CN113625439B - Digital scanning structured light super-resolution microscopic imaging system and method for flat field illumination - Google Patents
Digital scanning structured light super-resolution microscopic imaging system and method for flat field illumination Download PDFInfo
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Abstract
The invention provides a flat-field illumination digital scanning structured light super-resolution microscopic imaging system and a method, wherein the system comprises the following components: a laser light source; the beam expanding and shaping reflection module is used for shaping the Gaussian-distribution laser beam generated by the laser source into uniformly-distributed flat top light; modulating uniformly distributed flat-top light according to the imported excitation mode switched at equal intervals to generate a digital micromirror device of a sparse focusing lattice which moves along with time; projecting the sparse focusing lattice onto a sample surface, and exciting the sample to generate a fluorescent 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 which move along with time, and simultaneously excites the sample to generate fluorescent signals through the plurality of focusing points, thereby improving the imaging range of the image scanning microscope system, reducing the fluorescent signal acquisition time and realizing super-resolution microscopic imaging with high resolution and wide field of view.
Description
Technical Field
The invention belongs to the technical field of optical imaging, and particularly relates to a digital scanning structured light super-resolution microscopic imaging system and method for flat field illumination.
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 a confocal microscopic system, only information from a focal plane is received by the system through a pair of conjugated precise pinholes and a single focusing point razor scanning mode, and out-of-focus information outside the focal plane is filtered, so that the signal-to-noise ratio is improved and the chromatographic capability is good.
The resolution of the confocal microscope depends on the pinhole size, with smaller pinholes having higher resolution, but at the same time smaller pinholes limit 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 (image scanning microscopy, ISM) and a multi-focus structured light illumination microscope (multifocal structuredillumination microscopy, MSIM), but the existing microscopic imaging system has low imaging speed, uneven illumination light field intensity, and reduced imaging resolution and field of view.
Accordingly, there is a need for further improvements in the art.
Disclosure of Invention
In view of the shortcomings in the prior art, the invention aims to provide a digital scanning structure light super-resolution microscopic imaging system and method for flat field illumination, which overcome the defects that the existing microscopic imaging system is low in imaging speed and uneven in illumination light field intensity and reduces imaging resolution and field of view.
The first embodiment disclosed in the invention is a digital scanning structured light super-resolution microscopic imaging system with flat field illumination, which comprises: the system comprises a laser light source, a beam expanding and shaping reflection module, a digital micro-mirror device, an objective lens, a detector and a control terminal;
the beam expanding and shaping reflection module is used for receiving Gaussian-distributed laser beams generated by the laser source, shaping the Gaussian-distributed laser beams into uniformly-distributed flat top lights, and enabling the uniformly-distributed flat top lights to be incident to the digital micromirror device at a preset angle;
the digital micro-mirror device is used for receiving the uniformly distributed flat-top light incident by the beam expanding and shaping reflection module, modulating the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals, and generating a sparse focusing lattice 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 fluorescent signal;
the detector is used for collecting the fluorescent signals to obtain a plurality of image data;
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 flat field illumination digital scanning structured light super-resolution microscopic imaging system, wherein the beam expanding, shaping and reflecting module comprises: a beam expanding and collimating unit, a beam shaper and a first reflecting mirror;
the beam expanding and collimating unit is used for receiving the Gaussian-distributed laser beams generated by the laser source and carrying out beam expanding and collimation on the Gaussian-distributed laser beams;
the beam shaper is used for receiving the Gaussian-distributed laser beams after beam expansion collimation and shaping the Gaussian-distributed laser beams into uniformly distributed flat top lights;
the first reflecting mirror is used for receiving the uniformly distributed flat top light and making the uniformly distributed flat top light incident to the digital micro-mirror 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 sheet are arranged between the digital micro-mirror device and the objective lens;
the 4f system is used for receiving the sparse focusing lattice and filtering stray light in the sparse focusing lattice;
the first lens is used for receiving the sparse focusing lattice after stray light is filtered, and projecting the sparse focusing lattice to the bicolor sheet;
the bicolor sheet 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 second lens, a diaphragm and a third lens which are sequentially arranged along a light path; the digital micro-mirror device is arranged on the front focal plane of the second lens, the diaphragm is arranged on the back focal plane of the second lens, the back focal plane of the second lens is overlapped with the front focal plane of the third lens, and the back focal plane of the third lens is overlapped with the front focal plane of the first lens.
The flat field illumination digital scanning structure light super-resolution microscopic imaging system is characterized in that the control terminal is connected with the digital micro-mirror device and the detector at the same time, 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 fluorescent signals.
In the flat-field illumination digital scanning structured light super-resolution microscopic imaging system, the angle of incidence of the uniformly distributed flat top light received by the digital micromirror device is 24 degrees with the horizontal plane.
The second embodiment disclosed by the invention is a digital scanning structured light super-resolution microscopic imaging method for flat field illumination, which comprises the following steps:
shaping a Gaussian-distributed laser beam generated by a laser source, and shaping the Gaussian-distributed laser beam into uniformly-distributed flat top light;
modulating the uniformly distributed flat-top light according to the imported excitation mode switched at equal intervals to generate a sparse focusing lattice which moves along with time;
receiving the sparse focusing lattice, projecting the sparse focusing lattice to a sample surface, and exciting a sample to generate a fluorescent signal;
collecting the fluorescence signals to obtain a 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 flat-field illumination digital scanning structured light super-resolution microscopic imaging method, wherein the step of performing image reconstruction on the plurality of image data to obtain a super-resolution image of a sample comprises the following steps:
constructing a multi-vector detection problem according to the plurality of image data;
solving the multi-vector detection problem to obtain a vector estimated value;
and obtaining a super-resolution image of the sample according to the vector estimation value.
The flat field illumination digital scanning structured light super-resolution microscopic imaging method, wherein the step of solving the multi-vector detection problem to obtain a vector estimated value comprises the following steps:
initializing a hyper-parameter of the multi-vector detection problem, and calculating an expected value and variance of a posterior probability density of the multi-vector detection problem according to the hyper-parameter;
maximizing the posterior probability density through an expectation maximization algorithm to obtain updated superparameters, and continuously executing the steps of calculating the expectation and variance of the posterior probability density of the multi-vector detection problem according to the superparameters when the updated superparameters do not converge to a superparameter vector, and maximizing the posterior probability density through the expectation maximization algorithm to obtain updated superparameters until the updated superparameters converge to a superparameter vector;
and obtaining a vector estimated value according to the updated super-parameters.
The flat field illumination digital scanning structured light super-resolution microscopic imaging method, 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 values to obtain a superposition vector;
and converting the superposition vector into a super-resolution image with a preset size to obtain the super-resolution image of the sample.
The invention has the beneficial effects that the digital micro-mirror device modulates the evenly distributed flat-top light to generate a plurality of focusing points which move along with time, and the sample is excited to generate fluorescent signals through the plurality of focusing points, so that the imaging range of the image scanning microscopic system is improved, the fluorescent signal acquisition time is shortened, and compared with the traditional image scanning microscopic system, the digital micro-mirror device has higher signal-to-noise ratio, and can realize super-resolution microscopic imaging with high resolution and wide view field.
Drawings
FIG. 1 is a schematic diagram of a digital scanning structured light super-resolution microscopic imaging system with flat field illumination according to 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 microscopic imaging system according to an embodiment of the present invention;
FIG. 3 is a graph showing the intensity distribution of excitation light generated by a flat-field illuminated digital scanning structured light super-resolution microscopic imaging system on a uniform dye sample according to an embodiment of the present invention;
FIG. 4 is a super-resolution image obtained by performing super-resolution microscopic imaging on a Hela cell microtubule sample by using the flat-field illuminated digital scanning structured light super-resolution microscopic imaging system provided by the embodiment of the invention;
fig. 5 is a flowchart of an embodiment of a digital scanning structured light super-resolution microscopic imaging method with flat field illumination according to an embodiment of the present invention.
The marks in the drawings are as follows: 1. a laser light source; 2. a beam expanding and shaping reflection module; 3. a digital micromirror device; 4. an objective lens; 5. sample surface; 6. a detector; 7. a control terminal; 8. 4f system; 9. a first lens; 10. a bicolor sheet; 11. a tube mirror; 21. a beam expansion collimation unit; 22. a beam shaper; 23. a first mirror; 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 more clear and clear, the present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the description and claims, unless the context clearly dictates otherwise, the terms "a" and "an" and "the" may refer to either a single or a plurality.
In addition, if there is a description of "first", "second", etc. in the embodiments 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 a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present invention.
The resolution of the confocal microscope depends on the pinhole size, with smaller pinholes having higher resolution, but at the same time smaller pinholes limit 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, an image scanning microscope (image scanning microscopy, ISM) has been proposed in which a photomultiplier tube in a conventional confocal microscope is replaced with a CCD, and acquired signals are subjected to data processing to obtain
The resolution of the multiple is improved. However, the weak signal capability of the CCD detector itself results in a slow imaging speed of the ISM, which takes 60 seconds to scan a sample area of size 8 μm by 8 μm. In order to solve the problem of slow imaging speed, a multi-focus scanning structure light microscope (multifocal structuredillumination microscopy, MSIM) is proposed, which uses a digital micromirror device to generate a sparse two-dimensional excitation mode in a light path to scan a sample, as a parallel ISM, while retaining good chromatographic capacity, can realize a field size of 50 μm×50 μm and an imaging speed of 1Hz, and performs super-resolution processing on acquired data to obtain a resolution of about 145nm in the transverse direction and about 400nm in the axial direction. However, MSIM adopts dot matrix scanning, so that the imaging speed is still relatively low, and the intensity of an illumination light field is uneven, thereby reducing the resolution and the field of view of imaging.
In order to solve the above problems, the present invention provides a digital scanning structured light super-resolution microscopic imaging system for flat field illumination, as shown in fig. 1, the system comprises: the device comprises a laser light source 1, a beam expanding and shaping reflection module 2, a digital micro-mirror device 3, an objective lens 4, a detector 6 and a control terminal 7; the laser light source 1 is used for generating a Gaussian-distributed laser beam; the beam expanding and shaping reflection module 2 is configured to receive a gaussian-distributed laser beam generated by the laser source 1, shape the gaussian-distributed laser beam into uniformly distributed flat top light, and make the uniformly distributed flat top light incident to the digital micromirror device 3 at a preset angle; the digital micro-mirror device 3 is used for receiving the uniformly distributed flat-top light incident by the beam expanding and shaping reflection module 2, modulating the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals, and generating a sparse focusing lattice moving along with time; the objective lens 4 is used for receiving the sparse focusing lattice, projecting the sparse focusing lattice onto the sample surface 5, and exciting the sample to generate a fluorescent signal; the detector 6 is used for collecting the fluorescent signals to obtain a plurality of image data; the control terminal 7 is configured to receive the plurality of image data, and perform 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 and shaping reflection module 2 receives a Gaussian distribution laser beam generated by the laser source 1, shapes the Gaussian distribution laser beam into a uniformly distributed flat top light, and makes the uniformly distributed flat top light incident to the digital micro-mirror device 3 at a preset angle, after the digital micro-mirror device 3 receives the uniformly distributed flat top light, the uniformly distributed flat top light is modulated according to an imported excitation mode switched at equal intervals to generate a sparse focusing lattice moving along with time, the sparse focusing lattice moving along with time is projected onto the sample surface 5 through the objective lens 4, a fluorescent signal is excited to generate a fluorescent signal, the fluorescent signal is acquired through the detector 6 to obtain a plurality of image data, and the control terminal 7 performs image reconstruction on the plurality of received image data to obtain a super-resolution image of the sample. In the embodiment, the digital micro-mirror device 3 modulates the flat-top light which is uniformly distributed to generate a plurality of focusing points which move along with time, and simultaneously excites a sample to generate fluorescent signals through the plurality of focusing points, so that the imaging range of an image scanning microscopic system is improved, the fluorescent signal acquisition time is shortened, and compared with the traditional image scanning microscopic system, the super-resolution microscopic imaging with high resolution and wide field of view can be realized.
Specifically, the excitation mode of the present invention is a 1024×768 binary image, as shown in fig. 2, the illumination mode is a series of black-and-white dot matrix images, black squares represent pixels, when the digital micromirror device 3 starts to switch the excitation mode according to the order of loading the image, the positions of the pixels will change along with the track of the arrow, and each time the pixels are shifted and switched at equal intervals, so that a periodically arranged sparse focusing dot matrix is generated on the sample surface 5, as shown in fig. 3. According to the embodiment, samples are excited through the periodically arranged sparse focusing dot matrix, the imaging range of an image scanning microscope system is improved, the sample acquisition time is shortened, for example, when the sparse focusing dot matrix is 4 multiplied by 1 pixel points, compared with a traditional MSIM1 multiplied by 1 pixel point scanning template, the signal to noise ratio is higher, and the scanning step length is shortened to be 1/4 of the original scanning step length.
In a specific embodiment, the beam expanding and shaping reflection 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, where 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 gaussian-distributed laser beam and perform beam expanding and collimating on the gaussian-distributed laser beam; the beam shaper 22 is configured to receive the gaussian-distributed laser beam after beam expansion and collimation, and shape the gaussian-distributed laser beam into uniformly distributed flat top light; the first reflecting mirror 23 is configured to receive the uniformly distributed flat top light and to make the uniformly distributed flat top light incident on the center of the dmd 3 at a preset angle. In a specific embodiment, the angle of incidence of the uniformly distributed flat top light received by the dmd 3 is 24 ° from the horizontal. In a specific imaging process, the laser light source 1 is a 488nm solid laser, the laser light source 1 generates continuous laser with specific wavelength, after beam expansion and collimation are performed by a beam expansion collimation unit 21 formed by a fourth lens 211 and a fifth lens 212, a beam shaper 22 shapes a Gaussian-distributed laser beam after beam expansion collimation into uniformly-distributed flat top light, and then the uniformly-distributed flat top light is incident to the beam expansion shaping reflection module 2 at a preset angle by a first reflection mirror 23. In this embodiment, the focal lengths of the fourth lens 211 and the fifth lens 212 are changed to adjust the expansion ratio of the gaussian-distributed laser beam, and the beam shaper 22 shapes the gaussian-distributed laser beam into uniformly distributed flat top light, so that the illumination light field of the sample is uniform in intensity, and the resolution and the field of view of imaging are improved.
In a specific embodiment, a 4f system 8, a first lens 9 and a bicolor patch 10 are arranged between the digital micromirror device 3 and the objective lens 4; the 4f system 8 is configured to receive the sparse focusing lattice and filter stray light in the sparse focusing lattice; the first lens 9 is configured to receive the sparse focusing lattice after stray light is filtered, and project the sparse focusing lattice to the bicolor patch 10; the bicolor plate 10 is used for receiving the sparse focusing lattice projected by the first lens 9 and projecting the sparse focusing lattice onto the objective lens 4, so that the sparse focusing lattice is projected onto a sample surface through the objective lens 4 in a subsequent step, and the sample is excited to generate a fluorescent signal. In a specific imaging process, after stray light is filtered by a 4f system 8, the sparse focusing lattice generated by the digital micro-mirror device 3 is projected to a bicolor 10 by a first lens 9, and the sparse focusing lattice is projected to an objective lens 4 by the bicolor 10.
Further, the 4f system 8 includes a second lens 81, a diaphragm 82 and a third lens 83 sequentially disposed along the optical path, the digital micromirror device 3 is disposed on a front focal plane of the second lens 81, the diaphragm 82 is disposed on a back focal plane of the second lens 81, the diaphragm 82 is used for shielding reflected light of excessive diffraction orders, the back focal plane of the second lens 81 coincides with a front focal plane of the third lens 83, and the back focal plane of the third lens 83 coincides with a 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 process of collecting the fluorescent signal, the 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, so as 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 introduce a series of excitation modes into the digital micromirror device 3, and receive several image data obtained by the detector 6, where each pixel of the excitation modes corresponds to a micromirror on a panel of the digital micromirror device 3, a pixel value of 1 represents an "on" state of the micromirror, and 0 represents an "off" state of the micromirror. In a specific imaging process, the control terminal 7 sequentially guides a series of excitation modes into the internal memory of the digital micro-mirror device 3, and after the digital micro-mirror device 3 receives uniformly distributed flat top lights incident by the beam expanding and shaping reflection module 2, the excitation modes are switched at equal intervals through software setting, so that uniformly distributed flat top light is modulated into a sparse focusing lattice moving along with time.
In a specific implementation, the digital micromirror device 3 is in an equal interval switching excitation mode, so that the detector 6 can accurately acquire each image data when the digital micromirror device 3 switches the excitation mode, in this embodiment, the digital micromirror device 3 is in data connection with the detector 6, when the digital micromirror device 3 switches the illumination mode, a rising edge signal with a voltage of 3V is sent to the detector 6, and the detector 6 acquires the image data synchronously after receiving the rising edge signal, so as to obtain a series of image data I 1 ，I 2 …I n Wherein the exposure time of the detector 6 is the interval between two rising edge signals.
In the specific implementation, the detector 6 acquires a plurality of pieces of image data obtained by exciting the surface of the sample by the laser beam, and the obtained image data is required to be further subjected to image reconstruction 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 plurality of image data to the control terminal 7 for image reconstruction. In a specific image reconstruction process, the control terminal 7 first converts an image reconstruction problem of a plurality of image data into a multi-vector detection problem:
wherein Y=HX，y m The system is a column vector obtained by converting the mth lattice data, H is a measurement matrix obtained by a system PSF, and X=S.E, S and E are a sample real structure and an excitation mode respectively; then solving a Multi-vector detection problem by utilizing a Multi-measurement vector sparse Bayesian learning (Multi-vectors Sparse Bayesian Learning, MSBL) algorithm to obtain a vector estimation value; then adding all column vectors of the vector estimation values to obtain a superposition vector; finally, the superposition vector is converted into m 0 ×n 0 The super-resolution image of the sample is obtained as shown in fig. 4.
Further, when solving the multi-vector detection problem by using a multi-measurement vector sparse Bayesian learning algorithm, the control terminal 7 firstly initializes a hyper-parameter of the multi-vector detection problem, and calculates an expected value and a variance of a posterior probability density of the multi-vector detection problem according to the hyper-parameter; and then maximizing the posterior probability density through an expectation maximization algorithm (EM algorithm) to obtain updated superparameters, and continuously executing the steps of calculating the expected value and the variance of the posterior probability density of the multi-vector detection problem according to the superparameters when the updated superparameters do not converge to one superparameter vector, and maximizing the posterior probability density through the expected value maximization algorithm to obtain updated superparameters until the updated superparameters converge to one superparameter vector. In this embodiment, by performing image reconstruction on the image data, resolution improvement of about 2 times of the wide field can be achieved.
The invention also provides a digital scanning structure light super-resolution microscopic imaging method of flat field illumination based on the digital scanning structure light super-resolution microscopic imaging system of flat field illumination, as shown in fig. 5, comprising the following steps:
s1, shaping Gaussian-distributed laser beams generated by a laser source, and shaping the Gaussian-distributed laser beams into uniformly distributed flat top lights;
s2, modulating the uniformly distributed flat-top light according to the imported excitation mode switched at equal intervals to generate a sparse focusing lattice which moves along with time;
s3, receiving the sparse focusing lattice, projecting the sparse focusing lattice to a sample surface, and exciting the sample to generate a fluorescent signal;
s4, collecting the fluorescence signals to obtain a plurality of image data;
s5, performing image reconstruction on the plurality of image data to obtain a super-resolution image of the sample.
In the specific implementation, the beam expanding and shaping reflection module shapes the Gaussian-distributed laser beam generated by the laser source, after the Gaussian-distributed laser beam is shaped into uniformly-distributed flat top light, the digital micro-mirror device carries out phase modulation on the uniformly-distributed flat top light according to the imported excitation mode which is switched at equal intervals, a sparse focusing lattice which moves along with time is generated by continuously switching the excitation mode on the digital micro-mirror device, the sparse focusing lattice is projected onto a sample surface by an objective lens after being received, high-precision random addressing scanning is carried out on the sample surface until all selected areas on the sample surface are excited to generate fluorescent signals, the fluorescent signals are acquired by a detector, a plurality of image data are obtained and 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. In the embodiment, the digital micro-mirror device modulates the evenly distributed flat-top light to generate a plurality of focusing points which move along with time, and simultaneously excites the sample to generate fluorescent signals through the plurality of focusing points, so that the imaging range of the image scanning microscopic system is improved, the fluorescent signal acquisition time is shortened, and compared with the traditional image scanning microscopic system, the digital micro-mirror device has higher signal-to-noise ratio, and can realize super-resolution microscopic imaging with high resolution and wide field of view.
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 estimated value;
and S53, obtaining a super-resolution image of the sample according to the vector estimation value.
In particular, image reconstruction of several image dataCan be described as y=h·s·e, where Y represents the image data, H represents the measurement matrix obtained by the system PSF, S and E represent the sample real structure and excitation pattern, respectively, let the matrix x=s·e, where s=diag (S1, …, sN) be a non-negative diagonal matrix, represent the sample structure of N-pixel gridding, X and S have the same non-zero rows, so the image reconstruction problem for several pieces of image data can be expressed as a multi-vector detection problem:
at this time y=h·x, y= [ Y ] 1 ,y 2 ,…y N ]，y m Is a column vector converted from the mth dot matrix data.
After converting an image reconstruction problem of a plurality of image data into a Multi-vector detection problem, solving the Multi-vector detection problem by utilizing a Multi-measurement vector sparse Bayesian learning (Multi-vectors Sparse Bayesian Learning, MSBL) algorithm:
and obtaining a vector estimated value X ', and finally obtaining a super-resolution image of the sample according to the vector estimated value X'.
In one embodiment, step S52 specifically includes:
s521, initializing a hyper-parameter of the multi-vector detection problem, and calculating an expected value and variance of a posterior probability density of the multi-vector detection problem according to the hyper-parameter;
s522, maximizing the posterior probability density through an expected value maximizing algorithm to obtain updated super-parameters, and continuously executing the steps of calculating the expected value and the variance of the posterior probability density of the multi-vector detection problem according to the super-parameters when the updated super-parameters are not converged to a super-parameter vector, and maximizing the posterior probability density through the expected value maximizing algorithm to obtain updated super-parameters until the updated super-parameters are converged to the super-parameter vector;
s522, obtaining a vector estimated value according to the updated super-parameters.
In particular, according to Bayesian reasoning,multi-vector detection problem:
the solution X' of (2) can be estimated from the maximum a posteriori probability: />
When the MSBL algorithm is utilized to solve the multi-vector detection problem, firstly, initializing the superparameter of the multi-vector detection problem, calculating the expected value and variance of the posterior probability density p (X|Y) of the multi-vector detection problem according to the superparameter, then maximizing the posterior probability density p (X|Y) through an expected value maximizing algorithm (EM), obtaining updated superparameter, when the updated superparameter does not converge to a superparameter vector, continuously executing the steps of calculating the expected value and variance of the posterior probability density of the multi-vector detection problem according to the superparameter, updating the superparameter according to the expected value and variance of the posterior probability density until the updated superparameter converges to a superparameter vector, and obtaining the solution of the multi-vector detection problem, namely a vector estimation value X'.
In one embodiment, step S53 specifically includes:
s531, adding all column vectors of the vector estimation values to obtain a superposition vector;
s532, converting the superposition vector into a super-resolution image with a preset size, and obtaining the super-resolution image of the sample.
After the vector estimation value X 'is obtained, all column vectors in the vector estimation value X' are added to obtain a superposition vector, and then the superposition vector is converted into a super-resolution image with a preset size, so that the super-resolution image of the sample is obtained. For example, the superimposed vector is converted into a vector of size m 0 ×n 0 The super-resolution image of (2) has a size of m 0 ×n 0 The super-resolution image of the sample is obtained.
In summary, the present invention provides a flat-field illumination digital scanning structured light super-resolution microscopic imaging system and method, the system includes: a laser light source; the method comprises the steps of shaping Gaussian-distributed laser beams generated by a laser source into uniformly distributed flat top lights, and enabling the uniformly distributed flat top lights to be incident to a beam expanding shaping reflection module of a digital micro-mirror device at a preset angle; modulating uniformly distributed flat-top light according to the imported excitation mode switched at equal intervals to generate a digital micromirror device of a sparse focusing lattice which moves along with time; projecting the sparse focusing lattice onto a sample surface, and exciting the sample to generate a fluorescent signal; and carrying out image reconstruction on the plurality of image data to obtain a control terminal of the super-resolution image of the sample. The invention modulates the evenly distributed flat-top light through the digital micro-mirror device to generate a plurality of focusing points which move along with time, and simultaneously excites the sample through the plurality of focusing points to generate fluorescent signals, thereby improving the imaging range of the image scanning microscopic system, reducing the fluorescent signal acquisition time, having higher signal-to-noise ratio compared with the traditional image scanning microscopic system and realizing super-resolution microscopic imaging with high resolution and wide field of view.
It is to be understood that the system application of the present invention is not limited to the examples described above, and that modifications and variations may be made by those skilled in the art in light of the above teachings, all of which are intended to be within the scope of the invention as defined in the appended claims.
Claims (8)
1. A flat-field illuminated digital scanning structured light super-resolution microscopy imaging system, comprising: the system comprises a laser light source, a beam expanding and shaping reflection module, a digital micro-mirror device, an objective lens, a detector and a control terminal;
the beam expanding and shaping reflection module is used for receiving Gaussian-distributed laser beams generated by the laser source, shaping the Gaussian-distributed laser beams into uniformly-distributed flat top lights, and enabling the uniformly-distributed flat top lights to be incident to the digital micromirror device at a preset angle;
the digital micro-mirror device is used for receiving the uniformly distributed flat-top light incident by the beam expanding and shaping reflection module, modulating the uniformly distributed flat-top light according to the introduced excitation mode switched at equal intervals, and generating a sparse focusing lattice 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 fluorescent signal;
the detector is used for collecting the fluorescent signals to obtain a plurality of image data;
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 image reconstruction is performed on the plurality of image data to obtain a super-resolution image of the sample, including:
constructing a multi-vector detection problem according to the plurality of image data;
solving the multi-vector detection problem to obtain a vector estimated value;
obtaining a super-resolution image of the sample according to the vector estimation value;
the solving the multi-vector detection problem to obtain a vector estimated value comprises the following steps:
initializing a hyper-parameter of the multi-vector detection problem, and calculating an expected value and variance of a posterior probability density of the multi-vector detection problem according to the hyper-parameter;
maximizing the posterior probability density through an expectation maximization algorithm to obtain updated superparameters, and continuously executing the steps of calculating the expectation and variance of the posterior probability density of the multi-vector detection problem according to the superparameters when the updated superparameters do not converge to a superparameter vector, and maximizing the posterior probability density through the expectation maximization algorithm to obtain updated superparameters until the updated superparameters converge to a superparameter vector;
and obtaining a vector estimated value according to the updated super-parameters.
2. The flat-field illuminated digital scanning structured light super-resolution microscopy imaging system according to claim 1, wherein said beam expanding, shaping and reflecting module comprises: a beam expanding and collimating unit, a beam shaper and a first reflecting mirror;
the beam expanding and collimating unit is used for receiving the Gaussian-distributed laser beams generated by the laser source and carrying out beam expanding and collimation on the Gaussian-distributed laser beams;
the beam shaper is used for receiving the Gaussian-distributed laser beams after beam expansion collimation and shaping the Gaussian-distributed laser beams into uniformly distributed flat top lights;
the first reflecting mirror is used for receiving the uniformly distributed flat top light and making the uniformly distributed flat top light incident to the digital micro-mirror device at a preset angle.
3. The flat-field illuminated digital scanning structured light super-resolution microscopic imaging system according to claim 1, wherein 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 lattice and filtering stray light in the sparse focusing lattice;
the first lens is used for receiving the sparse focusing lattice after stray light is filtered, and projecting the sparse focusing lattice to the bicolor sheet;
the bicolor sheet 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 said 4f system comprises a second lens, a stop, and a third lens arranged in sequence along the optical path; the digital micro-mirror device is arranged on the front focal plane of the second lens, the diaphragm is arranged on the back focal plane of the second lens, the back focal plane of the second lens is overlapped with the front focal plane of the third lens, and the back focal plane of the third lens is overlapped with the front focal plane of the first lens.
5. The flat-field illuminated digital scanning structured light super-resolution microscopy imaging system of claim 1, wherein the control terminal is simultaneously connected to the digital micromirror device and the detector, the digital micromirror device is connected to the detector, and the detector synchronously collects the fluorescent signal when the digital micromirror device switches excitation modes.
6. The flat-field illuminated digital scanning structured light super-resolution microscopy imaging system of 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 illuminated digital scanning structured light super-resolution microscopic imaging method, comprising:
shaping a Gaussian-distributed laser beam generated by a laser source, and shaping the Gaussian-distributed laser beam into uniformly-distributed flat top light;
modulating the uniformly distributed flat-top light according to the imported excitation mode switched at equal intervals to generate a sparse focusing lattice which moves along with time;
receiving the sparse focusing lattice, projecting the sparse focusing lattice to a sample surface, and exciting a sample to generate a fluorescent signal;
collecting the fluorescence signals to obtain a plurality of image data;
performing image reconstruction on the plurality of image data to obtain a super-resolution image of the sample;
the step of reconstructing the image data to obtain the super-resolution image of the sample comprises the following steps:
constructing a multi-vector detection problem according to the plurality of image data;
solving the multi-vector detection problem to obtain a vector estimated value;
obtaining a super-resolution image of the sample according to the vector estimation value;
the step of solving the multi-vector detection problem to obtain a vector estimated value comprises the following steps:
initializing a hyper-parameter of the multi-vector detection problem, and calculating an expected value and variance of a posterior probability density of the multi-vector detection problem according to the hyper-parameter;
maximizing the posterior probability density through an expectation maximization algorithm to obtain updated superparameters, and continuously executing the steps of calculating the expectation and variance of the posterior probability density of the multi-vector detection problem according to the superparameters when the updated superparameters do not converge to a superparameter vector, and maximizing the posterior probability density through the expectation maximization algorithm to obtain updated superparameters until the updated superparameters converge to a superparameter vector;
and obtaining a vector estimated value according to the updated super-parameters.
8. The flat-field illuminated digital scanning structured light super-resolution microscopic imaging method according to claim 7, wherein said step of obtaining a super-resolution image of a sample based on said vector estimation comprises:
adding all column vectors of the vector estimation values to obtain a superposition vector;
and converting the superposition vector into a super-resolution image with a preset size to obtain the super-resolution image of the sample.
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