterized in that, The method includes the following steps: The S1 laser emits an excitation beam, which is expanded twice by a double convex lens and then illuminates the digital micromirror device. The reflected beam modulated by the digital micromirror device converges on the back focal plane of the 4f filter system to form a sparse excitation lattice, and its position coincides with the front focal plane of the excitation optical path tube mirror to ensure that the optical reflection surface of the digital micromirror device is conjugate with the front focal plane of the excitation optical path tube mirror. The excitation lattice then passes through a microscopic system composed of the excitation optical path tube mirror and the objective lens, and finally obtains a sparse multifocal excitation lattice conjugate with the optical reflection surface of the digital micromirror device on the back focal plane of the objective lens. After the Gaussian spot focused by S2 exits from the objective lens, due to the effect of the mirror, the part of the focused spot that is not incident on the mirror surface and the part reflected from the mirror surface interfere with each other, forming an interference enhancement point on the surface of the mirror where the axial distribution size of the light field is compressed by 4 times and the light field intensity is increased by 3.5 times. The S3 uses fluorescent probes to label samples and places them on the surface of a mirror. By controlling the digital micromirror device to switch the excitation array position, the camera synchronously records the fluorescence signal at each position, achieving the effect of multi-focal parallel scanning. Due to the mirror interference enhancement effect, the image signal-to-noise ratio is improved, which can effectively reduce the number of scanning steps and improve the temporal resolution by 2-3 times. Digital pinhole filtering, pixel relocation, image superposition, and deconvolution processing are performed on a series of multi-focal excited sample images to reconstruct a high signal-to-noise ratio super-resolution image.

2. The multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement according to claim 1, characterized in that, Due to the effect of the mirrors, the portion of the focused spot that is not incident on the mirror surface and the portion reflected from the mirror surface in the sparse multifocal excitation array self-interfere on the sample surface, forming an interference enhancement point with an axial size compressed by 4 times on the mirror surface. The smaller axial size of the spot greatly reduces the defocus background signal and effectively improves the optical tomography capability of the multifocal wide-field microscope, achieving an axial resolution of 110 nm.

3. The multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement according to claim 1, characterized in that, The reconstruction of the multifocal excited sample image in step S3 includes: ① Locate the positions of all excitation points and add digital pinholes for spatial filtering; ② Perform pixel relocation on the image after pinhole filtering, that is, interpolate to obtain an image with twice the number of original pixels; ③ Overlay all the images processed in steps ① and ② to obtain a reconstructed image with 1.5 times higher resolution than a wide-field microscope; ④ The superimposed images are deconvolved to obtain a super-resolution image with a resolution improvement of 2 times compared to a wide-field microscope.

4. An apparatus for implementing the multifocal wide-field super-resolution microscopy imaging method based on mirror interference enhancement as described in any one of claims 1 to 3, characterized in that, The device includes a multifocal excitation array generation module, a wide-field microscopic imaging module, a reflector sample module, and a signal acquisition module. The multifocal excitation array generation module is used to generate a sparse multifocal excitation array and switch different excitation array positions. After passing through the reflector sample module, the excitation array undergoes interference enhancement. The signal acquisition module acquires the fluorescence signal generated by the sample being scanned and excited.

5. The apparatus according to claim 4, characterized in that, The multifocal excitation lattice generation module includes a continuous laser, a biconvex lens beam expander system, a reflector, a digital micromirror device (DMD), and a 4f filtering system. The excitation beam emitted from the continuous laser is expanded to twice its original diameter by the beam expander system, and then the reflector changes the beam direction, causing it to illuminate the DMD at a 24° incident angle. The DMD consists of several miniature square reflectors, which can be rapidly switched between ±12° via hinges below the reflectors. When the incident light forms a 24° angle with the normal to the DMD surface, the micro-reflectors reflect the illumination light into the subsequent 4f filtering system, filtering out excess diffraction-level reflected light to prevent stray light interference. The controlled reflected beam converges on the back focal plane of the 4f filtering system lens to form a sparse excitation lattice, and its position coincides with the front focal plane of the excitation optical path tube of the wide-field microscopy imaging module, ensuring that the optical reflecting surface of the DMD is conjugate to the front focal plane of the excitation optical path tube.

6. The apparatus according to claim 4, characterized in that, The wide-field microscopy imaging module includes an excitation optical path tube, a dichroic mirror, and an objective lens; the sparse lattice generated by the multifocal excitation lattice generation module is focused by the excitation optical path tube and then by the objective lens onto the fluorescent probe-labeled reflector sample module, which can be used to separate the excitation light and fluorescence. The sparse lattice generated by the multifocal excitation lattice generation module is conjugate with the sample surface. After passing through a microscope system consisting of an excitation optical path tube and an objective lens, the size is reduced to 1 / 167 of the original and then imaged onto the sample surface.

7. The apparatus according to claim 4, characterized in that, The reflector sample module consists of, from top to bottom, a glass or silicon substrate, a silver or gold plating layer, a silicon dioxide protective layer, a sample labeled with a fluorescent probe, and a cover glass.

8. The apparatus according to claim 6, characterized in that, The signal acquisition module includes a coaxially placed fluorescence optical path tube, a filter, and a camera. After the sample is excited by the enhanced excitation mode, the fluorescence signal is filtered by a two-way mirror and a filter, and then focused onto the detection surface by the fluorescence optical path tube. The camera acquires the signal and sends it to the computer. The position of the excitation array is switched to scan the sample, and a series of multifocal excited sample images are collected. Finally, the computer performs digital pinhole filtering, pixel relocation, image overlay, and deconvolution processing on the sample images to reconstruct a high signal-to-noise ratio super-resolution image.

Images