A multi-layer three-dimensional single molecule localization super-resolution imaging method and system based on a zoom lens

Abstract

This invention discloses a multi-layer three-dimensional single-molecule localization super-resolution imaging method and system based on a zoom lens, including step one: pre-experimental preparation, completing the calibration of the electrically adjustable lens zoom parameters and obtaining the astigmatism calibration curve; step two: fixing the sample, focusing, and activating the anti-drift function and synchronous control program; step three: acquiring multi-layer three-dimensional single-molecule scintillation images using a frame-by-frame scanning mode; and step four: performing three-dimensional localization and stitching reconstruction on all acquired single-molecule fluorescence scintillation images. This invention overcomes the problems of slow scanning speed and mechanical inertia associated with traditional axial scanning relying on the mechanical movement of the microscope objective or stage. Furthermore, due to the rapid zooming and accurate resetting capabilities of ETL, a novel frame-by-frame scanning method can be used, solving the problem in traditional layer-by-layer scanning modes where the focal plane of later scans is irradiated by laser for a longer time than the focal plane of earlier scans, thus minimizing the impact of photobleaching on the imaging quality of different layers.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy imaging technology, specifically to a multi-layer three-dimensional single-molecule localization super-resolution imaging method and system based on a zoom lens. Background Technology

[0002] Optical microscopy, with its advantages of high sensitivity, high spatiotemporal resolution, and non-invasiveness, has become a core tool in biomedical research. However, traditional optical microscopy is limited by diffraction effects, with a resolution of only 250-300 nm, which cannot meet the needs of observing ultra-fine structures in biological samples. The emergence of super-resolution fluorescence microscopy has broken through this limitation. Among them, single molecule localization microscopy (SMLM) is widely used in the biomedical field due to its high resolution (20-50 nm) and good system compatibility (it can be modified based on a traditional inverted fluorescence microscope).

[0003] The core principle of SMLM (Super-Resolution Image Processing) is to collect and precisely locate dispersed single-molecule images by randomly and sparsely emitting fluorescent probes within the same diffraction limit range. This information is then integrated with the localization information from tens of thousands of original images to reconstruct a super-resolution image of the sample. Since fluorescent molecules can be considered as point sources, their point spread function (PSF) formed by the imaging system presents an approximately Gaussian distribution in two-dimensional imaging. The localization process involves determining the coordinates of the Gaussian peak. However, biological samples are mostly three-dimensional structures, and the axial changes of the three-dimensional PSF are slow and symmetrical, making axial localization significantly more difficult than lateral localization. Axial encoding is required to achieve this.

[0004] Astigmatism is currently the mainstream three-dimensional localization method. By introducing a cylindrical lens in the detection optical path, the magnification in the x and y directions differs. Fluorescent molecules at different z-axis positions present elliptical PSFs with different orientations and ellipticity on the two-dimensional image. The axial encoding is completed using the major and minor axis widths of the PSFs, thereby obtaining the three-dimensional coordinates of the fluorescent molecules (Reference 1: Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy, Science, 2008, 319 (5864): 810-813). This method is simple to operate, allows for high single-molecule density, and has a relatively fast imaging speed. However, it can only achieve accurate three-dimensional localization in the range of 600-800 nm near the focal plane. Imaging of thick samples requires the combination of axial multi-layer scanning and stitching reconstruction.

[0005] Traditional multilayer 3D STORM imaging employs a "layer-by-layer scanning" mode: after acquiring a sufficient number of sparse single-molecule images on a single focal plane, the microscope objective or stage is mechanically moved to switch to the next focal plane (Reference 2: Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution, Nature methods, 2008, 5 (12): 1047-1052). This mode has two major problems: first, the photobleaching effect is uneven, and the fluorescent molecules on the later acquired focal plane are exposed to light for a longer time, which significantly reduces the positioning accuracy and imaging quality, resulting in distortion of the reconstructed image structure of thick samples; second, the mechanical inertia effect, the mechanical movement of the objective or stage can easily lead to interlayer stitching deviations, further reducing the imaging quality.

[0006] To address the issue of uneven photobleaching, Lin Danying et al. proposed the probe-refreshSTORM (prSTORM) technology in 2018 (Reference 3: Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM, Biophysical Journal, 2018, 114 (8): 1980-1987). This technology uses single-stranded DNA coupled with antibody-labeled samples and utilizes breakable DNA links to refresh fluorescent probes, eliminating differences in photobleaching between different layers. However, this technology relies on specific probes, limiting its application scope, and still uses mechanical methods to achieve axial scanning, failing to solve the reconstruction problem caused by mechanical inertia.

[0007] In summary, existing 3D SMLM technology still faces challenges in imaging thick samples, including uneven photobleaching, mechanical inertia affecting interlayer stitching, and axial drift reducing positioning accuracy.

[0008] Therefore, we propose a multi-layer three-dimensional single-molecule localization super-resolution imaging method and system based on zoom lenses. Summary of the Invention

[0009] The purpose of this invention is to provide a multi-layer three-dimensional single-molecule localization super-resolution imaging method and system based on a zoom lens, which solves the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a multi-layer three-dimensional single-molecule localization super-resolution imaging method based on a zoom lens, comprising the following method steps:

[0011] Step 1: Pre-experiment preparation, including calibration of the electrically adjustable lens zoom parameters and acquisition of astigmatism calibration curves. For zoom parameter calibration, the zoom range must be determined first, and the correspondence between simulated values, current values, and the focal length of the electrically adjustable lens must be established. Specifically, this is achieved by correlating voltage with zoom step size and repeatedly measuring and averaging the values ​​to improve calibration accuracy. The astigmatism calibration curve is used to establish the correspondence between the major and minor axis widths of the diffusion function of fluorescent molecules and their axial position. Specifically, this is achieved by acquiring data on the full width at half maximum (FWHM) of the diffusion function of fluorescent beads in the x and y directions as a function of the z-axis, and then performing polynomial fitting on the data to generate the calibration curve.

[0012] Step 2: Place the sample to be imaged on the stage of the inverted microscope and fix it in place. Adjust the optical paths of the excitation module, the inverted microscope, and the anti-drift module. During the adjustment process, first activate the laser of the corresponding wavelength in the excitation module (e.g., activate the 640nm laser for the AlexaFluo647 fluorescent probe) to focus and find a suitable imaging area. Then activate the laser used for detection in the anti-drift module (i.e., the 785nm laser), adjust the optical path, and ensure that the anti-drift module can detect axial drift normally, while ensuring that the excitation light can effectively irradiate the sample and that the sample fluorescence signal can be effectively collected. Then, start the LabVIEW synchronous control program running on the computer terminal to complete the parameter settings and preparation work before imaging.

[0013] Step 3: Using the LabVIEW control program running on the computer terminal, set the number of scanning layers of the electrically adjustable lens, the axial zoom step size, the acquisition frequency of the electron multiplier charge-coupled device camera, the exposure time, and the total number of acquisition frames; first adjust the laser power to make the fluorescent molecules in the sample reach a sparse scintillation state, then start the frame-by-frame scanning mode, control the electrically adjustable lens to switch the focal plane in a step zoom manner according to the set parameters, and simultaneously control the electron multiplier charge-coupled device camera to perform multi-plane cyclic exposure. After tens of thousands of exposures, save the acquired single-molecule fluorescence scintillation images as stacked data.

[0014] Step 4: Process all the collected single-molecule fluorescence scintillation image stack data; first, perform asymmetric two-dimensional Gaussian fitting on the spread function of the elliptical points in each frame image to obtain the center coordinates and the width of the major and minor axes; then, based on the astigmatism calibration curve obtained in Step 1, convert the width of the major and minor axes into the corresponding z-axis coordinates to achieve single-molecule three-dimensional localization; finally, integrate the three-dimensional spatial coordinates (x, y, z) and brightness information of all single-molecule localization points to complete multi-layer image stitching and reconstruction, and obtain a three-dimensional super-resolution image of the thick sample.

[0015] In a preferred embodiment of the present invention, the specific process of calibrating the zoom parameters of the electrically adjustable lens in step one is as follows: using a thin object as the calibration sample, firstly, the microscope objective of the inverted microscope is adjusted to find the focal plane of the sample; then, the microscope objective is moved by a preset scanning step length by the axial nano-displacement stage installed on the objective base, so that the sample is in a defocused state; then, the control current of the electrically adjustable lens is adjusted so that the imaging plane returns to the original focal plane, and the analog value output by the data acquisition card and the actual current value of the electrically adjustable lens are recorded at this time; the above defocusing, focusing and recording steps are repeated until the preset zoom range is covered, thus completing the calibration of the zoom parameters of the electrically adjustable lens.

[0016] In a preferred embodiment of the present invention, the specific process of obtaining the astigmatism calibration curve in step one is as follows: using fluorescent beads as calibration samples, the fluorescent beads are controlled to move gradually from a preset depth below the focal plane to a preset depth above the focal plane in the axial direction using an axial nano-displacement stage, with a movement step size of 10 nm; at each axial position of the fluorescent beads, the electron multiplication charge-coupled device camera is controlled to acquire a preset number of point spread function images; asymmetric two-dimensional Gaussian fitting is performed on each acquired point spread function image to calculate the full width at half maximum (FWHM) in the x and y directions; the full WHM corresponding to all axial positions is polynomially fitted with the z-axis coordinate of that position to generate the astigmatism calibration curve.

[0017] In a preferred embodiment of the present invention, the specific process of the frame-by-frame scanning mode in step three is as follows: Assume the number of focal planes to be scanned is M, and the number of image frames to be acquired for each focal plane is N; during the first exposure, the electrically adjustable lens first switches to the first focal plane position, and the electron multiplier charge-coupled device (ECCDP) camera simultaneously acquires one frame of image. Subsequently, the electrically adjustable lens sequentially switches to the second to the Mth focal plane positions, and the ECCDP camera acquires one frame of image for each focal plane, completing the first round of M frame image acquisition; the electrically adjustable lens returns to the first focal plane position, and the second exposure begins, repeating the above focal plane switching and image acquisition process; after N exposures, NM frames of images are acquired, and the illumination time for all focal planes is consistent.

[0018] In a preferred embodiment of the present invention, during the data processing in step four, when performing asymmetric two-dimensional Gaussian fitting on the elliptical point spread function, it is necessary to obtain the center coordinates of the point spread function and the full width at half maximum (FWHM) in the x direction and the full WHM in the y direction; when converting the axial coordinates based on the astigmatic calibration curve, it is necessary to determine the axial position of each fluorescent molecule by querying the correspondence between the full WHM and the z-axis coordinates in the calibration curve; when stitching and reconstructing multilayer images, it is necessary to integrate the three-dimensional positioning results of each focal plane according to the actual position of the focal plane to eliminate interlayer position deviations.

[0019] In a preferred embodiment of the present invention, the specific process of adjusting the optical path of the anti-drift module in step two is as follows: turn on the 785nm laser in the anti-drift module, after the laser is reflected by the mirror and expanded and collimated by the beam expander lens group, it enters the inverted microscope in sequence through the beam splitter and the tube mirror; adjust the laser optical path so that the laser is focused on the back focal plane of the microscope objective after being reflected by the dichroic mirror of the inverted microscope, and the laser undergoes total internal reflection at the interface between the sample and the coverslip after exiting the microscope objective; adjust the angle of the beam splitter so that the laser reflected back by the total internal reflection can be accurately incident on the four-quadrant detector after being transmitted through the beam splitter, ensuring that the four-quadrant detector can detect a stable spot signal.

[0020] This invention also relates to a multi-layer three-dimensional single-molecule localization super-resolution imaging system based on a zoom lens, comprising an excitation module, an inverted microscope, a zoom and three-dimensional astigmatism detection module, an anti-drift module, and a computer terminal; the computer terminal runs a LabVIEW control program for synchronously controlling the working status and data transmission of each module; the excitation module is used to provide multi-wavelength excitation light, the inverted microscope is used to transmit excitation light and collect sample fluorescence signals, the zoom and three-dimensional astigmatism detection module is used to realize non-mechanical zoom and single-molecule fluorescence image acquisition, and the anti-drift module is used to detect and correct axial drift in real time.

[0021] In a preferred embodiment of the present invention, the excitation module includes a 640nm laser, a 488nm laser, a 405nm laser, two dichroic mirrors, a filter wheel, a beam expander lens group, a tube mirror, and several reflectors. The lasers emitted by the three lasers are reflected by the reflectors and combined by the dichroic mirrors. The laser power is adjusted by the filter wheel, then expanded and collimated by the beam expander lens group, and the height and angle are adjusted by the reflectors. Finally, the lasers enter the inverted microscope through the tube mirror. The 640nm laser is used to excite fluorescent probes such as AlexaFluo647 and Cy5, the 488nm laser is used to excite fluorescent probes such as AlexaFluo488 and GFP, and the 405nm laser is used to activate the fluorescent probes to achieve a scintillation effect.

[0022] In a preferred embodiment of the present invention, the zoom and three-dimensional astigmatism detection module includes a relay lens group, an electrically adjustable lens, a data acquisition card, a cylindrical lens, and an electron multiplication charge-coupled device (ECCDP) camera. The cylindrical lens is disposed between the relay lens group and the ECCDP camera to enable fluorescent molecules at different z-axis positions to form elliptical point diffusion functions with different ellipticity and orientation on the detection surface of the ECCDP camera. The analog output port of the data acquisition card is connected to the analog input port of the electrically adjustable lens and the trigger port of the ECCDP camera, respectively, to output analog signals to control the zoom of the electrically adjustable lens and trigger the synchronous exposure of the ECCDP camera. The computer terminal is communicatively connected to the data acquisition card, the electrically adjustable lens drive controller, and the ECCDP camera, respectively, to transmit control commands and receive image data.

[0023] In a preferred embodiment of the present invention, the anti-drift module includes a 785nm laser, a beam expander lens group, a beam splitter, a four-quadrant detector, a tube mirror, and several reflecting mirrors; the four-quadrant detector is communicatively connected to a computer terminal to transmit the detected spot position signal to the computer terminal; an axial nano-displacement stage is installed on the objective lens base of the inverted microscope, and the axial nano-displacement stage is electrically connected to the computer terminal. The computer terminal calculates the axial drift based on the change in the spot position of the four-quadrant detector and outputs a control signal to drive the axial nano-displacement stage to move in the opposite direction to counteract the drift.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention achieves non-mechanical axial multi-layer scanning by introducing an electrically adjustable lens (ETL) into a traditional astigmatic 3D SMLM imaging system. This not only overcomes the problems of slow scanning speed and mechanical inertia associated with traditional axial scanning that relies on the mechanical movement of microscope objectives or stages, but also allows for a novel frame-by-frame scanning method due to the ETL's rapid zooming and accurate resetting capabilities. This method rapidly traverses each focal plane during each exposure cycle, thus solving the problem that later-scanned focal planes are illuminated by laser for longer periods than earlier-scanned focal planes in traditional layer-by-layer scanning modes. This minimizes the impact of photobleaching on the imaging quality of different layers. Furthermore, the combination of the ETL zoom module and the anti-drift module overcomes the axial drift problem caused by long-term scanning imaging. Attached Figure Description

[0026] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0027] Figure 1 This is the system optical path diagram of the present invention;

[0028] Figure 2This is a schematic diagram of the synchronous scanning mode of the present invention;

[0029] Figure 3 This is a schematic diagram of the frame-by-frame scanning synchronization control signal of the present invention;

[0030] Figure 4 This is a schematic diagram of the data reconstruction method of the present invention;

[0031] Figure 5 This is a flowchart of the method of the present invention.

[0032] Figure 1 The English tags in Chinese are defined as follows:

[0033] 640, 488, 405, 785: Lasers of different wavelengths; M: Mirror; DM: Dichroic mirror; FW: Filter wheel; L: Lens; TL: Tube lens; QD: Quadrant detector; BS: Beam splitter; OL: Objective lens; SS: Stage; PZT: Axial nano-displacement stage; F: Filter; ETL: Motorized adjustable lens; CL: Cylindrical lens; EMCCD: Electron multiplier charge-coupled device; DAQ: Data acquisition card; PC: Computer. Detailed Implementation

[0034] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0035] like Figure 1-5 As shown, a multi-layer three-dimensional single-molecule localization super-resolution imaging system based on a zoom lens includes an excitation module, an inverted microscope, a zoom and three-dimensional astigmatism detection module, an anti-drift module, and a computer terminal. The computer terminal runs a LabVIEW control program to synchronously control the working status and data transmission of each module. The excitation module provides multi-wavelength excitation light, the inverted microscope transmits the excitation light and collects sample fluorescence signals, the zoom and three-dimensional astigmatism detection module realizes non-mechanical zoom and single-molecule fluorescence image acquisition, and the anti-drift module detects and corrects axial drift in real time.

[0036] The excitation module includes a 640nm laser, a 488nm laser, a 405nm laser, two dichroic mirrors, a filter wheel, a beam expander lens group, a tube mirror, and several reflectors. The lasers emitted by the three lasers are reflected by the reflectors and combined by the dichroic mirrors. The laser power is adjusted by the filter wheel, then expanded and collimated by the beam expander lens group, and the height and angle are adjusted by the reflectors. Finally, the lasers enter the inverted microscope through the tube mirror. The 640nm laser is used to excite fluorescent probes such as AlexaFluo647 and Cy5, the 488nm laser is used to excite fluorescent probes such as AlexaFluo488 and GFP, and the 405nm laser is used to activate the fluorescent probes to achieve a scintillation effect.

[0037] The zoom and 3D astigmatism detection module includes a relay lens group, an electrically adjustable lens, a data acquisition card, a cylindrical lens, and an electron multiplier charge-coupled device (ECCDP) camera. The cylindrical lens is positioned between the relay lens group and the ECCDP camera to enable fluorescent molecules at different z-axis positions to form elliptic point diffusion functions with varying ellipticity and orientation on the ECCDP camera's detection surface. The analog output port of the data acquisition card is connected to the analog input port of the electrically adjustable lens and the trigger port of the ECCDP camera, respectively, to output analog signals to control the zoom of the electrically adjustable lens and trigger synchronous exposure of the ECCDP camera. The computer terminal is communicatively connected to the data acquisition card, the electrically adjustable lens drive controller, and the ECCDP camera, respectively, for transmitting control commands and receiving image data.

[0038] The anti-drift module includes a 785nm laser, a beam expander lens group, a beam splitter, a four-quadrant detector, a tube mirror, and several reflecting mirrors. The four-quadrant detector is connected to a computer terminal to transmit the detected spot position signal to the computer terminal. An axial nano-displacement stage is installed on the objective lens base of the inverted microscope. The axial nano-displacement stage is electrically connected to the computer terminal. The computer terminal calculates the axial drift based on the change in the spot position of the four-quadrant detector and outputs a control signal to drive the axial nano-displacement stage to move in the opposite direction to counteract the drift.

[0039] The specific implementation method is as follows:

[0040] The first step is the preparation work before the actual experiment, including the calibration of ETL zoom parameters and the acquisition of astigmatism calibration curves.

[0041] Calibrate the ETL zoom parameters, which involves determining the correspondence between the input analog voltage value and the actual scanning step size. Assuming the desired zoom range is -5μm to 5μm and the desired zoom step size is 500nm, a 0-5V stepped analog voltage can be set in 0.25V steps. This means the 0.25V analog voltage matches the current value at an actual zoom distance of 500nm. Place a thin sample (e.g., a 100nm diameter TetraSpeck™ microsphere sample) on the sample stage. First, adjust the microscope's focus knob to find the sample's focal plane. Then, use the axial nanostage to move the objective lens downwards by 5μm. In the ETL control program, select the current control mode and change the ETL control current value to refocus the sample. Record the current value at this point. Next, control the nanostage to move upwards in 500nm steps and repeat the above steps until calibration is complete. This process can be repeated multiple times to calculate the average value for more accurate ETL axial zoom parameter calibration.

[0042] Astigmatism calibration curves are obtained, which calibrate the correspondence between the widths wx and wy of the elliptical PSF in two directions and the z-axis position of the fluorescent molecule, for subsequent axial localization. Generally, fluorescent beads with a diameter of 100 nm are still used as samples. Specifically, a nanostage is controlled to move gradually from a certain depth below the focal plane to a certain depth above the focal plane in 10 nm steps, acquiring PSF images of the fluorescent beads at different axial positions. Then, through asymmetric two-dimensional Gaussian fitting, the full width at half maximum (FWHM) of these elliptical PSFs in the x and y directions are obtained, denoted as wx and wy, respectively. x and w y Then, a polynomial fit is performed between this curve and the known axial position z to obtain the astigmatic calibration curve. Sufficient single-molecule PSF samples need to be collected at each axial position to reduce error.

[0043] After completing the above preparations, multi-slice 3D SMLM imaging can be performed on the actual sample. The following uses a BSC cell microtubule standard sample labeled with the AlexaFluo647 probe (located in a glass-bottomed culture dish) as an example to illustrate the specific experimental steps:

[0044] Step 1: Imaging Preparation and Wide-Field Image Recording. Place the sample on the stage and fix it in place. Add imaging buffer to the culture dish. Turn on the 640nm laser and emit the laser. Adjust the filter wheel and illuminate the sample with a low-power laser. Adjust the microscope objective to find the focal plane of the sample, and then move the stage to find a suitable imaging area. Turn on the 785nm laser and fine-tune the incident position of the laser so that the laser is fully emitted at the interface between the sample and the coverslip at the bottom of the culture dish and returns along the original optical path. The reflected laser should be able to enter the four-quadrant detector to ensure that the anti-drift module is working properly. Open the control software that comes with the EMCCD, set the field of view of the camera to 256×256, the acquisition frequency to 10Hz, and the number of frames to 10. Click "Acquire" in the software interface to complete the acquisition of the wide-field image and save the image. Open the data acquisition card and LabVIEW synchronization control program. In the synchronization control program, set the EMCCD acquisition frequency to 100Hz, the ETL scanning layer number to 20 layers, and the axial zoom step size to 500nm. Also, select the correct signal output port. In addition, you need to configure the control software that comes with the EMCCD and ETL. Set the EMCCD to external trigger mode, set the ETL zoom frequency to 1kHz, and set the ETL to work in sensor control mode to achieve analog voltage signal control of ETL axial zoom.

[0045] Step Two: Acquisition of Sparse Single-Molecular Scintillation Images. Adjust the filter wheel to increase the laser power, causing the fluorescent molecules in the sample to be rapidly bleached and then begin random scintillation. Click the "Run Button" in the LabVIEW control program. The ETL will begin zooming according to the pre-set frame-by-frame scanning method, while the camera begins synchronous exposure. That is, first, a frame of scintillation image is acquired on the first focal plane. Then, the data acquisition card outputs a stepped wave signal to control the ETL to zoom and achieve axial movement of the focal plane, and then a frame of scintillation image is acquired on the second focal plane. This process continues until scintillation images have been acquired on all focal planes, ending the first round of exposure. Then, the system returns to the position of the first focal plane to begin the second round of exposure. This process is repeated tens of thousands of times to complete the acquisition of all scintillation images. The images are saved as stacked data for subsequent processing and reconstruction.

[0046] Step 3: Data Processing and Super-Resolution Reconstruction. Asymmetric 2D Gaussian fitting is performed on the elliptical PSFs in each frame of the scintillation image to obtain the center coordinates (x, y) of each PSF and its full width at half maximum (WHM) in the x and y directions. x ,w y Then, based on the pre-established astigmatism calibration curve, the width information is converted into the corresponding axial position foot coordinates (z) to obtain the three-dimensional positioning result of each PSF; for the axial positioning obtained from different layers, it is only necessary to introduce the known focal plane position information for correction to complete the multi-layer stitching reconstruction; finally, by integrating the three-dimensional spatial coordinates (x, y, z) and brightness information of all single-molecule positioning points, the three-dimensional super-resolution image of the sample is reconstructed.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0048] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A multi-layer three-dimensional single-molecule localization super-resolution imaging method based on a zoom lens, characterized in that: The methods and steps include the following: Step 1: Pre-experiment preparation, including calibration of the electrically adjustable lens zoom parameters and acquisition of astigmatism calibration curves. For zoom parameter calibration, the zoom range must be determined first, and the correspondence between simulated values, current values, and the focal length of the electrically adjustable lens must be established. Specifically, this is achieved by correlating voltage with zoom step size and repeatedly measuring and averaging the values ​​to improve calibration accuracy. The astigmatism calibration curve is used to establish the correspondence between the major and minor axis widths of the diffusion function of fluorescent molecules and their axial position. Specifically, this is achieved by acquiring data on the full width at half maximum (FWHM) of the diffusion function of fluorescent beads in the x and y directions as a function of the z-axis, and then performing polynomial fitting on the data to generate the calibration curve. Step 2: Place the sample to be imaged on the stage of the inverted microscope and fix it in place. Adjust the optical paths of the excitation module, the inverted microscope, and the anti-drift module. During the adjustment process, first activate the laser of the corresponding wavelength in the excitation module and focus on the 640nm laser of the AlexaFluo647 fluorescent probe to find a suitable imaging area. Then activate the laser used for detection in the anti-drift module and adjust the optical path of the 785nm laser to ensure that the anti-drift module can detect axial drift normally, while ensuring that the excitation light can effectively irradiate the sample and that the sample fluorescence signal can be effectively collected. Then, start the LabVIEW synchronous control program running on the computer terminal to complete the parameter settings and preparation work before imaging. Step 3: Using the LabVIEW control program running on the computer terminal, set the number of scanning layers of the electrically adjustable lens, the axial zoom step size, the acquisition frequency of the electron multiplier charge-coupled device camera, the exposure time, and the total number of acquisition frames; first adjust the laser power to make the fluorescent molecules in the sample reach a sparse scintillation state, then start the frame-by-frame scanning mode, control the electrically adjustable lens to switch the focal plane in a step zoom manner according to the set parameters, and simultaneously control the electron multiplier charge-coupled device camera to perform multi-plane cyclic exposure. After tens of thousands of exposures, save the acquired single-molecule fluorescence scintillation images as stacked data. The specific process of the frame-by-frame scanning mode is as follows: Let M be the number of focal planes to be scanned, and N be the number of image frames to be acquired for each focal plane; during the first exposure, the electrically adjustable lens first switches to the first focal plane position, and the electron multiplier charge-coupled device (ECCDP) camera simultaneously acquires 1 frame of image. Subsequently, the electrically adjustable lens switches sequentially to the second to the Mth focal plane positions, and the ECCDP camera acquires 1 frame of image for each focal plane, completing the first round of M frame image acquisition; the electrically adjustable lens returns to the first focal plane position, and the second exposure begins, repeating the above focal plane switching and image acquisition process; after N exposures, NM frames of images are acquired, and the illumination time for all focal planes is consistent; Step 4: Process all the collected single-molecule fluorescence scintillation image stack data; first, perform asymmetric two-dimensional Gaussian fitting on the spread function of the elliptical points in each frame image to obtain the center coordinates and the width of the major and minor axes; then, based on the astigmatism calibration curve obtained in Step 1, convert the width of the major and minor axes into the corresponding z-axis coordinates to achieve single-molecule three-dimensional localization; finally, integrate the three-dimensional spatial coordinates and brightness information of all single-molecule localization points to complete multi-layer image stitching and reconstruction, and obtain a three-dimensional super-resolution image of the thick sample; During data processing, when performing asymmetric two-dimensional Gaussian fitting on the elliptical point spread function, it is necessary to obtain the center coordinates of the point spread function as well as the full width at half maximum (FWHM) in the x-direction and the full WHM in the y-direction. When converting axial coordinates based on the astigmatic calibration curve, it is necessary to determine the axial position of each fluorescent molecule by querying the correspondence between the full WHM and the z-axis coordinates in the calibration curve. When stitching and reconstructing multilayer images, it is necessary to integrate the three-dimensional positioning results of each focal plane according to the actual position of the focal plane to eliminate interlayer positional deviations.

2. The method for multi-layer three-dimensional single-molecule localization super-resolution imaging based on a zoom lens according to claim 1, characterized in that: The specific process for calibrating the zoom parameters of the electrically adjustable lens in Step 1 is as follows: Using a thin object as the calibration sample, first adjust the microscope objective of the inverted microscope to find the focal plane of the sample; then, control the microscope objective to move by a preset scanning step length through the axial nano-displacement stage mounted on the objective base, so that the sample is in a defocused state; then adjust the control current of the electrically adjustable lens to bring the imaging plane back to the original focal plane, and record the analog value output by the data acquisition card and the actual current value of the electrically adjustable lens at this time; repeat the above defocusing, focusing and recording steps until the preset zoom range is covered, and the calibration of the zoom parameters of the electrically adjustable lens is completed.

3. The method for multi-layer three-dimensional single-molecule localization super-resolution imaging based on a zoom lens according to claim 1, characterized in that: The specific process for obtaining the astigmatism calibration curve in step one is as follows: using fluorescent beads as calibration samples, the fluorescent beads are controlled to move gradually from a preset depth below the focal plane to a preset depth above the focal plane in the axial direction using an axial nano-displacement stage, with a movement step size of 10 nm; at each axial position of the fluorescent beads, the electron multiplication charge-coupled device camera is controlled to acquire a preset number of point spread function images; asymmetric two-dimensional Gaussian fitting is performed on each acquired point spread function image to calculate the full width at half maximum (FWHM) in the x and y directions; the FWHM corresponding to all axial positions is polynomially fitted with the z-axis coordinate of that position to generate the astigmatism calibration curve.

4. The method for multi-layer three-dimensional single-molecule localization super-resolution imaging based on a zoom lens according to claim 1, characterized in that: The specific process of adjusting the optical path of the anti-drift module in step two is as follows: Turn on the 785nm laser in the anti-drift module. After the laser is reflected by the mirror and expanded and collimated by the beam expander lens group, it enters the inverted microscope in sequence through the beam splitter and the tube mirror. Adjust the laser optical path so that the laser is focused on the back focal plane of the microscope objective after being reflected by the dichroic mirror of the inverted microscope, and the laser undergoes total internal reflection at the interface between the sample and the coverslip after exiting the microscope objective. Adjust the angle of the beam splitter so that the laser reflected back by the total internal reflection can be accurately incident on the four-quadrant detector after being transmitted through the beam splitter, ensuring that the four-quadrant detector can detect a stable spot signal.

5. A multi-layer three-dimensional single-molecule localization super-resolution imaging system based on a zoom lens, applicable to the multi-layer three-dimensional single-molecule localization super-resolution imaging method based on a zoom lens as described in any one of claims 1-4, characterized in that: It includes an excitation module, an inverted microscope, a zoom and three-dimensional astigmatism detection module, an anti-drift module, and a computer terminal. The computer terminal runs a LabVIEW control program to synchronously control the working status of each module and data transmission. The excitation module is used to provide multi-wavelength excitation light, the inverted microscope is used to transmit excitation light and collect sample fluorescence signals, the zoom and three-dimensional astigmatism detection module is used to realize non-mechanical zoom and single-molecule fluorescence image acquisition, and the anti-drift module is used to detect and correct axial drift in real time.

6. The multi-layer three-dimensional single-molecule localization super-resolution imaging system based on a zoom lens according to claim 5, characterized in that: The excitation module includes a 640nm laser, a 488nm laser, a 405nm laser, two dichroic mirrors, a filter wheel, a beam expander lens group, a tube mirror, and several reflectors. The lasers emitted by the three lasers are reflected by the reflectors and combined by the dichroic mirrors. The laser power is adjusted by the filter wheel, then expanded and collimated by the beam expander lens group, and the height and angle are adjusted by the reflectors. Finally, the lasers enter the inverted microscope through the tube mirror. The 640nm laser is used to excite the AlexaFluo647 and Cy5 fluorescent probes, the 488nm laser is used to excite the AlexaFluo488 and GFP fluorescent probes, and the 405nm laser is used to activate the fluorescent probes to achieve a scintillation effect.

7. The multi-layer three-dimensional single-molecule localization super-resolution imaging system based on a zoom lens according to claim 5, characterized in that: The zoom and 3D astigmatism detection module includes a relay lens group, an electrically adjustable lens, a data acquisition card, a cylindrical lens, and an electron multiplication charge-coupled device (ECCDP) camera. The cylindrical lens is positioned between the relay lens group and the ECCDP camera to enable fluorescent molecules at different z-axis positions to form elliptical point diffusion functions with different ellipticity and orientation on the detection surface of the ECCDP camera. The analog output port of the data acquisition card is connected to the analog input port of the electrically adjustable lens and the trigger port of the ECCDP camera, respectively, to output analog signals to control the zoom of the electrically adjustable lens and trigger the synchronous exposure of the ECCDP camera. The computer terminal is communicatively connected to the data acquisition card, the electrically adjustable lens drive controller, and the ECCDP camera, respectively, to transmit control commands and receive image data.

8. A multi-layer three-dimensional single-molecule localization super-resolution imaging system based on a zoom lens according to claim 5, characterized in that: The anti-drift module includes a 785nm laser, a beam expander lens group, a beam splitter, a four-quadrant detector, a tube mirror, and several reflecting mirrors. The four-quadrant detector is connected to a computer terminal to transmit the detected spot position signal to the computer terminal. An axial nano-displacement stage is installed on the objective lens base of the inverted microscope. The axial nano-displacement stage is electrically connected to the computer terminal. The computer terminal calculates the axial drift based on the change in the spot position of the four-quadrant detector and outputs a control signal to drive the axial nano-displacement stage to move in the opposite direction to counteract the drift.

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