阅读说明：本技术 平场照明的数字扫描结构光超分辨显微成像系统及方法 (Flat-field illumination digital scanning structure optical super-resolution microscopic imaging system and method ) 是由 于斌 葛阳阳 何灼奋 屈军乐 林丹樱 曹慧群 于 2021-08-16 设计创作，主要内容包括：本发明提供了一种平场照明的数字扫描结构光超分辨显微成像系统及方法,所述系统包括：激光光源；将激光光源产生的高斯分布的激光光束整形为均匀分布的平顶光的扩束整形反射模块；根据导入的等间隔切换的激发模式对均匀分布的平顶光进行调制,产生随时间移动的稀疏聚焦点阵的数字微镜器件；将稀疏聚焦点阵投射至样品面上,激发样品产生荧光信号的探测器；对若干幅图像数据进行图像重构的控制终端。本发明通过对均匀分布的平顶光进行调制产生随时间移动的多个聚焦点,并通过多个聚焦点同时激发样品产生荧光信号,提高了图像扫描显微系统的成像范围,减少荧光信号采集时间,可以实现高分辨率和宽视场的超分辨显微成像。(The invention provides a flat field illumination digital scanning structure light super-resolution microscopic imaging system and a method thereof, wherein the system comprises: a laser light source; the beam expanding and shaping reflection module is used for shaping a laser beam with Gaussian distribution generated by a laser light source into uniformly distributed flat top light; the digital micromirror device modulates the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time; a detector for projecting the sparse focusing lattice onto a sample surface to excite the sample to generate a fluorescence signal; and the control terminal is used for carrying out image reconstruction on the plurality of image data. The invention modulates the uniformly distributed flat-top light to generate a plurality of focusing points moving along with time, and simultaneously excites the sample to generate the fluorescence signal through the plurality of focusing points, thereby improving the imaging range of the image scanning microscope system, reducing the acquisition time of the fluorescence signal and realizing the super-resolution microscopic imaging with high resolution and wide view field.)

1. A flat field illuminated digital scanning structured light super-resolution microscopy imaging system, comprising: the device comprises a laser light source, a beam expanding and shaping reflection module, a digital micromirror device, an objective lens, a detector and a control terminal;

the beam expanding, shaping and reflecting module is used for receiving the laser beams with Gaussian distribution generated by the laser light source, shaping the laser beams with Gaussian distribution into flat top light with uniform distribution, and enabling the flat top light with uniform distribution to be incident to the digital micromirror device at a preset angle;

the digital micromirror device is used for receiving the uniformly distributed flat-top light incident from the beam expanding and shaping reflection module, and modulating the uniformly distributed flat-top light according to a guided excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time;

the objective lens is used for receiving the sparse focusing lattice, projecting the sparse focusing lattice onto a sample surface and exciting a sample to generate a fluorescence signal;

the detector is used for collecting the fluorescence signals to obtain a plurality of image data;

and the control terminal is used for receiving the plurality of image data and carrying out image reconstruction on the plurality of image data to obtain a super-resolution image of the sample.

2. The flat-field illuminated digital scanning structured light super-resolution microscopy imaging system according to claim 1, wherein the beam expanding and shaping reflector module comprises: the beam expanding and collimating unit, the beam shaper and the first reflector;

the beam expanding and collimating unit is used for receiving the laser beams with Gaussian distribution generated by the laser light source and expanding and collimating the laser beams with Gaussian distribution;

the beam shaper is used for receiving the laser beams with the Gaussian distribution after beam expanding and collimating and shaping the laser beams with the Gaussian distribution into uniformly distributed flat top light;

the first reflector is used for receiving the uniformly distributed flat top light and enabling the uniformly distributed flat top light to be incident to the digital micromirror device at a preset angle.

3. The flat-field illuminated digital scanning structured light super-resolution micro-imaging system according to claim 1, wherein a 4f system, a first lens and a dichroic sheet are arranged between the digital micromirror device and the objective lens;

the 4f system is used for receiving the sparse focusing dot matrix and filtering stray light in the sparse focusing dot matrix;

the first lens is used for receiving the sparse focusing dot matrix after stray light is filtered out and projecting the sparse focusing dot matrix to the double-color sheet;

the double-color plate is used for receiving the sparse focusing lattice projected by the first lens and projecting the sparse focusing lattice to the objective lens.

4. The flat-field illuminated digital scanning structured light super-resolution microscopy imaging system according to claim 3, wherein the 4f system comprises a second lens, a diaphragm and a third lens arranged in sequence along the light path; the digital micromirror device is arranged on the front focal plane of the second lens, the diaphragm is arranged on the rear focal plane of the second lens, the rear focal plane of the second lens coincides with the front focal plane of the third lens, and the rear focal plane of the third lens coincides with the front focal plane of the first lens.

5. The flat-field illuminated digital scanning structured light super-resolution microscopy imaging system according to claim 1, wherein the control terminal is connected to the digital micro-mirror device and the detector simultaneously, the digital micro-mirror device is connected to the detector, and the detector synchronously collects the fluorescence signal when the digital micro-mirror device switches excitation modes.

6. The flat-field illuminated digital scanning structured light super-resolution microscopy imaging system as claimed in claim 1, wherein the angle of incidence of the uniformly distributed flat top light received by the digital micromirror device is 24 ° from horizontal.

7. A flat field illumination digital scanning structure light super-resolution microscopic imaging method is characterized by comprising the following steps:

shaping a laser beam with Gaussian distribution generated by a laser light source, and shaping the laser beam with Gaussian distribution into uniformly distributed flat top light;

modulating the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time;

receiving the sparse focusing lattice, projecting the sparse focusing lattice onto a sample surface, and exciting a sample to generate a fluorescence signal;

collecting the fluorescence signals to obtain a plurality of image data;

and performing image reconstruction on the plurality of image data to obtain a super-resolution image of the sample.

8. The method of claim 7, wherein the step of performing image reconstruction on the plurality of image data to obtain a super-resolution image of the sample comprises:

constructing a multi-vector detection problem according to the plurality of image data;

solving the multi-vector detection problem to obtain a vector estimation value;

and obtaining a super-resolution image of the sample according to the vector estimation value.

9. The method of claim 8, wherein the step of solving the multi-vector detection problem to obtain vector estimates comprises:

initializing hyper-parameters of the multi-vector detection problem, and calculating an expected value and a variance of the posterior probability density of the multi-vector detection problem according to the hyper-parameters;

maximizing the posterior probability density through an expected value maximum algorithm to obtain an updated hyperparameter, when the updated hyperparameter does not converge to a hyperparameter vector, continuing to execute the steps of calculating the expected value and the variance of the posterior probability density of the multi-vector detection problem according to the hyperparameter, and maximizing the posterior probability density through the expected value maximum algorithm to obtain the updated hyperparameter until the updated hyperparameter converges to a hyperparameter vector;

and obtaining a vector estimation value according to the updated hyper-parameter.

10. The method of claim 8, wherein the step of obtaining a super-resolution image of the sample from the vector estimates comprises:

adding all column vectors of the vector estimation value to obtain a superposition vector;

and converting the superposition vector into a super-resolution image with a preset size to obtain a super-resolution image of the sample.

Technical Field

The invention belongs to the technical field of optical imaging, and particularly relates to a flat-field illumination digital scanning structure optical super-resolution microscopic imaging system and method.

Background

The laser scanning confocal microscope is an effective technical means for realizing microscopic imaging and has very wide application in the biomedical field. In the confocal microscope system, the system only receives information from a focal plane through a razor scanning mode of a pair of conjugate precise pinholes and a single focus point, and defocused information outside the focal plane is filtered, so that the signal-to-noise ratio is improved and the system has good chromatographic capacity.

The resolution of a confocal microscope depends on the size of the pinhole, and the smaller the pinhole, the higher the resolution, but at the same time, the smaller pinhole limits the optical signal collected by the optical system, resulting in lower signal-to-noise ratio. In recent years, in order to obtain good resolution and signal-to-noise ratio at the same time, researchers have proposed an Image Scanning Microscope (ISM) and a multi-focal structured light illumination microscope (MSIM), but the conventional microscopic imaging system has a slow imaging speed, and the intensity of an illumination light field is not uniform, which reduces the imaging resolution and the field of view.

Therefore, the prior art is subject to further improvement.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide a flat-field illumination digital scanning structure light super-resolution microscopic imaging system and method, which overcome the defects of low imaging speed, non-uniform illumination light field intensity and reduced imaging resolution and field of view of the existing microscopic imaging system.

The first embodiment disclosed by the invention is a flat-field illumination digital scanning structure light super-resolution microscopic imaging system, which comprises: the device comprises a laser light source, a beam expanding and shaping reflection module, a digital micromirror device, an objective lens, a detector and a control terminal;

the beam expanding, shaping and reflecting module is used for receiving the laser beams with Gaussian distribution generated by the laser light source, shaping the laser beams with Gaussian distribution into flat top light with uniform distribution, and enabling the flat top light with uniform distribution to be incident to the digital micromirror device at a preset angle;

the digital micromirror device is used for receiving the uniformly distributed flat-top light incident from the beam expanding and shaping reflection module, and modulating the uniformly distributed flat-top light according to a guided excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time;

the objective lens is used for receiving the sparse focusing lattice, projecting the sparse focusing lattice onto a sample surface and exciting a sample to generate a fluorescence signal;

the detector is used for collecting the fluorescence signals to obtain a plurality of image data;

and the control terminal is used for receiving the plurality of image data and carrying out image reconstruction on the plurality of image data to obtain a super-resolution image of the sample.

The digital scanning structure light super-resolution microscopic imaging system with flat field illumination comprises a beam expanding, shaping and reflecting module, a beam expanding and shaping module and a light source module, wherein the beam expanding, shaping and reflecting module comprises: the beam expanding and collimating unit, the beam shaper and the first reflector;

the beam expanding and collimating unit is used for receiving the laser beams with Gaussian distribution generated by the laser light source and expanding and collimating the laser beams with Gaussian distribution;

the beam shaper is used for receiving the laser beams with the Gaussian distribution after beam expanding and collimating and shaping the laser beams with the Gaussian distribution into uniformly distributed flat top light;

the first reflector is used for receiving the uniformly distributed flat top light and enabling the uniformly distributed flat top light to be incident to the digital micromirror device at a preset angle.

The flat field illumination digital scanning structure light super-resolution microscopic imaging system is characterized in that a 4f system, a first lens and a bicolor patch are arranged between the digital micromirror device and the objective lens;

the 4f system is used for receiving the sparse focusing dot matrix and filtering stray light in the sparse focusing dot matrix;

the first lens is used for receiving the sparse focusing dot matrix after stray light is filtered out and projecting the sparse focusing dot matrix to the double-color sheet;

the double-color plate is used for receiving the sparse focusing lattice projected by the first lens and projecting the sparse focusing lattice to the objective lens.

The flat field illumination digital scanning structure light super-resolution microscopic imaging system comprises a 4f system, a light source and a light source, wherein the 4f system comprises a second lens, a diaphragm and a third lens which are sequentially arranged along a light path; the digital micromirror device is arranged on the front focal plane of the second lens, the diaphragm is arranged on the rear focal plane of the second lens, the rear focal plane of the second lens coincides with the front focal plane of the third lens, and the rear focal plane of the third lens coincides with the front focal plane of the first lens.

The system comprises a control terminal, a digital micro-mirror device, a detector and a digital scanning structure light super-resolution microscopic imaging system, wherein the control terminal is simultaneously connected with the digital micro-mirror device and the detector, the digital micro-mirror device is connected with the detector, and when the digital micro-mirror device switches an excitation mode, the detector synchronously collects the fluorescence signals.

The flat-field illumination digital scanning structured light super-resolution microscopic imaging system is characterized in that the included angle between the incident angle of the uniformly distributed flat top light received by the digital micro-mirror device and the horizontal plane is 24 degrees.

The second embodiment disclosed by the invention is a flat-field illumination digital scanning structure optical super-resolution microscopic imaging method, which comprises the following steps:

shaping a laser beam with Gaussian distribution generated by a laser light source, and shaping the laser beam with Gaussian distribution into uniformly distributed flat top light;

modulating the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time;

receiving the sparse focusing lattice, projecting the sparse focusing lattice onto a sample surface, and exciting a sample to generate a fluorescence signal;

collecting the fluorescence signals to obtain a plurality of image data;

and performing image reconstruction on the plurality of image data to obtain a super-resolution image of the sample.

The method for performing super-resolution microscopic imaging by using the flat-field illuminated digital scanning structure light comprises the following steps of:

constructing a multi-vector detection problem according to the plurality of image data;

solving the multi-vector detection problem to obtain a vector estimation value;

and obtaining a super-resolution image of the sample according to the vector estimation value.

The method for the flat-field illumination digital scanning structure light super-resolution microscopic imaging comprises the following steps of:

initializing hyper-parameters of the multi-vector detection problem, and calculating an expected value and a variance of the posterior probability density of the multi-vector detection problem according to the hyper-parameters;

maximizing the posterior probability density through an expected value maximum algorithm to obtain an updated hyperparameter, when the updated hyperparameter does not converge to a hyperparameter vector, continuing to execute the steps of calculating the expected value and the variance of the posterior probability density of the multi-vector detection problem according to the hyperparameter, and maximizing the posterior probability density through the expected value maximum algorithm to obtain the updated hyperparameter until the updated hyperparameter converges to a hyperparameter vector;

and obtaining a vector estimation value according to the updated hyper-parameter.

The method for the flat-field illumination digital scanning structured light super-resolution microscopic imaging, wherein the step of obtaining the super-resolution image of the sample according to the vector estimation value comprises the following steps:

adding all column vectors of the vector estimation value to obtain a superposition vector;

and converting the superposition vector into a super-resolution image with a preset size to obtain a super-resolution image of the sample.

The invention has the advantages that the uniformly distributed flat-top light is modulated by the digital micro-mirror device to generate a plurality of focusing points moving along with time, and the plurality of focusing points simultaneously excite the sample to generate fluorescence signals, thereby improving the imaging range of the image scanning microscope system, reducing the acquisition time of the fluorescence signals, having higher signal-to-noise ratio compared with the traditional image scanning microscope system, and realizing the super-resolution microscopic imaging with high resolution and wide view field.

Drawings

FIG. 1 is a schematic structural diagram of a flat-field illuminated digital scanning structured light super-resolution microscopy imaging system provided by an embodiment of the present invention;

FIG. 2 is a schematic diagram of an excitation mode of a flat-field illuminated digital scanning structured light super-resolution micro-imaging system according to an embodiment of the present invention;

FIG. 3 is a graph of the distribution of the excitation light intensity produced by a flat-field illuminated digital scanning structured light super-resolution microscopy imaging system on a uniform dye sample according to an embodiment of the present invention;

FIG. 4 is a super-resolution image obtained by super-resolution microscopic imaging of a Hela cell microtubule sample by the flat-field illuminated digital scanning structure optical super-resolution microscopic imaging system provided by the embodiment of the present invention;

fig. 5 is a flowchart of an embodiment of a method for flat-field illuminated digital scanning structured light super-resolution microscopy imaging according to an embodiment of the present invention.

The various symbols in the drawings: 1. a laser light source; 2. a beam expanding and shaping reflection module; 3. a digital micromirror device; 4. an objective lens; 5. a sample face; 6. a detector; 7. a control terminal; 8. 4f system; 9. a first lens; 10. a two-color patch; 11. a tube mirror; 21. a beam expanding and collimating unit; 22. a beam shaper; 23. a first reflector; 81. a second lens; 82. a diaphragm; 83. a third lens; 211. a fourth lens; 212. and a fifth lens.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the embodiments and claims, the terms "a" and "an" can mean "one or more" unless the article is specifically limited.

In addition, if there is a description of "first", "second", etc. in an embodiment of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.

The resolution of a confocal microscope depends on the size of the pinhole, and the smaller the pinhole, the higher the resolution, but at the same time, the smaller pinhole limits the optical signal collected by the optical system, resulting in lower signal-to-noise ratio. In recent years, in order to obtain a good resolution and a good signal-to-noise ratio at the same time, an Image Scanning Microscope (ISM) has been proposed, in which a photomultiplier tube in a conventional confocal microscope is replaced with a CCD, and data processing is performed on an acquired signal to obtain an imageThe resolution is increased by times. However, the CCD detector is self-poweredThe slower imaging speed of ISM, which takes 60s to scan a sample area of 8 μm x 8 μm size, is caused by the weaker signal capability of the body. In order to solve the problem of slow imaging speed, a multi-focal scanning structure optical microscope (MSIM) is proposed, which uses a digital micromirror device to generate a sparse two-dimensional excitation pattern in an optical path to scan a sample, and as a parallel ISM, the MSIM can realize a field size of 50 μm × 50 μm and an imaging speed of 1Hz while maintaining good chromatographic capability, and perform super-resolution processing on acquired data to obtain a resolution of about 145nm in a transverse direction and about 400nm in an axial direction. However, in the MSIM, due to the adoption of lattice scanning, the imaging speed is still relatively slow, and the intensity of an illumination light field is not uniform, so that the imaging resolution and the imaging field are reduced.

In order to solve the above problems, the present invention provides a flat-field illuminated digital scanning structured light super-resolution microscopy imaging system, as shown in fig. 1, comprising: the device comprises a laser light source 1, a beam expanding and shaping reflection module 2, a digital micromirror device 3, an objective lens 4, a detector 6 and a control terminal 7; the laser light source 1 is used for generating a laser beam with Gaussian distribution; the beam expanding, shaping and reflecting module 2 is configured to receive a laser beam with gaussian distribution generated by the laser light source 1, shape the laser beam with gaussian distribution into uniformly distributed flat top light, and inject the uniformly distributed flat top light into the digital micromirror device 3 at a preset angle; the digital micromirror device 3 is used for receiving the uniformly distributed flat-top light incident from the beam expanding and shaping reflection module 2, and modulating the uniformly distributed flat-top light according to a guided excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time; the objective lens 4 is used for receiving the sparse focusing lattice, projecting the sparse focusing lattice onto a sample surface 5, and exciting a sample to generate a fluorescence signal; the detector 6 is used for collecting the fluorescence signals to obtain a plurality of image data; and the control terminal 7 is used for receiving the plurality of image data and carrying out image reconstruction on the plurality of image data to obtain a super-resolution image of the sample. In the specific imaging process, the beam expanding, shaping and reflecting module 2 receives a laser beam with Gaussian distribution generated by a laser light source 1, shapes the laser beam with Gaussian distribution into flat top light with uniform distribution, and makes the flat top light with uniform distribution incident to the digital micro-mirror device 3 at a preset angle, after the digital micro-mirror device 3 receives the flat top light with uniform distribution, the flat top light with uniform distribution is modulated according to an introduced excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time, the sparse focusing dot matrix moving along with time is projected onto a sample surface 5 through an objective lens 4 to excite a sample to generate a fluorescence signal, the fluorescence signal is collected through a detector 6 to obtain a plurality of image data, and a control terminal 7 performs image reconstruction on the received plurality of image data to obtain a super-resolution image of the sample. In the embodiment, the uniformly distributed flat top light is modulated by the digital micro-mirror device 3 to generate a plurality of focusing points moving along with time, and the plurality of focusing points simultaneously excite the sample to generate the fluorescent signal, so that the imaging range of the image scanning microscope system is improved, the acquisition time of the fluorescent signal is reduced, and compared with the traditional image scanning microscope system, the super-resolution microscope imaging with high resolution and wide view field can be realized.

Specifically, the excitation pattern of the present invention is a 1024 × 768 binary image, as shown in fig. 2, the illumination pattern is a series of black and white dot matrix images, the black squares represent pixels, when the digital micromirror device 3 starts to switch the excitation pattern according to the order of loading the images, the positions of the pixels will change along with the trajectory of the arrow, and each time, the pixels are shifted by one pixel and switched at equal intervals, so that a sparse focusing dot matrix with periodic arrangement is generated on the sample surface 5, as shown in fig. 3. In the embodiment, the samples are excited by the periodically arranged sparse focusing dot matrix, the imaging range of the image scanning microscope system is improved, and the sample acquisition time is reduced, for example, when the sparse focusing dot matrix is 4 × 1 pixel points, compared with the traditional MSIM1 × 1 pixel point scanning template, the signal-to-noise ratio is higher, and the scanning step length is shortened to be 1/4.

In a specific embodiment, the beam expanding and shaping reflective module 2 includes: a beam expanding and collimating unit 21, a beam shaper 22 and a first mirror 23. The beam expanding and collimating unit 21 includes a fourth lens 211 and a fifth lens 212, a back focal plane of the fourth lens 211 coincides with a front focal plane of the fifth lens 212, and the beam expanding and collimating unit 21 is configured to receive the laser beam with gaussian distribution and perform beam expanding and collimating on the laser beam with gaussian distribution; the beam shaper 22 is configured to receive the laser beam with gaussian distribution after beam expansion and collimation, and shape the laser beam with gaussian distribution into uniformly distributed flat top light; the first reflector 23 is configured to receive the uniformly distributed flat top light, and to make the uniformly distributed flat top light incident to the center of the digital micromirror device 3 at a preset angle. In one embodiment, the angle of incidence of the uniformly distributed flat top light received by the dmd 3 is 24 ° from the horizontal plane. In a specific imaging process, the laser light source 1 is a 488nm solid laser, the laser light source 1 generates continuous laser with a specific wavelength, after beam expansion and collimation are performed by the beam expansion and collimation unit 21 composed of the fourth lens 211 and the fifth lens 212, the laser beam with gaussian distribution after beam expansion and collimation is shaped into even-distribution flat-top light by the beam shaper 22, and then the even-distribution flat-top light is incident to the beam expansion and shaping reflection module 2 at a preset angle by the first reflection mirror 23. In this embodiment, the beam expansion multiple of the laser beam with gaussian distribution can be adjusted by changing the focal lengths of the fourth lens 211 and the fifth lens 212, and the laser beam with gaussian distribution is shaped into flat top light with uniform distribution by the beam shaper 22, so that the intensity of the illumination light field of the sample is uniform, and the imaging resolution and the imaging field of view are improved.

In one embodiment, a 4f system 8, a first lens 9 and a dichroic filter 10 are disposed between the digital micromirror device 3 and the objective lens 4; the 4f system 8 is used for receiving the sparse focusing dot matrix and filtering stray light in the sparse focusing dot matrix; the first lens 9 is configured to receive the sparse focusing dot matrix after the stray light is filtered out, and project the sparse focusing dot matrix to the two-color sheet 10; the two-color sheet 10 is configured to receive the sparse focusing lattice projected by the first lens 9, and project the sparse focusing lattice onto the objective lens 4, so that in a subsequent step, the sparse focusing lattice is projected onto a sample surface through the objective lens 4, and a sample is excited to generate a fluorescence signal. In the specific imaging process, after stray light is filtered by the 4f system 8, the sparse focusing dot matrix generated by the digital micromirror device 3 is projected to the two-color sheet 10 by the first lens 9, and the sparse focusing dot matrix is projected to the objective lens 4 by the two-color sheet 10.

Further, the 4f system 8 includes a second lens 81, a diaphragm 82 and a third lens 83 sequentially arranged along the optical path, the dmd 3 is arranged on the front focal plane of the second lens 81, the diaphragm 82 is arranged on the rear focal plane of the second lens 81, the diaphragm 82 is used for shielding the reflected light of the redundant diffraction order, the rear focal plane of the second lens 81 coincides with the front focal plane of the third lens 83, and the rear focal plane of the third lens 83 coincides with the front focal plane of the first lens 9.

In a specific embodiment, a tube lens 11 is disposed between the objective lens 4 and the detector 6, and in the fluorescent signal collecting process, a fluorescent signal generated by the sample is further amplified by the objective lens 4 and the tube lens 11 and then collected by the detector 6 to obtain a plurality of image data.

In a specific embodiment, the control terminal 7 is connected to the detector 6 and the digital micromirror device 3 at the same time, the control terminal 7 is configured to sequentially guide a series of excitation patterns into the digital micromirror device 3, and receive a plurality of image data obtained by the detector 6, each pixel of the excitation patterns corresponds to a micromirror on the panel of the digital micromirror device 3, a pixel value 1 represents an "on" state of the micromirror, and a pixel value 0 represents an "off" state of the micromirror. In a specific imaging process, the control terminal 7 guides a series of excitation modes into a memory of the digital micromirror device 3 in sequence, and after the digital micromirror device 3 receives the uniformly distributed flat top light incident from the beam expanding and shaping reflection module 2, the excitation modes are switched at equal intervals through software setting, so that the uniformly distributed flat top light is modulated into a sparse focusing dot matrix moving along with time.

In specific implementation, the digital micromirror device 3 switches the excitation mode at equal intervals, and in order to enable the detector 6 to accurately acquire each image data of the digital micromirror device 3 when the excitation mode is switched, the number of the digital micromirror device 3 and the number of the detector 6 are in this embodimentAccording to the connection, when the digital micro-mirror device 3 switches the illumination mode, a rising edge signal with 3V voltage is sent to the detector 6, the detector 6 synchronously collects image data after receiving the rising edge signal, and a series of image data I are obtained₁，I₂…I_nWherein the exposure time of the detector 6 is the interval between two rising edge signals.

In specific implementation, the detector 6 acquires a plurality of pieces of image data obtained by exciting the surface of the sample with the laser beam, and further image reconstruction needs to be performed on the acquired image data to obtain a super-resolution sample two-dimensional information image. In this embodiment, the control terminal 7 is in data connection with the detector 6, and the detector 6 transmits the acquired image data to the control terminal 7 for image reconstruction. In the specific image reconstruction process, the control terminal 7 firstly converts the image reconstruction problem of a plurality of image data into a multi-vector detection problem:wherein Y is H.X, Y_mThe measurement matrix is a column vector obtained by converting the mth piece of lattice data, H is a measurement matrix obtained by a system PSF, X is S.E, and S and E are a real structure and an excitation mode of a sample respectively; then, solving a Multi-vector detection problem by using a Multi-vector Sparse Bayesian Learning (MSBL) algorithm to obtain a vector estimation value; then adding all column vectors of the vector estimation value to obtain a superposition vector; finally, the superposition vector is converted into the size m₀×n₀The super-resolution image of the sample is obtained, as shown in fig. 4.

Further, when the control terminal 7 solves the multi-vector detection problem by using the multi-measurement vector sparse Bayesian learning algorithm, firstly, the hyper-parameters of the multi-vector detection problem are initialized, and the expected value and the variance of the posterior probability density of the multi-vector detection problem are calculated according to the hyper-parameters; and then maximizing the posterior probability density through an expected value maximum algorithm (EM algorithm) to obtain an updated hyperparameter, when the updated hyperparameter does not converge to a hyperparameter vector, continuing to execute the steps of calculating the expected value and the variance of the posterior probability density of the multi-vector detection problem according to the hyperparameter, and maximizing the posterior probability density through the expected value maximum algorithm to obtain the updated hyperparameter until the updated hyperparameter converges to a hyperparameter vector. In this embodiment, resolution improvement of about 2 times of a wide field can be achieved by performing image reconstruction on image data.

The invention also provides a flat field illumination based digital scanning structure optical super-resolution microscopic imaging system, and a flat field illumination based digital scanning structure optical super-resolution microscopic imaging method, as shown in fig. 5, the system comprises the following steps:

s1, shaping the laser beam with Gaussian distribution generated by the laser light source, and shaping the laser beam with Gaussian distribution into uniformly distributed flat top light;

s2, modulating the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time;

s3, receiving the sparse focusing lattice, projecting the sparse focusing lattice onto a sample surface, and exciting the sample to generate a fluorescence signal;

s4, collecting the fluorescence signals to obtain a plurality of image data;

and S5, carrying out image reconstruction on the plurality of image data to obtain a super-resolution image of the sample.

In specific implementation, the beam expanding, shaping and reflecting module shapes the laser beam with Gaussian distribution generated by the laser source, and after the laser beam with Gaussian distribution is shaped into uniformly distributed flat top light, the digital micromirror device phase-modulates the uniformly distributed flat top light according to the introduced excitation mode switched at equal intervals, generating a sparse focusing lattice moving along with time by continuously switching an excitation mode on the digital micromirror device, projecting the sparse focusing lattice onto a sample surface by an objective lens after the sparse focusing lattice is received by the objective lens, scanning the sample surface with high-precision random addressing until all the selected area on the sample surface is excited to generate fluorescence signals, the fluorescence signal is collected by a detector to obtain a plurality of image data and is transmitted to a control terminal, and the control terminal carries out image reconstruction on the received images to obtain a super-resolution image of the sample. The embodiment modulates the uniformly distributed flat-top light through the digital micro-mirror device to generate a plurality of focusing points moving along with time, and simultaneously excites the sample to generate the fluorescent signal through the plurality of focusing points, so that the imaging range of the image scanning microscope system is improved, the acquisition time of the fluorescent signal is reduced, and compared with the traditional image scanning microscope system, the image scanning microscope system has higher signal-to-noise ratio and can realize the super-resolution microscopic imaging with high resolution and wide view field.

In one embodiment, step S5 includes the steps of:

s51, constructing a multi-vector detection problem according to the plurality of image data;

s52, solving the multi-vector detection problem to obtain a vector estimation value;

and S53, obtaining a super-resolution image of the sample according to the vector estimation value.

Specifically, the image reconstruction of several pieces of image data may be described as Y ═ H · S · E, where Y denotes the image data, H denotes a measurement matrix obtained by the system PSF, S and E denote the sample true structure and excitation pattern, respectively, and let matrix X ═ S · E, where S ═ diag (S1, …, sN) is a non-negative diagonal matrix, which denotes the sample structure of an N-pixel gridded, and X and S have the same non-zero rows, so the image reconstruction problem of several pieces of image data can be expressed as a multi-vector detection problem:in this case, Y is H.X, and Y is [ Y ]₁,y₂,…y_N]，y_mIs the column vector converted from the m-th piece of dot matrix data.

After the image reconstruction problem of a plurality of image data is converted into a Multi-vector detection problem, the Multi-vector detection problem is solved by using a Multi-vector Sparse Bayesian Learning (MSBL) algorithm:obtain the vectorAnd estimating the value X ', and finally obtaining a super-resolution image of the sample according to the vector estimation value X'.

In one embodiment, step S52 specifically includes:

s521, initializing hyper-parameters of the multi-vector detection problem, and calculating expected values and variances of the posterior probability density of the multi-vector detection problem according to the hyper-parameters;

s522, maximizing the posterior probability density through an expected value maximum algorithm to obtain an updated hyperparameter, when the updated hyperparameter does not converge to a hyperparameter vector, continuing to execute the steps of calculating the expected value and the variance of the posterior probability density of the multi-vector detection problem according to the hyperparameter, and maximizing the posterior probability density through the expected value maximum algorithm to obtain the updated hyperparameter until the updated hyperparameter converges to a hyperparameter vector;

and S522, obtaining a vector estimation value according to the updated hyper-parameter.

Specifically, according to bayesian inference, the multi-vector detection problem:the solution X' of (a) can be estimated from the maximum a posteriori probability:when solving a multi-vector detection problem by using an MSBL algorithm, firstly initializing hyper-parameters of the multi-vector detection problem, calculating expected values and variances of posterior probability densities p (X | Y) of the multi-vector detection problem according to the hyper-parameters, then maximizing the posterior probability densities p (X | Y) by using an expected value maximum algorithm (EM) to obtain updated hyper-parameters, when the updated hyper-parameters do not converge to a hyper-parameter vector, continuously executing the steps of calculating the expected values and the variances of the posterior probability densities of the multi-vector detection problem according to the hyper-parameters, updating the hyper-parameters according to the expected values and the variances of the posterior probability densities, and obtaining the multi-vector detection problem according to the updated hyper-parameters after the updated hyper-parameters converge to a hyper-parameter vectorThe solution to the problem, i.e. the vector estimate X', is detected.

In one embodiment, step S53 specifically includes:

s531, adding all column vectors of the vector estimation value to obtain a superposition vector;

and S532, converting the superposition vector into a super-resolution image with a preset size to obtain the super-resolution image of the sample.

And after the vector estimation value X 'is obtained, adding all column vectors in the vector estimation value X' to obtain a superposition vector, and then converting the superposition vector into a super-resolution image with a preset size to obtain the super-resolution image of the sample. For example, the overlay vector is converted to a size of m₀×n₀The super-resolution image of (1), the size being m₀×n₀The super-resolution image is the super-resolution image of the sample.

In summary, the present invention provides a system and a method for flat-field illuminated digital scanning structured light super-resolution microscopy imaging, wherein the system comprises: a laser light source; shaping a laser beam with Gaussian distribution generated by a laser light source into uniformly distributed flat top light, and enabling the uniformly distributed flat top light to be incident to a beam expanding and shaping reflection module of a digital micromirror device at a preset angle; the digital micromirror device modulates the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals to generate a sparse focusing dot matrix moving along with time; a detector for projecting the sparse focusing lattice onto a sample surface to excite the sample to generate a fluorescence signal; and the control terminal is used for carrying out image reconstruction on the plurality of image data to obtain a super-resolution image of the sample. The invention modulates the uniformly distributed flat-top light by the digital micro-mirror device to generate a plurality of focusing points moving along with time, and simultaneously excites the sample to generate the fluorescence signal by the plurality of focusing points, thereby improving the imaging range of the image scanning microscope system, reducing the acquisition time of the fluorescence signal, having higher signal-to-noise ratio compared with the traditional image scanning microscope system, and realizing the super-resolution microscopic imaging with high resolution and wide view field.

It is to be understood that the system of the present invention is not limited to the above examples, and that modifications and variations may be made by one of ordinary skill in the art in light of the above teachings, and all such modifications and variations are intended to fall within the scope of the appended claims.