Addressing scanning super-resolution microscopy imaging method and related equipment

Abstract

The present invention discloses an addressing scanning super-resolution microscopy method and related equipment. The method includes: obtaining a wide-field fluorescence image of a sample, and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image; generating a plurality of multi-focus scanning arrays required for multi-focus structured light microscopy based on the starting point coordinate information of the circumscribed rectangle of the selected region of interest and the size of the rectangular area; controlling a two-dimensional acousto-optic deflector to scan the region of interest according to the generated multi-focus scanning array, and controlling an image detector to synchronously acquire multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence; and reconstructing the acquired multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample. The present invention can quickly achieve super-resolution microscopy of any number of regions of interest using an addressing scanning method, without mechanical inertia, so that the obtained super-resolution image will not have artifacts.

Description

Technical Field

[0001] The present invention relates to the field of optical imaging technology, and in particular to an addressing scanning super-resolution microscopic imaging method and related equipment. Background Art

[0002] Optical microscopy, particularly fluorescence microscopy, is widely used in life sciences and biomedicine. However, due to the diffraction of light waves by the limited aperture of optical components, the resolution of optical microscopy is limited, typically to 250-300 nm. Structured illumination microscopy (SIM) utilizes cosine fringes to shift high-frequency information that would otherwise be blocked from the system into the observable frequency range, achieving super-resolution imaging and doubling the resolution. However, SIM suffers from poor imaging depth, making it difficult to perform three-dimensional imaging of thick samples. Image scanning microscopy (ISM), based on point scanning imaging, addresses the poor imaging depth of SIM, but single-point scanning results in slow imaging speeds. Multifocal structured illumination microscopy (MSIM), using a parallel excitation mode, improves imaging speed over ISM. However, when imaging thick samples, sample scattering and out-of-focus background significantly affect the imaging results. Two-photon multi-focus structured light illumination microscopy (2P-MSIM) utilizes the optical tomography capability and deep-depth imaging advantages of two-photon microscopy to further enhance the imaging performance of MSIM. While achieving a resolution twice that of wide-field microscopy, it is capable of three-dimensional imaging of thick samples. At the same time, its excitation light source uses a femtosecond laser in the near-infrared band, which can minimize damage to living cells.

[0003] However, the traditional 2P-MSIM, which uses a microlens array and a scanning galvanometer to achieve two-photon excitation array generation and scanning, has poor flexibility and mechanical inertia, resulting in artifacts in the reconstructed image. While the 2P-MSIM, which uses a high-speed phase-type spatial light modulator (SLM), can avoid mechanical inertia and artifacts, it still suffers from slow imaging due to the limited refresh rate of the SLM.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an address scanning super-resolution microscopy method and related equipment to solve the problems of mechanical inertia and slow imaging speed in the existing microscopy using two-photon multi-focus structured illumination.

[0006] The technical solutions of the present invention are as follows:

[0007] An address scanning super-resolution microscopy method comprises the following steps:

[0008] Acquiring a wide-field fluorescence image of the sample, and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image;

[0009] generating a plurality of multi-focus scanning arrays required for multi-focus structured illumination microscopy imaging according to the selected starting point coordinate information of the circumscribed rectangle of the region of interest and the size of the rectangular region;

[0010] controlling the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and controlling the image detector to synchronously acquire the multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence;

[0011] The collected multi-focus fluorescence image sequence is reconstructed to obtain a super-resolution image of the region of interest in the sample.

[0012] In a further arrangement of the present invention, the step of acquiring a wide-field fluorescence image of the sample and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image comprises:

[0013] Control the output of low-power continuous laser to excite the sample in a wide field;

[0014] Acquire and save wide-field fluorescence images of the sample;

[0015] The wide-field fluorescence image is selected to obtain at least one region of interest for super-resolution imaging.

[0016] According to a further configuration of the present invention, the step of generating a plurality of multi-focus scanning arrays based on the selected starting point coordinate information of the circumscribed rectangle of the region of interest and the size of the rectangular region comprises:

[0017] Obtaining X and Y coordinate information of a starting point of a circumscribed rectangle of the rectangular area of ​​the region of interest, as well as width information Lx along the X direction and width information Ly along the Y direction;

[0018] The plurality of multi-focus scanning arrays are generated according to the X and Y coordinate information of the starting point, the width information Lx along the X direction and the width information Ly along the Y direction, and the pre-input X direction point number information Nx and scanning step information M.

[0019] In a further configuration of the present invention, each multi-focus scanning array includes Nx times Ny points, the point spacing D is obtained by dividing the width information Lx along the X direction by Nx and rounding it up, and the number of points in the Y direction Ny is obtained by dividing the width information Ly along the Y direction by the point spacing D and rounding it up, and two adjacent multi-focus scanning arrays move in the X and Y directions respectively with a scanning step size M.

[0020] In a further configuration of the present invention, the steps of controlling the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and controlling the image detector to synchronously acquire the multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence include:

[0021] The control data acquisition card generates digital signals and analog signals, wherein the digital signal is used to control the acousto-optic deflector to scan the region of interest according to the multiple multi-focus scanning arrays, and the analog signal is used to control the image detector to synchronously acquire a multi-focus fluorescence image sequence corresponding to the region of interest.

[0022] According to a further configuration of the present invention, the acousto-optic deflector scans the specific pixel coordinates in the region of interest according to the sound wave frequency; wherein the sound wave frequency is obtained by converting the digital signal, the specific pixel coordinates in the region of interest are obtained by the coordinates of each point in the multi-focus scanning array, and the corresponding sound wave frequency is calculated one by one according to the corresponding relationship between the pixel coordinates and the sound wave frequency.

[0023] According to a further configuration of the present invention, the digital signal and the analog signal have the same initial phase, and the frequency of the digital signal is Nx times Ny times the analog signal; when the digital signal received by the acousto-optic deflector is a rising edge, the acousto-optic deflector performs addressing scanning according to the pixel coordinates corresponding to the digital signal; when the image detector receives the rising edge of the analog signal, it starts exposure, and ends exposure when the next rising edge of the analog signal arrives, and enters the next exposure at the same time.

[0024] An address scanning super-resolution microscopy imaging device, used to implement the above-mentioned address scanning super-resolution microscopy imaging method, comprising:

[0025] an acquisition module, the acquisition module being configured to acquire a wide-field fluorescence image of the sample and select at least one region of interest for super-resolution imaging based on the wide-field fluorescence image;

[0026] A generation module, the generation module is used to generate a plurality of multi-focus scanning arrays required for multi-focus structured illumination microscopic imaging according to the starting point coordinate information of the circumscribed rectangle of the selected region of interest and the size of the rectangular region;

[0027] a control module, the control module being configured to control the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and to control the image detector to synchronously acquire multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence;

[0028] An image processing module is used to reconstruct the collected multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample.

[0029] A computer device comprises a memory and a processor, wherein the memory stores a computer program, and the processor implements the steps of the above-mentioned address scanning super-resolution microscopy imaging method when executing the computer program.

[0030] A computer-readable storage medium stores a computer program, which, when executed by a processor, implements the steps of the above-mentioned address scanning super-resolution microscopy imaging method.

[0031] The present invention provides an addressing scanning super-resolution microscopy method and related equipment, which includes the following steps: obtaining a wide-field fluorescence image of a sample, and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image; generating several multi-focus scanning arrays required for multi-focus structured light microscopy based on the starting point coordinate information of the circumscribed rectangle of the selected region of interest and the size of the rectangular area; controlling an acousto-optic deflector to scan the region of interest according to the several multi-focus scanning arrays, and controlling an image detector to synchronously acquire multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence; and reconstructing the acquired multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample. The present invention obtains a wide-field fluorescence image, selects one or more regions of interest that require super-resolution imaging on the wide-field fluorescence image and generates several multi-focus scanning arrays, then controls the acousto-optic deflector to scan the region of interest, and synchronously collects a multi-focus fluorescence image sequence corresponding to the region of interest. Finally, the collected multi-focus fluorescence image sequence is reconstructed to obtain a super-resolution image of the region of interest, thereby quickly achieving super-resolution microscopic imaging of any number of regions of interest in an addressing scanning manner, without mechanical inertia, and ensuring that the reconstructed super-resolution image is free of artifacts. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary personnel in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0033] Figure 1 It is a schematic flow chart of the addressing scanning super-resolution microscopy imaging method of the present invention.

[0034] Figure 2 This is a schematic diagram of the synchronous control principle of digital signals and analog signals in the present invention.

[0035] Figure 3 It is a schematic diagram of the acousto-optic deflector addressing scanning in the present invention to generate a multi-focus scanning array and perform MSIM imaging.

[0036] Figure 4 It is a diagram of a software control interface in one embodiment of the present invention.

[0037] Figure 5 This is a diagram showing the implementation results of AOD-MSIM imaging of an arbitrary area in one embodiment of the present invention.

[0038] Figure 6 This is a principle block diagram of the addressing scanning super-resolution microscopy imaging device of the present invention. DETAILED DESCRIPTION

[0039] The present invention provides an address-scanning super-resolution microscopy method and related equipment. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is further described below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0040] In the embodiments and patent claims, unless otherwise specified herein, the words "a," "an," "the," and "the" may include plural forms. If the embodiments of the present invention include descriptions of "first," "second," etc., such descriptions are for descriptive purposes only and should not be understood as indicating or implying their relative importance or implicitly specifying the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include at least one of such features.

[0041] It should be further understood that the term "comprising" as used in the description of the present invention refers to the presence of the stated features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. It should be understood that when we refer to an element as being "connected" or "coupled" to another element, it can be directly connected or coupled to the other element, or there can be intermediate elements. In addition, "connected" or "coupled" as used herein can include wireless connections or wireless couplings. The term "and / or" as used herein includes all or any units and all combinations of one or more associated listed items.

[0042] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0043] In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in this field can implement them. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0044] The inventors discovered that the traditional 2P-MSIM method, which uses a microlens array and a scanning mirror to generate and scan two-photon excitation arrays, suffers from poor flexibility and mechanical inertia, leading to artifacts in the reconstructed super-resolution images. To address these issues, a two-photon multi-focus structured light illumination super-resolution microscopy method based on a high-speed phase-type spatial light modulator (SLM) was proposed. This method enables the generation of multi-focus arrays and high-precision parallel digital random addressing scanning and excitation imaging on the sample surface. Combining pixel relocation and deconvolution techniques, it achieves three-dimensional two-photon multi-focus structured light super-resolution imaging. This method overcomes the mechanical inertia of the scanning mirror in 2P-MSIM imaging, while reducing system complexity and improving flexibility. However, this method suffers from slow imaging speed: for an imaging area of ​​26.1μm × 26.1μm, the imaging time is 6.25s. Furthermore, since a series of phase maps corresponding to the two-photon excitation array must be pre-programmed according to the scanning area and then imported into the SLM for scanning, the process is cumbersome and hinders real-time selection of regions of interest for addressing and scanning.

[0045] In response to the above technical problems, the present invention provides an addressing scanning super-resolution microscopy method and related equipment. After obtaining a wide-field fluorescence image, one or more regions of interest that require super-resolution imaging are selected on the wide-field fluorescence image and several multi-focus scanning arrays required for multi-focus structured light illumination microscopy are generated. Thereafter, the acousto-optic deflector is controlled to scan the region of interest, and a multi-focus fluorescence image sequence corresponding to the region of interest is synchronously acquired. Finally, the acquired multi-focus fluorescence image sequence is reconstructed to obtain a super-resolution image of the region of interest. In this way, super-resolution microscopy of any number of regions of interest can be quickly achieved in an addressing scanning manner, and there will be no mechanical inertia, so that the reconstructed super-resolution image will not have artifacts.

[0046] Please also see Figures 1 to 5 The present invention provides a preferred embodiment of an address scanning super-resolution microscopy imaging method.

[0047] like Figure 1 As shown, the present invention provides an address scanning super-resolution microscopy imaging method, which includes the steps of:

[0048] S100, acquiring a wide-field fluorescence image of a sample, and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image;

[0049] Specifically, the sample can be a fixed cell or a living cell, such as an animal cell or a plant cell. First, the computer device controls the image detector to collect a wide-field fluorescence image of the sample, which can be a wide-field fluorescence image of a complete cell or a local cell structure, and transmits it to the computer device. The captured image can be saved in any file path for later use. Thereafter, the wide-field fluorescence image is imported into the LabVIEW program, and one or more regions of interest are selected based on the obtained wide-field fluorescence image for MSIM imaging, wherein the region of interest can be manually selected by the user, and the region shape can be any shape, for example, a rectangle.

[0050] In some embodiments, the image detector may be, but is not limited to, an electron-multiplying charge coupled device (EMCCD), a CMOS camera, or other high-sensitivity area array detector.

[0051] S200, generating a plurality of multi-focus scanning arrays required for multi-focus structured illumination microscopic imaging according to the selected starting point coordinate information of the circumscribed rectangle of the region of interest and the size of the rectangular region;

[0052] S300, controlling the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and controlling the image detector to synchronously acquire the multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence;

[0053] Specifically, a computer device controls the acousto-optic deflector (AOD) to quickly deflect and focus the incident pulsed laser to specific pixel coordinates in the area of ​​interest, and the image detector collects data synchronously. That is, every time the acousto-optic deflector scans a dot matrix, the image detector completes an exposure to obtain a multi-focal fluorescence image. Finally, a series of multi-focal fluorescence images are captured.

[0054] S400 , reconstructing the collected multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample.

[0055] Specifically, after a computer device obtains a sequence of fluorescence images corresponding to the region of interest, it reconstructs the image using a multiple measurement vector sparse Bayesian learning (MSBL) algorithm to produce a super-resolution image of the region of interest. The MSBL process involves the following steps: 1. Initialize hyperparameters; 2. Calculate the expected value and variance of the posterior probability density p(X│Y); 3. Update the estimated hyperparameters using a maximization algorithm; and 4. Iterate steps 2 and 3 until convergence to a single hyperparameter vector.

[0056] In the above technical solution, after obtaining a widefield fluorescence image through an image detector, the present invention selects one or more regions of interest (ROIs) requiring super-resolution imaging on the widefield fluorescence image and generates several multi-focus scanning arrays required for multi-focus structured illumination microscopy. The acousto-optic deflector is then controlled to scan the region of interest, and a multi-focus fluorescence image sequence corresponding to the region of interest is simultaneously acquired. Finally, the acquired multi-focus fluorescence image sequence is reconstructed to obtain a super-resolution image of the region of interest. This allows for rapid super-resolution microscopy of any number of regions of interest using an addressable scanning method, without mechanical inertia, and without artifacts in the reconstructed super-resolution image. Furthermore, the present invention first performs rapid widefield fluorescence imaging on the sample, then selects the region of interest on the fluorescence image for 2P-MSIM super-resolution imaging. This not only allows for rapid and accurate acquisition of local super-resolution images that can be contrasted with the surrounding widefield fluorescence image, but also reduces phototoxicity to other parts of the sample, enabling super-resolution imaging monitoring of the physiological activities of specific intracellular structures.

[0057] In a further embodiment of the present invention, the step of acquiring a wide-field fluorescence image of the sample and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image includes:

[0058] S110, controlling the output of low-power continuous laser wide-field excitation of the sample;

[0059] S120, acquiring and saving a wide-field fluorescence image of the sample;

[0060] S130 , selecting an area from the wide-field fluorescence image to obtain at least one region of interest for super-resolution imaging.

[0061] Specifically, a continuous light laser (wavelength of 488nm) is controlled to output continuous light laser and excite the sample in a wide field under low-power conditions. At the same time, the image detector is controlled to record the fluorescence image of the sample in real-time shooting mode. The sample is focused by adjusting the objective lens, and the stage is adjusted to find a suitable area to be measured. A wide-field fluorescence image of the sample is collected to obtain a wide-field fluorescence image of the complete cell or local cell structure. The captured wide-field fluorescence image can be saved in any file path for calling in the software. The wide-field fluorescence image is imported into the LabVIEW program for selection, that is, one or more areas (i.e., regions of interest) to be subjected to MSIM super-resolution imaging are selected on the obtained wide-field fluorescence image.

[0062] In a further implementation of an embodiment, the step of generating a plurality of multi-focus scanning arrays required for multi-focus structured light microscopy imaging based on the starting point coordinate information of the selected circumscribed rectangle of the region of interest and the size of the rectangular region includes:

[0063] S210, obtaining X and Y coordinate information of a starting point of a circumscribed rectangle of a rectangular area of ​​the region of interest, as well as width information Lx along the X direction and width information Ly along the Y direction;

[0064] Each multifocal array contains Nx times Ny points. The point spacing is obtained by dividing the width information Lx along the X direction by N and rounding it up. The number of points in the Y direction Ny is obtained by dividing the width information Ly along the Y direction by the point spacing D and rounding it up. Two adjacent multifocal arrays move in the X and Y directions respectively with a scanning step size M. The number of multifocal arrays is related to the point spacing and the scanning step size M.

[0065] S220 , generating the plurality of multi-focus scanning arrays according to the X and Y coordinate information of the starting point, the width information Lx along the X direction, the width information Ly along the Y direction, and the pre-input X direction point number information Nx and the scanning step length M.

[0066] Specifically, after selecting a region of interest (ROI), the pixel coordinate information of the selected ROI and its bounding rectangle is saved. Subsequently, the X and Y coordinates (x1, y1) of the starting point of the bounding rectangle of interest and the width information Lx and Ly of the bounding rectangle in the X direction and Y direction are read. Based on these two sets of information and the previously entered number of X-direction points Nx, the point spacing D (determined by dividing Lx by Nx and rounding) and the number of Y-direction points Ny (determined by dividing Ly by D and rounding) are automatically calculated to generate the first multi-focus scanning array required by MSIM, which contains Nx times Ny points. Subsequently, based on the scan step size M, the multi-focus array is shifted M pixels in the X direction to obtain a second multi-focus array, until the multi-focus array scan in the X direction is completed. The multi-focus array is then shifted M pixels in the Y direction and continues to scan in the reverse direction in the X direction. This cycle continues until the X and Y directions of the ROI are scanned. The number of multi-focus scanning arrays generated in this way is related to the point spacing D and the scanning step size M. Generally, when M is 1, the required number of scans is D^2, so the multi-focus scanning array includes D^2 different multi-focus arrays. The pulsed laser light generated by the pulsed laser (usually in the red or infrared band, such as a wavelength of 800nm) passes through the acousto-optic deflector and scans the selected area of ​​interest according to the multi-focus scanning array.

[0067] In a further implementation of an embodiment, the steps of controlling the acousto-optic deflector to scan the region of interest according to the multi-focus scanning array, and controlling the image detector to synchronously acquire a multi-focus fluorescence image sequence corresponding to the region of interest include:

[0068] S310. Control the data acquisition card to generate digital signals and analog signals, wherein the digital signal is used to control the acousto-optic deflector to scan the region of interest according to the multi-focus scanning array, and the analog signal is used to control the image detector to synchronously acquire a multi-focus fluorescence image sequence corresponding to the region of interest.

[0069] Specifically, the acousto-optic deflector scans the specific pixel coordinates of the region of interest according to the frequency of the acoustic wave. The acoustic wave frequency is obtained by converting the 32-bit digital signal output by the data acquisition card, and the specific pixel coordinates of each pixel in the region of interest are determined by the coordinates of each point in the multi-focus scanning array, and the corresponding acoustic wave frequency is calculated one by one according to the correspondence between the pixel coordinates and the acoustic wave frequency. By changing the frequency of the acoustic wave loaded on the acousto-optic deflector, the deflection direction of the incident light beam can be quickly changed, thereby changing the position where the pulsed laser converges on the sample. It should be noted that a two-dimensional acousto-optic deflector composed of a pair of orthogonal acousto-optic deflectors can realize the deflection of the incident light beam in the X and Y directions by respectively changing the frequency of the acoustic wave loaded in the X and Y directions, thereby realizing addressing scanning of any point in the XY plane.

[0070] The digital signal and the analog signal have the same initial phase, and the frequency of the digital signal is Nx times Ny times the analog signal. When the digital signal received by the acousto-optic deflector is a rising edge, the acousto-optic deflector performs addressing and scanning according to the pixel coordinates corresponding to the digital signal. When the image detector receives a rising edge of the analog signal, exposure begins and ends when the next rising edge of the analog signal arrives, and the next exposure begins. In other words, the single-frame exposure time of the image detector is determined by the number of points in a single multi-focus scanning array (i.e., Nx times Ny).

[0071] That is, when the data from the multifocal scanning array is converted into a series of digital signals to control the acousto-optic deflector to rapidly deflect and focus the incident pulsed laser light to specific coordinate positions within the selected region of interest, the data acquisition card outputs an analog signal to control the exposure of the image detector, thereby synchronously capturing a sequence of multifocal fluorescence images. That is, each time the acousto-optic deflector scans a multifocal array, the image detector completes an exposure, ultimately capturing a series of multifocal fluorescence images corresponding to the plurality of multifocal arrays. The number of frames captured by the image detector is determined by the number of multifocal scanning arrays (which is related to the point spacing D and the scanning step size M).

[0072] The working principle of the present invention is further explained below: the pulsed laser outputs a light path for exciting the sample to scan the region of interest and perform two-photon excitation, and the continuous laser outputs a wide-field fluorescence imaging light path to perform wide-field illumination and excitation of the sample, so that the image detector can obtain a wide-field fluorescence image, wherein the two light paths can be controlled separately using different programs. In a specific implementation, after observing that the sample is focused using the microscope's built-in bright field light path (white light illumination), the image detector is controlled to capture a bright field image to obtain the cell's contour information, and then after turning off the bright field illumination, the continuous laser in the wide-field illumination excitation light path is controlled to irradiate the sample at a lower power, and the wide-field fluorescence image is captured and saved by the image detector for use in selecting any number of regions of interest for super-resolution imaging. If the bright field image is clear enough, both the bright field image and the wide-field fluorescence image can be used as the basis for selecting the area where super-resolution imaging is desired. After selecting the region of interest, the computer device will obtain the starting point coordinate information and the width information in the X and Y directions of the circumscribed rectangle of the selected single region, and then input the number of points Nx in the X direction of the multi-focus array to calculate the point spacing D and the number of points Ny in the Y direction based on the starting point coordinate information and the number of points Nx to generate the first multi-focus array. Then, the dot matrix scanning step size M is input to generate several multi-focus scanning arrays that are translated by M pixels in the X and Y directions respectively. The number of multi-focus scanning arrays is determined by the point spacing D and the scanning step size M. When M is generally 1, a total of D^2 multi-focus scanning arrays are generated. The computer equipment converts the coordinate information corresponding to all points in the multi-focus scanning array into the sound wave frequency loaded on the acousto-optic deflector in turn, and then a series of digital signals control the acousto-optic deflector to deflect the incident pulsed laser to the specific coordinate position of the selected area of ​​interest. At the same time, an analog signal synchronized with the acousto-optic deflector is generated to enable the acousto-optic deflector to scan a multi-focus array, and the image detector synchronously captures a frame of multi-focus fluorescence image, thereby achieving the purpose of multi-focus array scanning. Finally, a series of multi-focus fluorescence images corresponding to several multi-focus scanning arrays are obtained. The multi-focus fluorescence image sequence is input into the MSBL program to reconstruct a super-resolution image, thereby realizing the combination of the fast addressing characteristics of the acousto-optic deflector with MSIM, and realizing fast super-resolution imaging of any area of ​​interest in the sample. The relationship between digital signals and analog signals is as follows: Figure 2 As shown, the frequency of the digital signal is Nx times Ny times the analog signal, and the number of periods of the analog signal is equal to the number of multi-focus arrays, that is, when the scanning step length M is 1, it is D^2. The generation and scanning process of the multi-focus array is as follows Figure 3 As shown, Figure 3 Figure a is a schematic diagram of generating a single multi-focus array using AOD addressing scanning within a single exposure time of the image detector. Figure 3 b is the single multi-focus array image generated, Figure 3The diagram c in the figure is a schematic diagram of a multi-focus scanning array generated by the multi-focus array being translated several times according to the scanning step number M. Figure 3 Figure d in the middle is a diagram of the MSIM imaging process (detecting multi-focus fluorescence images in multi-focus illumination mode to obtain raw image data).

[0073] The following explanation is given using the standard sample of lily of the valley rhizome as an example.

[0074] First, turn on the pulsed laser and adjust the wavelength to 800nm. Also, turn on the continuous wave (CW) laser. The CW laser control program or motorized filter wheel controls the illumination intensity. The image detector and AOD controller or module power is turned on. Next, the CW laser is controlled at a low power level, and the image detector is set to real-time capture mode to record a widefield fluorescence image of the sample. The objective lens is adjusted to focus the sample, and the stage is adjusted to locate the appropriate area to be measured. A widefield fluorescence image of the sample is acquired, which the program then uses to select the area for super-resolution imaging.

[0075] See also Figure 4 , load the acquired wide-field fluorescence image (or bright-field image with the same function) under "File Path", select one or more rectangular regions of interest of any shape in the image, and enter the number of points N in the X direction of the dot matrix. The computer device will obtain the coordinates of the starting point of the rectangle circumscribing the selected area and the width of the rectangular area in the X and Y directions, and then calculate the point spacing D and the number of points Ny in the Y direction based on the selection width and the number of points Nx in the X direction (represented by N in the figure). The computer device automatically generates the first multi-focus array based on the obtained starting point coordinates (x, y) and the point spacing D, and at the same time generates several other multi-focus scanning arrays that scan along the X and Y directions respectively according to the scanning step size M, and converts the coordinates of each point in these multi-focus arrays into the acoustic wave frequency of the input AOD in sequence. The AOD is controlled by a digital signal to make the incident pulsed laser undergo the required deflection, and finally converges at different positions in the sample's area of ​​interest through the objective lens. At the same time, the computer device also generates an analog signal synchronized with the AOD digital signal to control the exposure of the image detector and perform synchronous acquisition. That is, each time the AOD scans a multi-focus array, the image detector completes an exposure, and finally captures a series of multi-focus fluorescence images corresponding to several multi-focus scanning arrays. Finally, the collected multi-focus fluorescence image sequence is reconstructed to obtain a super-resolution image of the area of ​​interest in the sample.

[0076] The demonstration results are as follows Figure 5 As shown, Figure 5 (a) is the MSIM selection program interface. Figure 5 (b) is the multi-focus fluorescence image recorded by the image detector after the first multi-focus array scan of the selected area. Figure 5(c) is a wide-field fluorescence image obtained by superimposing a multi-focus fluorescence image sequence. Figure 5 (d) is the super-resolution image reconstructed by the MSBL algorithm. Figure 5 As can be seen from the results, only the selected area is excited by the pulsed laser and emits fluorescence. It can be seen that the AOD-MSIM program can easily realize the selection of specific areas and perform MSIM super-resolution imaging of the corresponding areas by controlling the AOD addressing scan.

[0077] See also Figure 6 In some embodiments, the present invention further provides an address scanning super-resolution microscopy imaging device for implementing the above-mentioned address scanning super-resolution microscopy imaging method, which comprises:

[0078] An acquisition module 100 is configured to acquire a wide-field fluorescence image of a sample and select at least one region of interest for super-resolution imaging based on the wide-field fluorescence image;

[0079] A generation module 200 is configured to generate a plurality of multi-focus scanning arrays required for multi-focus structured illumination microscopy imaging based on the starting point coordinate information of the circumscribed rectangle of the selected region of interest and the size of the rectangular region;

[0080] a control module 300 configured to control the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and to control the image detector to synchronously capture each multi-focus fluorescence image corresponding to the region of interest to obtain a multi-focus fluorescence image sequence;

[0081] The image processing module 400 is used to reconstruct the collected multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample.

[0082] In some embodiments, the present invention further provides a computer device comprising a memory and a processor, wherein the memory stores a computer program, and when the processor executes the computer program, the following steps are implemented:

[0083] S100, acquiring a wide-field fluorescence image of a sample, and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image;

[0084] S200, generating a plurality of multi-focus scanning arrays required for multi-focus structured illumination microscopic imaging according to the selected starting point coordinate information of the circumscribed rectangle of the region of interest and the size of the rectangular region;

[0085] S300, controlling the acousto-optic deflector to scan the region of interest according to the multi-focus scanning array, and controlling the image detector to synchronously acquire each multi-focus fluorescence image corresponding to the region of interest to obtain a multi-focus fluorescence image sequence;

[0086] S400 , reconstructing the collected multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample.

[0087] In some embodiments, the present invention further provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the following steps:

[0088] S100, acquiring a wide-field fluorescence image of a sample, and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image;

[0089] S200, generating a plurality of multi-focus scanning arrays required for multi-focus structured illumination microscopic imaging according to the selected starting point coordinate information of the circumscribed rectangle of the region of interest and the size of the rectangular region;

[0090] S300, controlling the acousto-optic deflector to scan the region of interest according to the multi-focus scanning array, and controlling the image detector to synchronously acquire each multi-focus fluorescence image corresponding to the region of interest to obtain a multi-focus fluorescence image sequence;

[0091] S400 , reconstructing the collected multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample.

[0092] In summary, the address scanning super-resolution microscopy method and related equipment provided by the present invention have the following beneficial effects:

[0093] After obtaining a wide-field fluorescence image through the image detector, one or more regions of interest requiring super-resolution imaging are selected on the wide-field fluorescence image and a multi-focus scanning array is generated. The acousto-optic deflector is then controlled to scan the region of interest, and a multi-focus fluorescence image sequence corresponding to the region of interest is simultaneously acquired. Finally, the acquired multi-focus fluorescence image sequence is reconstructed to obtain a super-resolution image of the region of interest. This allows for rapid, directional super-resolution microscopic imaging of any number of regions of interest in an addressing scanning manner, without mechanical inertia, and ensures that the reconstructed super-resolution image is artifact-free.

[0094] First, the sample is subjected to rapid wide-field fluorescence imaging, and the region of interest is selected on the fluorescence image for 2P-MSIM super-resolution imaging. This not only allows the local super-resolution image to be obtained quickly and accurately, and contrasted with the surrounding wide-field fluorescence image, but also reduces phototoxicity to other parts of the sample, thus realizing super-resolution imaging monitoring of the physiological activities of certain specific structures within the cell.

[0095] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for address scanning super-resolution microscopy, characterized in that: Including steps: The pulsed laser outputs a light path for exciting the sample to scan the region of interest and perform two-photon excitation, while the continuous laser outputs a wide-field fluorescence imaging light path to illuminate and excite the sample in a wide field, allowing the image detector to acquire a wide-field fluorescence image. Acquiring a wide-field fluorescence image of the sample, and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image; generating a plurality of multi-focus scanning arrays required for multi-focus structured illumination microscopy imaging according to the selected starting point coordinate information of the circumscribed rectangle of the region of interest and the size of the rectangular region; controlling the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and controlling the image detector to synchronously acquire the multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence, and controlling the data acquisition card to generate a digital signal and an analog signal, wherein the digital signal is used to control the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and the analog signal is used to control the image detector to synchronously acquire the multi-focus fluorescence image sequence corresponding to the region of interest; Reconstructing the acquired multi-focus fluorescence image sequence using a sparse Bayesian learning algorithm based on a multiple measurement vector model to obtain a super-resolution image of the region of interest in the sample; The step of generating a multi-focus scanning array required for multi-focus structured light microscopy imaging based on the starting point coordinate information of the selected circumscribed rectangle of the region of interest and the size of the rectangular region includes: Obtaining X and Y coordinate information of a starting point of a circumscribed rectangular area of ​​the region of interest, as well as width information Lx along the X direction and width information Ly along the Y direction; Generate the plurality of multi-focus scanning arrays according to the X and Y coordinate information of the starting point, the width information Lx along the X direction, the width information Ly along the Y direction, and the pre-input X direction point number information Nx and the scanning step information M; Each multi-focus scanning array contains Nx times Ny points. The point spacing D is obtained by dividing the width information Lx along the X direction by Nx and rounding it up. The number of points in the Y direction Ny is obtained by dividing the width information Ly along the Y direction by the point spacing D and rounding it up. Two adjacent multi-focus scanning arrays move in the X and Y directions respectively with a scanning step size M.

2. The address scanning super-resolution microscopy method according to claim 1, characterized in that: The step of acquiring a wide-field fluorescence image of the sample and selecting at least one region of interest for super-resolution imaging based on the wide-field fluorescence image comprises: Control the output of low-power continuous laser to excite the sample in a wide field; Acquire and save wide-field fluorescence images of the sample; The wide-field fluorescence image is selected to obtain at least one region of interest for super-resolution imaging.

3. The address scanning super-resolution microscopy method according to claim 1, characterized in that: The acousto-optic deflector scans the specific pixel coordinates in the region of interest according to the sound wave frequency; wherein the sound wave frequency is obtained by converting the digital signal, the specific pixel coordinates in the region of interest are obtained by the coordinates of each point in the multi-focus scanning array, and the corresponding sound wave frequency is calculated one by one according to the correspondence between the pixel coordinates and the sound wave frequency.

4. The address scanning super-resolution microscopy method according to claim 1, characterized in that: The digital signal and the analog signal have the same initial phase, and the frequency of the digital signal is Nx times Ny times the analog signal; when the digital signal received by the acousto-optic deflector is a rising edge, the acousto-optic deflector performs addressing scanning according to the pixel coordinates corresponding to the digital signal; when the image detector receives the rising edge of the analog signal, it starts exposure, and ends exposure when the next rising edge of the analog signal arrives, and enters the next exposure at the same time.

5. An address scanning super-resolution microscopy imaging device for implementing the address scanning super-resolution microscopy imaging method according to any one of claims 1 to 4, characterized in that: include: an acquisition module, the acquisition module being configured to acquire a wide-field fluorescence image of the sample and select at least one region of interest for super-resolution imaging based on the wide-field fluorescence image; A generating module, the generating module is used to generate a plurality of multi-focus scanning arrays according to the starting point coordinate information of the circumscribed rectangle of the selected region of interest and the size of the rectangular region; a control module, the control module being configured to control the acousto-optic deflector to scan the region of interest according to the plurality of multi-focus scanning arrays, and to control the image detector to synchronously acquire multi-focus fluorescence images corresponding to the region of interest to obtain a multi-focus fluorescence image sequence; An image processing module is used to reconstruct the collected multi-focus fluorescence image sequence to obtain a super-resolution image of the region of interest in the sample.

6. A computer device, characterized in that: The invention comprises a memory and a processor, wherein a computer program is stored in the memory, and when the processor executes the computer program, the steps of the address scanning super-resolution microscopy imaging method according to any one of claims 1 to 4 are implemented.

7. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the address scanning super-resolution microscopy imaging method according to any one of claims 1 to 4 are implemented.

Images