Arbitrary surface three-dimensional addressing scanning super-resolution microscopic imaging method, system and terminal

Abstract

The present invention provides a method, system, and terminal for super-resolution microscopy of arbitrary curved surfaces, specifically relating to the field of optical microscopy. Based on a wide-field fluorescence image of a target sample, this solution obtains the size and position coordinates of the circumscribed rectangle of a region of interest (ROI), generating a series of multi-focus 2D scanning dot arrays corresponding to the ROI. Based on the structural characteristics of the target sample, an axial coordinate is assigned to each scanning point in the multi-focus 2D scanning dot array, generating a series of 3D scanning dot arrays matching the selected surface template and converting them into a series of 2D dot array fluorescence images. The 2D dot array fluorescence images are then reconstructed to obtain a 3D super-resolution image of each ROI. This solution enables rapid, dynamic 3D super-resolution imaging of any curved surface within any ROI, without being limited by depth of field.

Description

Technical Field

[0001] The present invention relates to the field of optical microscopic imaging technology, and in particular to a method, system and terminal for super-resolution microscopic imaging of arbitrary curved surfaces using three-dimensional addressing and scanning. Background Art

[0002] Optical microscopy enables real-time, non-destructive, and non-contact observation of cells at the nanoscale, and is one of the most basic and important research tools in the fields of life sciences and biomedicine. Because structured illumination microscopy's super-resolution approach offers fast imaging speeds and has no special requirements for fluorescent probes or excitation light energy density, it has a natural advantage in live cell super-resolution imaging applications. In the development of structured illumination microscopy super-resolution imaging, the earliest technique to appear was SIM (Structured Illumination Microscopy), which uses cosine fringe structured light to illuminate samples for super-resolution imaging. This technique, compared to conventional widefield imaging, can achieve twice the resolution. However, the excitation light energy density under this illumination mechanism is low and cannot penetrate the sample surface, resulting in poor imaging depth and inability to perform three-dimensional imaging of thicker samples.

[0003] The subsequent emergence of Image Scanning Microscopy (ISM) combined traditional confocal microscopy with improved tomography capabilities while maintaining the resolution-enhancing effect of SIM, enabling greater imaging depth. However, the single-point scanning method of ISM severely restricted the imaging speed. Building on this, Multifocal Structured Illumination Microscopy (MSIM) emerged, significantly improving ISM imaging speed by scanning samples at multiple focal points in parallel. However, when imaging thick samples, sample scattering and defocused background have a significant impact on the imaging results. Two-photon microscopy has the advantages of excellent optical tomography capabilities and deep imaging. The imaging performance of two-photon multifocal structured illumination microscopy (2P-MSIM), which combines two-photon excitation fluorescence, has been further improved, reducing the impact of sample scattering and defocused background on imaging. However, in practical applications, MSIM technology and 2P-MSIM technology still face the problem of mutual restriction between imaging depth and three-dimensional imaging speed. There is a bottleneck in the application of dynamic super-resolution imaging of thick biological samples, and they have not yet fully utilized their natural advantages in super-resolution imaging of living cells and living biological samples. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method, system and terminal for super-resolution microscopy of arbitrary curved surfaces with three-dimensional addressing and scanning, aiming to solve the problem of mutual restriction between imaging depth and three-dimensional imaging speed in the prior art three-dimensional super-resolution imaging technology.

[0005] To achieve the above objectives, the present invention provides, in a first aspect, a method for three-dimensional addressing and scanning super-resolution microscopy of an arbitrary curved surface, which is applied to a super-resolution imaging device. The device mainly includes a laser, an acousto-optic deflector, an adjustable acoustic gradient lens, and a microscope objective lens, which are arranged in sequence along the optical path. The method mainly includes the following steps:

[0006] Acquiring a wide-field fluorescence image of the target sample, and selecting at least one region of interest based on the wide-field fluorescence image;

[0007] Based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as the preset scanning point number information and scanning step length information, a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest are generated;

[0008] Based on the structural characteristics of the target sample, a preset curved surface template is selected, an axial coordinate is set for each scanning point in the multi-focus two-dimensional scanning dot matrix, and a series of three-dimensional scanning dot matrices matching the selected curved surface template are generated;

[0009] Based on a mapping relationship between a preset transverse coordinate and the acoustic wave frequency of the acousto-optic deflector, and a mapping relationship between a preset axial coordinate and the acoustic wave frequency of the adjustable acoustic gradient lens, the target sample is scanned according to the three-dimensional scanning dot matrix, and fluorescence signals emitted by the target sample are synchronously collected to obtain a series of two-dimensional dot matrix fluorescence images;

[0010] The two-dimensional dot matrix fluorescence image is reconstructed using a preset reconstruction algorithm to obtain a three-dimensional super-resolution image that matches the selected curved surface template in each region of interest.

[0011] Optionally, acquiring a wide-field fluorescence image of the target sample includes:

[0012] Adjusting the microscope objective lens to a focal plane, and controlling the acousto-optic deflector to scan the target sample according to each of the preset full-field scanning multi-focus two-dimensional scanning lattice coordinates and a mapping relationship between the preset lateral coordinates and the acoustic wave frequency of the acousto-optic deflector, thereby obtaining a series of two-dimensional lattice fluorescence images;

[0013] The two-dimensional dot matrix fluorescence images are superimposed to obtain a wide-field fluorescence image of the target sample.

[0014] Optionally, the step of generating a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as preset scanning point number information and scanning step length information, includes:

[0015] Obtaining, based on the size and position coordinate information of the selected bounding rectangle of the region of interest, the X and Y coordinate information of the starting point of the bounding rectangle of the region of interest, as well as the width information along the X direction and the width information along the Y direction;

[0016] Based on the X and Y coordinate information of the starting point, the width information along the X direction, the width information along the Y direction, and the preset number of scanning points and scanning step information in the X direction, a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest are obtained.

[0017] Optionally, based on the X and Y coordinate information of the starting point, the width information along the X direction, the width information along the Y direction, and the preset number of scanning points and scanning step length information in the X direction, a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest are generated, including:

[0018] Divide the width information along the X direction by the preset number of scanning points in the X direction and round down to obtain the point spacing;

[0019] Divide the width information along the Y direction by the point spacing and round up to obtain the scanning point number information in the Y direction;

[0020] Based on the X and Y coordinate information of the starting point and the preset number of scanning points in the X direction and the number of scanning points in the Y direction, a multi-focus two-dimensional scanning dot matrix is ​​obtained;

[0021] Based on the scanning step information, the X and Y coordinate information of the starting point are adjusted to obtain a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest.

[0022] Optionally, based on the structural features of the target sample, a preset curved surface template is selected, an axial coordinate is set for each scanning point in the multi-focus two-dimensional scanning dot matrix, and a series of three-dimensional scanning dot matrices matching the selected curved surface template are generated, including:

[0023] Based on the structural characteristics of the target sample, a preset curved surface template is selected, and according to the three-dimensional coordinate relationship of each point on the curved surface template, an axial coordinate is set for each scanning point in the multi-focus two-dimensional scanning lattice to generate a three-dimensional scanning lattice that matches the curved surface template;

[0024] Repeat the steps of generating a three-dimensional scanning dot matrix that matches the curved surface template until all scanning points in the multi-focus two-dimensional scanning dot matrix are set with axial coordinates, thereby generating a series of three-dimensional scanning dot matrices that match the curved surface template.

[0025] Optionally, based on the mapping relationship between the preset transverse coordinate and the acoustic wave frequency of the acousto-optic deflector, and the mapping relationship between the preset axial coordinate and the acoustic wave frequency of the adjustable acoustic gradient lens, the target sample is scanned according to the three-dimensional scanning dot matrix, and the fluorescence signal emitted by the target sample is synchronously collected to obtain a series of two-dimensional dot matrix fluorescence images, including:

[0026] Based on the mapping relationship between the preset lateral coordinates and the acoustic wave frequency of the acousto-optic deflector, the lateral position at which the fluorescence signal converges in the sample is adjusted. At the same time, based on the mapping relationship between the preset axial coordinates and the acoustic wave frequency of the adjustable acoustic gradient lens, the axial position at which the fluorescence signal converges in the sample is adjusted. The target sample is scanned according to the three-dimensional scanning dot matrix, and the fluorescence signal emitted by the target sample is synchronously collected to obtain a series of two-dimensional dot matrix fluorescence images that correspond one to one with the three-dimensional scanning dot matrix.

[0027] Optionally, reconstructing the two-dimensional dot matrix fluorescence image using a preset reconstruction algorithm to obtain a three-dimensional super-resolution image matching the selected curved surface template in each region of interest includes:

[0028] Reconstructing all two-dimensional dot matrix fluorescence images of the same region of interest on the target sample using a two-dimensional super-resolution reconstruction algorithm to obtain a two-dimensional super-resolution image;

[0029] A corresponding axial coordinate is set for each scanning point in the two-dimensional super-resolution image to obtain a three-dimensional super-resolution image that matches the selected curved surface template in each region of interest.

[0030] A second aspect of the present invention provides a three-dimensional addressing scanning super-resolution microscopic imaging system for arbitrary curved surfaces, the system comprising:

[0031] a region selection module, configured to acquire a wide-field fluorescence image of a target sample and select at least one region of interest based on the wide-field fluorescence image;

[0032] A two-dimensional scanning dot matrix generation module is used to generate a series of multi-focus two-dimensional scanning dot matrices corresponding to the region of interest based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as preset scanning point number information and scanning step length information;

[0033] A three-dimensional scanning dot matrix generation module is used to select a preset curved surface template based on the structural characteristics of the target sample, set an axial coordinate for each scanning point in the multi-focus two-dimensional scanning dot matrix, and generate a series of three-dimensional scanning dot matrices that match the selected curved surface template;

[0034] a two-dimensional dot matrix fluorescence image acquisition module, configured to scan the target sample according to the three-dimensional scanning dot matrix based on a mapping relationship between a preset transverse coordinate and the acoustic wave frequency of the acousto-optic deflector, and a mapping relationship between a preset axial coordinate and the acoustic wave frequency of the adjustable acoustic gradient lens, and synchronously collect fluorescence signals emitted by the target sample to obtain a series of two-dimensional dot matrix fluorescence images;

[0035] The three-dimensional super-resolution image generation module is used to reconstruct the two-dimensional dot matrix fluorescence image using a preset reconstruction algorithm to obtain a three-dimensional super-resolution image that matches the selected curved surface template in each region of interest.

[0036] A third aspect of the present invention provides an intelligent terminal, which includes a memory, a processor, and an arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program stored in the memory and runnable on the processor. When the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program is executed by the processor, any one of the steps of the above-mentioned arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging method is implemented.

[0037] A fourth aspect of the present invention provides a computer-readable storage medium, on which is stored a program for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface. When the program for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface is executed by a processor, the program implements any one of the steps of the above-mentioned method for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface.

[0038] Compared with the existing technology, the beneficial effects of this solution are as follows:

[0039] The present invention selects at least one region of interest of a wide-field fluorescence image of a target sample, and generates a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, preset scanning point number information, and scanning step length information; selects a preset curved surface template based on the structural characteristics of the target sample, and generates a series of three-dimensional scanning dot arrays matching the selected curved surface template; based on the mapping relationship between the preset lateral coordinates and the acoustic wave frequency of the acousto-optic deflector, and the mapping relationship between the preset axial coordinates and the acoustic wave frequency of the adjustable acoustic gradient lens, the target sample is scanned according to the three-dimensional scanning dot array, and the fluorescence signal emitted by the target sample is synchronously collected to obtain a series of two-dimensional dot array fluorescence images; the two-dimensional dot array fluorescence image is super-resolved and reconstructed in three dimensions based on the axial coordinates of each scanning point to obtain a three-dimensional super-resolved image of the curved surface corresponding to the region of interest.

[0040] Since both the acousto-optic deflector and the adjustable acoustic gradient lens have the characteristics of fast response, and their response rates are on the same order of magnitude, the present invention combines the two to perform addressing scanning, which can realize fast dynamic three-dimensional addressing scanning of any curved surface of the sample without being restricted by the depth of field. This effectively solves the problem of mutual restriction between imaging depth and three-dimensional imaging speed in the existing technology for super-resolution dynamic imaging of samples, and can perform fast dynamic three-dimensional super-resolution imaging of any curved surface in any area of ​​interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, 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 technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 This is a flow chart of the method for three-dimensional addressing and scanning super-resolution microscopy of arbitrary curved surfaces of the present invention;

[0043] Figure 2 Schematic diagram of the synchronous control signals of the control program of the present invention for the tunable acoustic gradient lens (TAG Lens), sCMOS camera and two-dimensional acousto-optic deflector (2D-AOD);

[0044] Figure 3 (a) is a schematic diagram of a transverse scan of the acousto-optic deflector (AOD) of the present invention;

[0045] Figure 3 (b) is a schematic diagram of the axial scanning of the TAG lens of the present invention;

[0046] Figure 3 (c) is a schematic diagram of the three-dimensional addressing scanning of the acousto-optic deflector and the adjustable acoustic gradient lens (i.e., AOD and TAG Lens) of the present invention;

[0047] Figure 4 (a) is a schematic diagram showing a three-dimensional scanning dot matrix corresponding to a selected curved surface generated in a region of interest of a sample according to the present invention;

[0048] Figure 4 (b) Schematic diagram showing the generation of a 2D dot matrix fluorescence image corresponding to a 3D scanning dot matrix during a single exposure process;

[0049] Figure 4 (c) Schematic diagram of a series of two-dimensional dot matrix fluorescence images obtained by multiple exposures;

[0050] Figure 5 (a) Schematic diagram of a wide-field fluorescence image of the sample region of interest obtained by directly superimposing a series of two-dimensional dot-matrix fluorescence images;

[0051] Figure 5 (b) Schematic diagram of the MSIM super-resolution image obtained by reconstructing a series of two-dimensional dot matrix fluorescence images;

[0052] Figure 5 (c) is a schematic diagram of the 3D super-resolution image of the corresponding scanned surface obtained by further reconstruction;

[0053] Figure 6 This is a schematic structural diagram of the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging system of the present invention;

[0054] Figure 7 It is a schematic diagram of the structure of the intelligent terminal of the present invention. DETAILED DESCRIPTION

[0055] In the following description, specific details such as particular system structures and techniques are provided for purposes of illustration, not limitation, to facilitate a thorough understanding of the embodiments of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present invention with unnecessary detail.

[0056] It will be understood that when used in this specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0057] It should also be understood that the terms used in the present specification are only for the purpose of describing particular embodiments and are not intended to limit the present invention. As used in the present specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0058] It should be further understood that the term "and / or" used in the present description and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0059] The following is a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0060] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0061] To address the mutual constraints between imaging depth and 3D imaging speed in existing super-resolution imaging systems, the present invention proposes a method for 3D addressable scanning super-resolution microscopy of arbitrary curved surfaces. This method first utilizes a femtosecond laser and a two-dimensional acousto-optic deflector (2D-AOD) for rapid multi-focus scanning imaging and generates a wide-field image of the sample. Based on this, one or more regions of interest are selected. By combining and controlling the acoustic wave frequencies of a tunable acoustic gradient index lens (TAG lens) and an acousto-optic deflector (AOD), a 3D scanning array is generated to perform 3D rapid MSIM imaging of selected surfaces in the selected regions, thereby obtaining super-resolution images containing surface information. This method can address the mutual constraints between imaging depth and 3D imaging speed in super-resolution dynamic imaging of living biological samples, aiming to provide a technology for life science research that can perform rapid MSIM super-resolution imaging of arbitrary curved surfaces in any region of interest, enabling super-resolution imaging monitoring of specific intracellular structures.

[0062] Exemplary Methods

[0063] The embodiment of the present invention provides a method for super-resolution microscopy of arbitrary curved surfaces using three-dimensional addressing scanning. The method is deployed on electronic devices such as computers and servers, and is applied in a super-resolution imaging device. The device mainly includes a laser, an acousto-optic deflector, an adjustable acoustic gradient lens, a microscope objective lens, etc. arranged in sequence according to the direction of the optical path. This embodiment proposes a method for super-resolution microscopy (3D-AS-MSIM) of arbitrary curved surfaces using three-dimensional addressing scanning. Specifically, as shown in FIG. Figure 1 As shown, it mainly includes the following steps:

[0064] Step S100: Acquire a wide-field fluorescence image of a target sample, and select at least one region of interest based on the wide-field fluorescence image.

[0065] Specifically, obtaining a wide-field fluorescence image of a target sample includes: adjusting the microscope objective to the focal plane, controlling the acousto-optic deflector to scan the target sample according to each preset full-field scanning multi-focus two-dimensional scanning lattice coordinate based on the preset full-field scanning multi-focus two-dimensional scanning lattice coordinates and the mapping relationship between the lateral coordinates and the acoustic wave frequency of the acousto-optic deflector, and obtaining a series of two-dimensional lattice fluorescence images that correspond one-to-one to the preset full-field scanning multi-focus two-dimensional scanning lattice coordinates; and superimposing the two-dimensional lattice fluorescence images to obtain a wide-field fluorescence image of the target sample.

[0066] In one embodiment, the adjustable acoustic gradient lens is not turned on and the microscope objective is adjusted to the focal plane of the sample. A control program is written in LabView to automatically superimpose a series of two-dimensional dot matrix fluorescence images of the obtained two-dimensional scanning dot matrix to obtain a large-field wide-field fluorescence image of the target sample. One or more arbitrary regions of interest can be manually selected on the wide-field fluorescence image displayed on the control program interface. The region shape can be selected as a default rectangle or an arbitrary shape. In order to simplify the difficulty of subsequent super-resolution reconstruction of the region of interest and improve processing efficiency, this embodiment automatically adds a bounding rectangle to the selected region of interest of rectangular shape or other arbitrary shape during actual operation, and scans the bounding rectangle area containing the region of interest. The control program written in LabView is used to automatically save the pixel coordinate information of the selected region of interest and its bounding rectangle.

[0067] It should be noted that LabView is selected to write the control program in this embodiment. As other preferred implementations, other existing graphic software development platforms can also be selected to write the control program according to actual applications.

[0068] Step S200: Based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as the preset scanning point number information and scanning step length information, a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest are generated.

[0069] Specifically, based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, the X- and Y-direction coordinate information of the starting point of the circumscribed rectangle of the region of interest, as well as the width information Lx along the X direction and the width information Ly along the Y direction are obtained; the number of scanning points Nx and the scanning step length M in the X direction are set based on experience; and based on the X- and Y-direction coordinate information of the starting point, the width information Lx along the X direction and the width information Ly along the Y direction, as well as the preset number of scanning points Nx and the scanning step length M in the X direction, a series of multi-focus two-dimensional scanning dot arrays are generated.

[0070] Based on 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, as well as the preset number of scanning points Nx and scanning step length M in the X direction, a series of multi-focus two-dimensional scanning dot arrays are generated, including:

[0071] The width information Lx along the X direction is divided by the preset number of scanning points in the X direction Nx and rounded down to obtain the point spacing D. The width information Ly along the Y direction is divided by the point spacing D and rounded up to obtain the number of scanning points in the Y direction Ny. Based on the X and Y coordinate information of the starting point, the preset number of scanning points in the X direction Nx, and the obtained number of scanning points in the Y direction Ny, a multi-focus two-dimensional scanning dot matrix consisting of Nx×Ny scanning points is generated to obtain the coordinates of each point in the two-dimensional scanning dot matrix. Based on the preset scanning step information M, the X and Y coordinate information of the starting point is adjusted to generate the next multi-focus two-dimensional scanning dot matrix. Based on the preset scanning step information M, the X and Y coordinate information of the starting point is continuously adjusted until a series of multi-focus two-dimensional scanning dot matrices that meet the MSIM super-resolution imaging requirements are generated. It is easy to understand that the width information, scanning point number information, scanning step information, and point spacing in this embodiment are all positive integers. Furthermore, the X direction and Y direction referred to in this embodiment represent two mutually orthogonal lateral directions parallel to the focal plane, corresponding to the deflection directions of the laser beam by two orthogonal acousto-optic deflectors, respectively, and the opposite Z direction represents the axial direction perpendicular to the focal plane.

[0072] In one embodiment, a control program written in LabView automatically generates the multi-focus two-dimensional scanning dot matrix required for MSIM imaging by reading the X and Y coordinate information of the starting point of the circumscribed rectangular area of ​​the region of interest, the width information Lx and Ly of the rectangular area along the X direction, the preset number of scanning points Nx in the X direction, and the preset scanning step size M. This automatically generates a coordinate sequence of the multi-focus two-dimensional scanning dot matrix corresponding to the focal plane. It is easy to understand that the coordinate sequence of the multi-focus two-dimensional scanning dot matrix here includes the order in which the scanning points are subsequently scanned, and the scanning order can be flexibly set according to actual needs.

[0073] Step S300: Based on the structural characteristics of the target sample, a preset surface template is selected, an axial coordinate is set for each scanning point in the multi-focus two-dimensional scanning dot matrix, and a series of three-dimensional scanning dot matrices matching the selected surface template are generated.

[0074] Specifically, based on the structural characteristics of the target sample, a preset curved surface template is selected. Based on the three-dimensional coordinate relationship of each point on the curved surface template, an axial coordinate is assigned to each scanning point in a multi-focus two-dimensional scanning lattice, thereby generating a three-dimensional scanning lattice that matches the curved surface template. The step of generating a three-dimensional scanning lattice that matches the curved surface template is repeated until all scanning points in the multi-focus two-dimensional scanning lattice are assigned axial coordinates, thereby generating a series of three-dimensional scanning lattices that match the curved surface template. It is understandable that since each scanning point in the multi-focus two-dimensional scanning lattice is assigned an axial coordinate based on the preset curved surface template, the axial coordinate values ​​of different scanning points are likely to be different. Therefore, the scanning lattice generated that matches the curved surface template is distributed on a curved surface, forming a layer of three-dimensional scanning lattices. The series of three-dimensional scanning lattices generated that match the curved surface template are a series of relatively moving three-dimensional scanning lattices that meet the requirements of multi-focus structured illumination microscopy (MSIM). The set of all scanning points in all three-dimensional scanning lattices will completely cover a surface corresponding to the selected curved surface template in the selected region of interest.

[0075] In one embodiment, a control program written in LabView selects a preset arbitrary curved surface template (e.g., an arbitrary angled slope, a hemispherical surface, a wavy surface, etc.) based on the sample's structural features in a widefield image of a selected region of interest. The control program automatically assigns an axial coordinate to each scan point in each 2D scanning dot matrix sequence based on the 3D coordinate relationship between each point on the selected surface template, thereby generating a series of 3D scanning dot matrices corresponding to the sample's region of interest and the surface template. Similarly, if there is more than one region of interest, a preset surface template suitable for each region of interest is selected based on the sample's structural features in the widefield image, thereby generating a series of 3D scanning dot matrices corresponding to each region of interest and the corresponding surface template.

[0076] Step S400: Based on the mapping relationship between the preset lateral coordinate and the acoustic wave frequency of the acousto-optic deflector, and the mapping relationship between the preset axial coordinate and the acoustic wave frequency of the adjustable acoustic gradient lens, the target sample is scanned according to the three-dimensional scanning dot matrix, and the fluorescence signal emitted by the target sample is synchronously collected to obtain a series of two-dimensional dot matrix fluorescence images.

[0077] Specifically, based on the mapping relationship between the preset lateral coordinates and the acoustic wave frequency of the acousto-optic deflector, the lateral position of the laser convergence in the sample is adjusted. At the same time, based on the mapping relationship between the preset axial coordinates and the acoustic wave frequency of the adjustable acoustic gradient lens, the axial position of the laser convergence in the sample is adjusted. The target sample is scanned according to the three-dimensional scanning dot matrix, and the fluorescence signal emitted by the target sample is synchronously collected to obtain a series of two-dimensional dot matrix fluorescence images that correspond one-to-one to the three-dimensional scanning dot matrix.

[0078] By simultaneously manipulating the acoustic wave frequency combination applied to the acousto-optic deflector and the adjustable acoustic gradient lens, the lateral and axial positions of the laser focus within the sample are rapidly altered. Different scanning points within the same 3D scanning array may correspond to different axial positions. However, because the adjustable acoustic gradient lens exerts opposing modulation effects on the transmitted pulsed laser beam and the returning fluorescence signal, the fluorescence signals generated by the pulsed laser at different axial positions on the sample are consistently focused on the image detector's detection surface. Therefore, the acquired fluorescence signal only reflects planar information parallel to the focal plane, excluding axial information at the scanning point. Consequently, a 2D lattice fluorescence image is acquired. Different 3D scanning lattices are generated by scanning the same surface as required by MSIM. Each 3D scanning lattice corresponds to the selected surface but is shifted along the surface relative to the previous lattice. Consequently, a series of 2D lattice fluorescence images corresponding to the 3D scanning lattice are acquired.

[0079] In one embodiment, a control program calculates the acoustic frequency combination of the AOD and TAG lens corresponding to each scanning point based on a pre-calibrated linear correspondence between pixel coordinates and acoustic wave frequencies. The program then controls the data acquisition card to synchronously output a corresponding 32-bit digital signal for regulating the 2D-AOD and another digital signal for regulating the TAG lens, thereby enabling the laser spot to scan a 3D scanning array of dots within the sample corresponding to the selected curved surface. Simultaneously, the program controls the data acquisition card to output an analog signal to synchronize the exposure frequency of the sCMOS camera with the scanning of the 3D scanning array. This ensures that the camera completes one exposure after each 3D scan of the array, thereby capturing a series of 2D array fluorescence images.

[0080] In the simulation experiment, the control program controls the synchronous control signals of TAG lens, sCMOS camera and 2D-AOD as follows: Figure 2 As shown, the pulsed laser passes through the 2D-AOD and TAG lens, spot-exciting the target sample. Each pulse of the TAG lens and 2D-AOD synchronous control signal corresponds to a scanning point. Each pulse sequence, consisting of Nx*Ny (Nx and Ny are both positive integers), forms a pulse sequence. Each pulse sequence corresponds to a single exposure of the sCMOS, resulting in a 2D scanned lattice image corresponding to the preset surface. By generating a continuous pulse sequence and subjecting the sCMOS to multiple exposures (Exposure 1, Exposure 2, ...), a series of 2D scanned lattice images corresponding to the preset surface are obtained.

[0081] Step S500: reconstructing the two-dimensional dot matrix fluorescence image using a preset reconstruction algorithm to obtain a three-dimensional super-resolution image corresponding to a preset curved surface in each region of interest.

[0082] Specifically, a two-dimensional super-resolution reconstruction algorithm is used to reconstruct all two-dimensional dot matrix fluorescence images of the same region of interest of the target sample to obtain a two-dimensional super-resolution image, which corresponds to the selected surface in the region of interest of the target sample but does not contain axial information; according to step S200, corresponding axial (i.e., Z direction) coordinates are set for each scanning point in the two-dimensional scanning dot matrix to perform three-dimensional reconstruction of the two-dimensional super-resolution image to obtain a three-dimensional super-resolution image of the surface corresponding to each region of interest.

[0083] The following further describes the principle of the three-dimensional addressing scanning of the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging method of the present invention. The principle of using a control program to control the acousto-optic deflector (AOD) and the adjustable acoustic gradient (TAG) lens to achieve three-dimensional addressing scanning is as follows: Figure 3 As shown. Among them, Figure 3(a) Schematic diagram of the transverse scanning of the AOD. The pulsed laser passes through the AOD located on the conjugate plane of the microscope objective lens, along the optical path, and then illuminates the sample plane (Sample plane) to achieve addressable scanning of single-point excitation. Figure 3 (b) Schematic diagram of the axial scanning of the TAG lens. The axial depth of the scan can be changed by adjusting the frequency of the acoustic wave loaded on the TAG lens. Figure 3 (c) is a schematic diagram of the three-dimensional addressing scanning of the AOD and TAG lenses. Since the response rate of TAG is of the same order of magnitude as that of AOD, by matching the acoustic wave frequency of the TAG lens with the acoustic wave frequency of the AOD, it is possible to quickly switch from one scanning point to another scanning point whose lateral and axial coordinates may be different, thereby achieving fast three-dimensional addressing scanning.

[0084] In the simulation experiment, by changing the combination of acoustic wave frequencies loaded on the 2D-AOD and TAG lens, and combining it with the synchronous acquisition control of the image detector, it is possible to perform 3D dot scanning on the selected surface in the selected region of interest of the target sample according to the above-mentioned 3D addressing scanning principle according to the needs of MSIM super-resolution imaging, thereby obtaining a series of corresponding 2D dot matrix fluorescence images. The schematic diagram of the 2D dot matrix fluorescence image acquired by using the control program to control the AOD and TAG lens to achieve MSIM multi-focus 3D scanning dot matrix excitation and acquisition of arbitrary-shaped surfaces in the sample is shown in the figure. Figure 4 As shown. Among them, Figure 4 (a) Schematic diagram of a 3D scanning dot matrix corresponding to a selected curved surface generated in a sample region of interest using 3D addressing scanning with a 2D-AOD and TAG lens. To meet the requirements of MSIM super-resolution imaging, the 3D scanning dot matrix also needs to be scanned along the curved surface. Each 3D dot matrix scan corresponds to a single exposure of the image detector, resulting in a corresponding 2D dot matrix fluorescence image. Figure 4 (b) Schematic diagram of the generation of a 2D dot matrix fluorescence image corresponding to a 3D scanning dot matrix during a single exposure. Each scanning point in the 3D scanning dot matrix performs rapid 3D addressing scanning and point excitation of the target sample according to its coordinates and sequence. The fluorescence signal generated by each point excitation of the target sample is recorded one by one as a spot in the same frame of the image, forming a 2D dot matrix fluorescence image. Figure 4 (c) shows a schematic diagram of a series of two-dimensional dot matrix fluorescence images obtained by multiple exposures. Using a preset reconstruction algorithm, the obtained series of two-dimensional dot matrix fluorescence images are reconstructed to achieve three-dimensional addressing scanning super-resolution microscopic imaging of the selected surface in the selected region of interest of the target sample. Specifically, a schematic diagram of the reconstruction of a three-dimensional super-resolution image using the obtained two-dimensional dot matrix fluorescence image sequence is shown in FIG. Figure 5 As shown. Among them, Figure 5(a) shows a schematic diagram of a wide-field fluorescence image of the sample region of interest obtained directly from a series of two-dimensional dot matrix fluorescence images. Figure 5 (b) shows a schematic diagram of a two-dimensional super-resolution image obtained by reconstructing a series of two-dimensional dot matrix fluorescence images using the MSIM reconstruction algorithm. Figure 5 (c) shows a schematic diagram of a three-dimensional super-resolution image of the corresponding scanning surface obtained by further reconstructing the two-dimensional super-resolution image in three dimensions in combination with the axial coordinate information of the preset surface.

[0085] In summary, the method of this embodiment first controls the data acquisition card to output digital and analog signals without turning on the TAG lens. The digital signal controls the AOD to generate and rapidly scan a dot matrix corresponding to full-field illumination. The analog signal controls the sCMOS camera to synchronously capture fluorescence images corresponding to each scanned dot matrix. The widefield image formed by superimposing the acquired series of fluorescence images is then imported into the control program to select a single or multiple regions of interest on the image for MSIM super-resolution imaging. Then, with the TAG lens turned on, the control program determines the two-dimensional coordinate information of each point in each scanned dot matrix based on the coordinates of the first pixel point of the circumscribed rectangle of the selected region of interest, the width of the circumscribed rectangle, and the dot matrix parameter information. It also determines the axial coordinate information of each point based on the preset curved surface shape. Based on the calibrated coordinate-acoustic frequency conversion relationship, the data acquisition card is controlled to generate and output corresponding digital and analog signals. The digital signal synchronously regulates the acoustic wave frequency of the AOD and TAG lens to scan the selected curved surface of the selected region of interest according to the three-dimensional scanning dot matrix coordinate information. The analog signal controls the sCMOS camera to synchronously capture the two-dimensional dot matrix fluorescence image corresponding to each three-dimensional scanning dot matrix. Finally, a series of two-dimensional dot matrix fluorescence images captured by the sCMOS camera are reconstructed using the MSIM reconstruction algorithm to obtain a two-dimensional super-resolution image. According to the axial coordinate information of the preset surface, corresponding axial coordinate information is set for each scanning point in the two-dimensional super-resolution image for three-dimensional reconstruction, thereby obtaining a three-dimensional super-resolution image corresponding to the selected surface in the selected area of ​​interest in the sample.

[0086] Since both the acousto-optic deflector and the adjustable acoustic gradient lens have fast response characteristics and their response rates are on the same order of magnitude, this solution can achieve fast dynamic three-dimensional addressing and scanning imaging of the target surface by combining their acoustic wave frequencies without being restricted by the depth of field. This effectively solves the problem of mutual restriction between imaging depth and three-dimensional imaging speed in the existing technology for super-resolution dynamic imaging of samples, and can quickly perform three-dimensional super-resolution imaging of any surface in any area of ​​interest.

[0087] Exemplary Systems

[0088] like Figure 6As shown, corresponding to the above-mentioned arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging method, an embodiment of the present invention further provides an arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging system, which includes:

[0089] A region selection module 610 is configured to obtain a wide-field fluorescence image of a target sample and select at least one region of interest based on the wide-field fluorescence image;

[0090] A two-dimensional scanning dot matrix generation module 620 is configured to generate a series of multi-focus two-dimensional scanning dot matrices corresponding to the selected region of interest based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as the preset number of scanning points and scanning step length information;

[0091] The 3D scanning dot matrix generation module 630 is used to select a preset curved surface template based on the target sample structure characteristics, set an axial coordinate for each scanning point in the multi-focus 2D scanning dot matrix, and generate a series of 3D scanning dot matrices that match the selected curved surface template;

[0092] A two-dimensional dot matrix fluorescence image acquisition module 640 is configured to scan a target sample according to a three-dimensional scanning dot matrix based on a mapping relationship between a preset transverse coordinate and the acoustic wave frequency of the acousto-optic deflector, and a mapping relationship between a preset axial coordinate and the acoustic wave frequency of the adjustable acoustic gradient lens, and simultaneously collect fluorescence signals emitted by the target sample to obtain a series of two-dimensional dot matrix fluorescence images;

[0093] The three-dimensional super-resolution image generation module 650 is used to reconstruct the two-dimensional dot matrix fluorescence image using a preset reconstruction algorithm to obtain a three-dimensional super-resolution image that matches the selected curved surface template in each region of interest.

[0094] Specifically, in this embodiment, the specific functions of the above-mentioned arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging system can also refer to the corresponding description in the above-mentioned arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging method, and will not be repeated here.

[0095] Based on the above embodiment, the present invention also provides an intelligent terminal, whose principle block diagram can be shown as follows: Figure 7As shown. The above-mentioned intelligent terminal includes a processor, a memory, a network interface and a display screen connected through a system bus. Among them, the processor of the intelligent terminal is used to provide computing and control capabilities. The memory of the intelligent terminal includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system and an arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program. The internal memory provides an environment for the operation of the operating system in the non-volatile storage medium and the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program. The network interface of the intelligent terminal is used to communicate with an external terminal through a network connection. When the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program is executed by the processor, the steps of any one of the above-mentioned arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging methods are implemented. The display screen of the intelligent terminal can be a liquid crystal display or an electronic ink display.

[0096] Those skilled in the art will understand that Figure 7 The principle block diagram shown in the figure is only a block diagram of a partial structure related to the solution of the present invention and does not constitute a limitation on the smart terminal to which the solution of the present invention is applied. The specific smart terminal may include more or fewer components than shown in the figure, or combine certain components, or have a different component arrangement.

[0097] In one embodiment, a smart terminal is provided, which includes a memory, a processor, and an arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program stored in the memory and runnable on the processor. When the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program is executed by the processor, the steps of any one of the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging methods provided in the embodiments of the present invention are implemented.

[0098] An embodiment of the present invention also provides a computer-readable storage medium, on which a program for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface is stored. When the program for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface is executed by a processor, the steps of any one of the methods for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface provided in an embodiment of the present invention are implemented.

[0099] It should be understood that the sequence numbers of the steps in the above embodiments do not imply a specific order of execution; the order of execution of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0100] Those skilled in the art can clearly understand that, for the convenience and brevity of description, only the division of the above-mentioned functional units and modules is used as an example for illustration. In actual applications, the above-mentioned functions can be distributed and completed by different functional units and modules as needed, that is, the internal structure of the above-mentioned device can be divided into different functional units or modules to complete all or part of the functions described above. The functional units and modules in the embodiment can be integrated into one processing unit, or each unit can exist physically alone, or two or more units can be integrated into one unit. The above-mentioned integrated unit can be implemented in the form of hardware or in the form of software functional units. In addition, the specific names of the functional units and modules are only for the convenience of distinguishing each other, and are not used to limit the scope of protection of the present invention. The specific working process of the units and modules in the above-mentioned system can refer to the corresponding process in the aforementioned method embodiment, and will not be repeated here.

[0101] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0102] Those skilled in the art will appreciate that the units and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Professionals and technicians may use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of the present invention.

[0103] In the embodiments provided by the present invention, it should be understood that the disclosed apparatus / terminal device and method can be implemented in other ways. For example, the apparatus / terminal device embodiments described above are merely illustrative. For example, the division of the modules or units described above is merely a logical functional division. In actual implementation, other division methods may be used. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not implemented.

[0104] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it should be understood by those skilled in the art that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. A method for three-dimensional addressing and scanning super-resolution microscopy of arbitrary curved surfaces, applied to a super-resolution imaging device, the device mainly comprising a laser, an acousto-optic deflector, an adjustable acoustic gradient lens, and a microscope objective lens arranged in sequence along an optical path, characterized in that: The main steps include: Acquiring a wide-field fluorescence image of the target sample, and selecting at least one region of interest based on the wide-field fluorescence image; Based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as the preset scanning point number information and scanning step length information, a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest are generated; Based on the structural characteristics of the target sample, a preset curved surface template is selected, and according to the three-dimensional coordinate relationship of each point on the curved surface template, an axial coordinate is set for each scanning point in the multi-focus two-dimensional scanning lattice to generate a three-dimensional scanning lattice that matches the curved surface template; Repeating the step of generating a three-dimensional scanning dot matrix that matches the curved surface template until all scanning points in the multi-focus two-dimensional scanning dot matrix are set with axial coordinates, thereby generating a series of three-dimensional scanning dot matrices that match the curved surface template; Based on a mapping relationship between a preset lateral coordinate and the acoustic wave frequency of the acousto-optic deflector, the lateral position at which the fluorescence signal converges in the sample is adjusted. Simultaneously, based on a mapping relationship between a preset axial coordinate and the acoustic wave frequency of the adjustable acoustic gradient lens, the axial position at which the fluorescence signal converges in the sample is adjusted. The target sample is scanned according to the three-dimensional scanning dot matrix, and the fluorescence signal emitted by the target sample is synchronously collected to obtain a series of two-dimensional dot matrix fluorescence images corresponding one-to-one to the three-dimensional scanning dot matrix. Reconstructing all two-dimensional dot matrix fluorescence images of the same region of interest on the target sample using a two-dimensional super-resolution reconstruction algorithm to obtain a two-dimensional super-resolution image; A corresponding axial coordinate is set for each scanning point in the two-dimensional super-resolution image to obtain a three-dimensional super-resolution image that matches the selected curved surface template in each region of interest.

2. The method for three-dimensional addressing scanning super-resolution microscopy of arbitrary curved surfaces according to claim 1, characterized in that: The step of obtaining a wide-field fluorescence image of a target sample comprises: Adjusting the microscope objective lens to a focal plane, and controlling the acousto-optic deflector to scan the target sample according to each of the preset full-field scanning multi-focus two-dimensional scanning lattice coordinates and a mapping relationship between the preset lateral coordinates and the acoustic wave frequency of the acousto-optic deflector, thereby obtaining a series of two-dimensional lattice fluorescence images; The two-dimensional dot matrix fluorescence images are superimposed to obtain a wide-field fluorescence image of the target sample.

3. The method for three-dimensional addressing scanning super-resolution microscopy of arbitrary curved surfaces according to claim 1, characterized in that: The step of generating a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as the preset scanning point number information and scanning step length information, includes: Obtaining, based on the size and position coordinate information of the selected bounding rectangle of the region of interest, the X and Y coordinate information of the starting point of the bounding rectangle of the region of interest, as well as the width information along the X direction and the width information along the Y direction; Based on the X and Y coordinate information of the starting point, the width information along the X direction, the width information along the Y direction, and the preset number of scanning points and scanning step information in the X direction, a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest are obtained.

4. The method for three-dimensional addressing scanning super-resolution microscopy of arbitrary curved surfaces according to claim 3, characterized in that: Based on the X and Y coordinate information of the starting point, the width information along the X direction, the width information along the Y direction, and the preset number of scanning points and scanning step information in the X direction, a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest are generated, including: Divide the width information along the X direction by the preset number of scanning points in the X direction and round down to obtain the point spacing; Divide the width information along the Y direction by the point spacing and round up to obtain the scanning point number information in the Y direction; Based on the X and Y coordinate information of the starting point and the preset number of scanning points in the X direction and the number of scanning points in the Y direction, a multi-focus two-dimensional scanning dot matrix is ​​obtained; Based on the scanning step information, the X and Y coordinate information of the starting point are adjusted to obtain a series of multi-focus two-dimensional scanning dot arrays corresponding to the region of interest.

5. A three-dimensional addressing scanning super-resolution microscopy system for arbitrary curved surfaces, characterized in that: The system is used to implement the steps of the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging method according to any one of claims 1 to 4, and the system comprises: a region selection module, configured to acquire a wide-field fluorescence image of a target sample and select at least one region of interest based on the wide-field fluorescence image; A two-dimensional scanning dot matrix generation module is used to generate a series of multi-focus two-dimensional scanning dot matrices corresponding to the region of interest based on the size and position coordinate information of the circumscribed rectangle of the selected region of interest, as well as preset scanning point number information and scanning step length information; A three-dimensional scanning dot matrix generation module is used to select a preset curved surface template based on the structural characteristics of the target sample, set an axial coordinate for each scanning point in the multi-focus two-dimensional scanning dot matrix, and generate a series of three-dimensional scanning dot matrices that match the selected curved surface template; a two-dimensional dot matrix fluorescence image acquisition module, configured to scan the target sample according to the three-dimensional scanning dot matrix based on a mapping relationship between a preset transverse coordinate and the acoustic wave frequency of the acousto-optic deflector, and a mapping relationship between a preset axial coordinate and the acoustic wave frequency of the adjustable acoustic gradient lens, and synchronously collect fluorescence signals emitted by the target sample to obtain a series of two-dimensional dot matrix fluorescence images; The three-dimensional super-resolution image generation module is used to reconstruct the two-dimensional dot matrix fluorescence image using a preset reconstruction algorithm to obtain a three-dimensional super-resolution image that matches the selected curved surface template in each region of interest.

6. An intelligent terminal, characterized in that: The intelligent terminal includes a memory, a processor, and an arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program stored in the memory and runnable on the processor. When the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging program is executed by the processor, the steps of the arbitrary curved surface three-dimensional addressing scanning super-resolution microscopy imaging method as described in any one of claims 1-4 are implemented.

7. A computer-readable storage medium, characterized in that The computer-readable storage medium stores a program for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface. When the program is executed by a processor, the steps of the method for three-dimensional addressing and scanning super-resolution microscopy imaging of an arbitrary curved surface are implemented as described in any one of claims 1 to 4.

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