41 results about "Single molecule localization" patented technology

The invention relates to the technical field of image data processing or generation, in particular to a monomolecular positioning super-resolution imaging chromatic aberration correction method and system, and the method comprises the following steps: removing misregistration points from a plurality of groups of transverse moving images of two different wavelength channels; the reserved points are registered to obtain a chromatic aberration correction mapping function, and single-molecule positioning super-resolution imaging transverse chromatic aberration correction is completed; gaussian fitting and linear least square fitting are carried out on the multiple sets of axial height data of each wavelength channel, the average deviation of the two channels at different axial heights is obtained and compensated to a single-molecule positioning super-resolution imaging chromatic aberration correction system, and single-molecule positioning super-resolution imaging axial chromatic aberration correction is completed. According to the method and the system provided by the invention, the correction of transverse and axial chromatic aberration in multi-color single-molecule positioning super-resolution imaging is realized, the registration precision and the correction speed of the system are improved, and the correction cost is reduced.

Method for single molecule localization microscopy (SMLM) based on conventional fluorescent binder probes, including primary or secondary antibodies, that are applied in a gradual stepwise fashion to a fixed biological sample. In each step of the process, a low dose of the probe is applied in solution to the fixed biological sample to sparsely label target epitopes followed by sample washing to remove the free probe. The remaining bound fluorescent probes that are sparsely distributed over the sample are then imaged until they are photobleached. The high light collection efficiency from each probe in this approach (limited only by photobleaching) enables few Ångstrom lateral resolution and few nm axial resolution. Photobleaching of the probes at the end of each step generates a clean slate for application of the next step in the process, as well as the possibility for applying one or more probes in a consecutive fashion to the same sample for serial SMLM.

Disclosed in the present invention are a prism-type total internal reflection imaging apparatus compatible with a commercial inverted microscope, and a method for using same. The method comprises the following steps: first injecting a sample into a sample cell; turning on a laser, and generated evanescent waves illuminating nearby fluorescent molecules to obtain an image; and processing the image to obtain the three-dimensional positions of all the fluorescent molecules in a visual field, obtaining the three-dimensional trajectories of the fluorescent molecules, calculating a mean square end-to-end distance and a diffusion factor of the trajectories, and obtaining a relative potential energy distribution curve on the basis of the relative positions of the molecules. The present invention can be compatible with a mainstream commercial inverted microscope, and does not need to carry out any refit on existing microscopes. A sample stage is mounted on the inverted microscope and an excitation light path module is mounted on the side edge of the sample stage, so that a total internal reflection light path having a specific wavelength can be generated. The apparatus can achieve single molecule positioning and three-dimensional tracking with a high signal-to-noise ratio, and the sample cell can be flexibly replaced before each test without influencing the total internal reflection light path. The apparatus is applied to the fields of life science, material analysis and the like.

The invention discloses a single molecule positioning super-resolution imaging method and device, equipment and a medium, and relates to the field of optical microscopic imaging, and the method comprises the steps: obtaining a currently collected single molecule image containing a plurality of PSF light spots; inputting the single molecule image into a single molecule positioning model based on a deep residual network, a feature pyramid network, a region generation network, a RoIAlign layer, a mask head and a key nodding so as to carry out sub-pixel-level positioning and brightness estimation on a PSF light spot in the current single molecule image to obtain a plurality of single molecule positioning points and positioning point brightness values, carrying out uncertainty prediction on the single molecule positioning point and the brightness value of the positioning point; and screening the single-molecule positioning points based on the positioning point uncertainty and the brightness uncertainty, and superposing and rendering the screened positioning points to perform super-resolution reconstruction on the single-molecule image to obtain a single-molecule super-resolution image. According to the invention, the precision and efficiency of single-molecule positioning super-resolution imaging can be improved.

The invention relates to the field of imaging correction, and provides a three-dimensional drift correction method and system for a single-molecule localization super-resolution image, and the method comprises the steps: carrying out the high-speed photographing, and obtaining imaging data; obtaining grouped imaging data including a reference group and a calibration group, and performing neighbor distance point matching in the reference group according to the search area to obtain matched fluorescent points; respectively obtaining a plane average offset and a vertical average offset, and carrying out iteration to obtain a rough correction plane relative displacement and a rough correction vertical relative displacement; performing coarse calibration on the fluorescent points in the calibration group to obtain coarse calibration fluorescent points, selecting the nearest fluorescent point closest to the coarse calibration fluorescent points, calculating a relative plane position difference and a relative vertical position difference, and solving a vector average sum to obtain fine calibration plane relative displacement and fine calibration vertical relative displacement; according to the method, a plane coordinate drift curve and a vertical coordinate drift curve are obtained respectively, imaging data are corrected, and a high-precision corrected fluorescence image can be obtained.

The invention discloses an interference single molecule positioning microscopic imaging device and method based on mirror reflection and a single objective lens. The device comprises a light source system, an illumination and modulation system, a fluorescence imaging system and a signal control and processing system. The exciting light passes through the objective lens and then is reflected back by the reflector positioned above the objective lens, and a standing wave interference field is formed at the sample to excite fluorescent molecules. The axial position of the reflector is accurately adjusted to modulate the illumination phase; based on the change of the fluorescence intensity in the collected single molecule image along with the interference fringes, the phase of the fluorescence intensity relative to the interference fringes is calculated, and analysis is carried out by combining the axial relative position information, so that the nanoscale axial positioning precision is realized. According to the invention, a light path structure combining a single objective lens with mirror reflection is adopted, so that high-precision pairing and complex alignment of traditional double objective lenses are avoided. The axial resolution of the device is close to that of a double-objective 4Pi single-molecule positioning microscope, and meanwhile, the system complexity, the installation and adjustment difficulty and the manufacturing cost are remarkably reduced.

The invention belongs to the technical field of data processing, and particularly relates to a single molecule tracing data processing method and system, computer equipment and a storage medium. The method integrally executes the following steps in a local graphical user interface environment: performing visual preprocessing operation on original single molecule tracing image data; based on the preprocessed original single-molecule tracing image data, detecting a single-molecule peak value and carrying out sub-pixel precision fitting to generate single-molecule positioning point data; associating the single-molecule positioning point data into single-molecule movement track data through a proximity search algorithm; and performing diffusion kinetic parameter analysis, limited radius estimation analysis and trajectory motion state classification analysis on the single-molecule motion trajectory data, and outputting multi-dimensional kinetic parameters. A data processing method is integrated in a local graphical user interface environment, and in addition, by executing diffusion kinetic parameter analysis, limited radius estimation analysis and track motion state classification analysis, a user can obtain richer multi-dimensional kinetic parameters.

The invention discloses a single molecule positioning method based on deep learning feature fusion, and the method comprises the following modules: a data processing module which uses an analog number sample generated by imageJ as a training set, and converts a generated positioning table into a corresponding image as a verification set; the Resnet module is used for extracting the characteristics of the data; the ASF module is a feature fusion module and is used for fusing features of different scales according to different weights; and the attention mechanism module can selectively pay attention to the positioning information of the single molecule and is beneficial to realizing more accurate positioning on the single molecule. According to the technology, the super-resolution positioning of the single molecule is completed through the deep learning network, and the accurate positioning of the single molecule image can be quickly formed. The technology not only is helpful to research and track experiments of single molecule dynamic behaviors, but also has a wide prospect in the aspects of assisting accurate analysis and early diagnosis of disease mechanisms.

The invention relates to the technical field of super-resolution optical imaging, in particular to a super-resolution imaging probe of GQDs-double fluorescent dye based on an FRET effect. The probe takes graphene quantum dots as donors and double fluorescent dyes as receptors, the surfaces of the graphene quantum dots are modified with polyethylene glycol, and the probe is used for super-resolution optical imaging of specific organelles through antibody specific targeting. According to the super-resolution imaging probe of the GQDs-double fluorescent dye based on the FRET effect, provided by the invention, single-laser excited double-channel super-resolution optical imaging is realized, and the super-resolution imaging probe can be used for super-resolution optical imaging based on a single-molecule positioning method.

Methods for single molecule localization microscopy may use a patterned illumination over the field of view. The patterned illumination may be dynamically adapted to the approximate positions of the detected fluorescent molecule emitters, allowing for increased signal-to-background for their single molecule localization.

The invention discloses a single-molecule positioning microscopic imaging device and method based on structured light illumination. In an excitation path, a modulated laser beam emitted by a spatial light modulator generates a dynamic stripe illumination pattern on a sample plane, and three phase shift patterns are adopted in each transverse direction, so that six different patterns are generated; in the emission path, the fluorescence signal is coherently collected by a dual objective, while four phase images are captured, each image corresponding to a particular phase delay. By using the interference effect in the excitation path and the emission path, the improvement of the two-time transverse resolution and the five-time axial resolution is simultaneously realized in the single-molecule positioning microscope, the difference between the axial resolution and the transverse resolution is effectively bridged, and the isotropic nano-scale three-dimensional resolution is realized.

The application relates to the technical field of biology, and discloses a method and device for analyzing the properties of a nucleic acid nanostructure, which comprises obtaining single-molecule positioning imaging data of the nucleic acid nanostructure, pre-processing the data to obtain spatial information and time information of the nucleic acid nanostructure, determining a profile curve of the nucleic acid nanostructure according to the spatial information, dividing the profile curve into a plurality of chain segments, calculating the length L of the chain segments through the spatial coordinates of the end points at both ends of the chain segments, calculating the angle correlation cos theta (L) through the unit vectors of the two end points with a distance of L, calculating the persistence length P of the nucleic acid nanostructure through the relationship between the chain segment length L and the average value <cos theta (L)> of the angle correlation when the chain segment length is L, determining quantitative analysis of structural rigidity, determining quantitative analysis of site accessibility, and performing correlation analysis of the structural rigidity and the site accessibility. The application provides accurate quantitative evaluation of the structural rigidity and / or site accessibility of the nucleic acid nanostructure.

The invention discloses an artifact evaluation method for three-dimensional super-resolution microscopic imaging, and belongs to the field of fluorescent microscopic imaging. According to the method, a high-resolution image is zoomed to obtain an optimal three-dimensional resolution zoomed image, linear transformation is carried out to obtain a linearly transformed three-dimensional resolution zoomed image, the three-dimensional resolution zoomed image and a low-resolution image are analyzed, and an output artifact evaluation index is solved; the authenticity of the high-resolution image is judged by analyzing the interpretable degree of the optical imaging model between the low-resolution image before super-resolution and the high-resolution image after super-resolution; the artifact condition in the three-dimensional super-resolution technology is quantitatively evaluated with low cost through a calculation method. The method is suitable for evaluating the super-resolution artifact condition of three-dimensional super-resolution microscopes such as a three-dimensional structured light illumination microscope and a three-dimensional single-molecule positioning super-resolution microscope, and can also be used for evaluating the super-resolution artifact condition of a three-dimensional deconvolution and three-dimensional deep learning network.

The invention discloses an axial stable shooting system and shooting method for a single molecule positioning microscope, and belongs to the technical field of microscopic imaging. The system comprises a sample axial drift real-time compensation light path, an infrared laser emits linear polarization parallel light, the linear polarization parallel light passes through a collimating lens L1, a half-wave plate H1 and a beam splitting cube BS1 to form two beams of parallel light, the two beams of parallel light are reflected by a polarization beam splitting cube PBS, then enter an objective lens through a half-wave plate H2, a lens L2, a beam splitting cube BS2 and a dichroscope DM1, and are focused at the position of a rear focal plane of the objective lens; the two beams of light reflected by the sample are transmitted out from a polarization beam splitting cube PBS along the original path, and enter a focus-locked camera camZ for imaging through a lens L3 and a band-pass optical filter F1. And during shooting, recording the difference delta x between the distance D of two light spots of a new image every time and the initial distance D0 in real time, controlling the axial position of the objective table to compensate by using an incremental PID (Proportion Integration Differentiation) algorithm, and controlling the delta x to be less than or equal to an allowable error. According to the system and the method, the problem of poor axial stability precision of the sample of the single-molecule positioning microscope is successfully solved.

The application discloses a three-dimensional single molecule positioning imaging system, method, device, medium and product, relates to the technical field of single molecule positioning microscopic imaging, and the system comprises a light source module, which is used for generating an excitation light beam for irradiating a single molecule sample; an intensity modulation module, which is used for intensity modulation on the excitation light beam to obtain a modulated excitation light beam; a depth modulation module, which is used for axial position modulation on the fluorescent pulse to obtain an encoded fluorescent pulse; an event detection module, which is used for generating a photon event sequence according to the encoded fluorescent pulse; and a positioning imaging module, which is used for solving three-dimensional space coordinates of each single molecule in the single molecule sample according to the photon event sequence, and reconstructing and generating a three-dimensional single molecule image corresponding to the single molecule sample according to the three-dimensional space coordinates of all single molecules in the single molecule sample. The application improves the utilization rate of single molecule positioning information, the fidelity of a three-dimensional image and the imaging flux.

The present invention relates to a chimeric nanobody, a single-molecule localization imaging probe and an imaging method. The chimeric nanobody of the present application sequentially comprises the following sequences from the N-terminus to the C-terminus: the NbGFPe nanobody sequence; a linker with a length of 36 amino acids; the Nb2 nanobody sequence; and a C-terminal linker containing cysteine. The chimeric nanobody of the present invention effectively avoids the problems of low affinity of a single antibody and multi-site labeling, and can achieve high-affinity labeling of 1:1. The probe obtained therefrom for DNA-PAINT super-resolution imaging can obtain high-quality images for imaging various fine structures in cells.

Disclosed are an interference single-molecule localization microscopic imaging apparatus based on specular reflection and a single objective lens, and an imaging method The apparatus comprises: a light source system, an illumination and modulation system, a fluorescence imaging system, and a signal control and processing system. After passing through an objective lens, excitation light is reflected by a reflector located above the objective lens, and forms a standing-wave interference field at the sample to excite fluorescent molecules. Precisely adjusting the axial position of the reflector to modulate the illumination phase; and on the basis of the fluorescence intensity changing with the interference fringes in the acquired single-molecule image, calculating the phase of the single-molecule image relative to the interference fringes, and analyzing in combination with the axial relative position information, thereby achieving nanoscale axial localization precision. In the present invention, an optical path structure using a single objective lens in combination with specular reflection is used, thereby avoiding high-precision pairing and complex alignment in conventional dual objective lenses. The present apparatus achieves axial resolution approaching that of a dual-objective 4Pi single-molecule localization microscopy, and also has significantly reduced system complexity, assembly and adjustment difficulty and manufacturing costs.

Provided herein are methods for analysis of single molecule localization microscopy (SMLM) data comprising a plurality of input data points in the form of molecular coordinates. These methods can identify clustering within input data points at all spatial scales within the input data set. These methods can comprise (i) grouping the plurality of input data points into clusters; (ii) discretizing the clusters into a discretized array; and (iii) measuring a spatial relationship between two species of localizations captured by the discretized array.

The application discloses a kind of super-resolution fluorescence microscopes for single molecule positioning, belong to the technical field of super-resolution microscopic imaging, including illumination module, laser emission module, lock focus optical path module, objective system module, cylindrical lens module and imaging module;Laser emission module includes first optical fiber head, first lens, first filter, adjustable diaphragm, first mirror, second mirror, first focusing lens, third mirror and fourth mirror;Wherein, first mirror is used to reflect the parallel light beam passing through first filter and adjustable diaphragm to second mirror;Fourth mirror is used to reflect light beam to objective system module, and objective system module is used to irradiate received light beam on sample.Laser emission module can change the propagation direction of light beam by multiple reflections, so as to reduce the transmission volume of light beam, and then make the layout of super-resolution fluorescence microscope more compact, which can reduce the volume of super-resolution fluorescence microscopic system.

The present invention relates to the field of image data processing or generation technology, and in particular to a method and system for chromatic aberration correction in single-molecule localization super-resolution imaging. The method comprises the following steps: eliminating misaligned points from multiple sets of lateral motion images of two different wavelength channels; performing registration between the retained points to obtain a chromatic aberration correction mapping function, thereby completing lateral chromatic aberration correction in single-molecule localization super-resolution imaging; performing Gaussian fitting and linear least squares fitting on multiple sets of axial height data for each wavelength channel to obtain the average deviation of the two channels at different axial heights, which is then compensated into the single-molecule localization super-resolution imaging chromatic aberration correction system to complete axial chromatic aberration correction in single-molecule localization super-resolution imaging. The method and system provided by the present invention achieve correction of lateral and axial chromatic aberration in multi-color single-molecule localization super-resolution imaging, improve the system's registration accuracy and correction speed, and reduce the cost of correction.

A 3D single-molecule super-resolution imaging technique is disclosed herein. In particular, the techniques described herein combine an optical platform including a tilted single-objective dithered light sheet illumination, a microfluidic system, and point spread function engineering to achieve precise 3D single-molecule super-resolution imaging of subcellular structures (e.g., a sample (110)) with easy control of the extracellular environment. In certain embodiments, the single-objective dithered light sheet (102) of the optical setup (100) includes a plurality of galvanometric mirrors (116, 118, 124) and a tunable lens (122) that may steer a light sheet (102) for optimized optical sectioning of the sample (110). In some embodiments, the sample (110) is positioned in the microfluidic device (160) during imaging. In this way, traditional challenges (e.g., high out-of-focus background fluorescence and lack of environmental control) generally facing single-molecule localization microscopy may be overcome.

The invention relates to the technical field of optical microscope imaging, in particular to a molecular scale resolution optical super-resolution microscopy method. The method comprises the following steps: constructing a single molecule polarization measurement module; performing single-molecule imaging on the sample, dividing single-molecule fluorescence into two beams by using the single-molecule polarization measurement module, and simultaneously imaging in different half regions of the EMCCD; carrying out single molecule identification and center calculation on the collected single molecule image; constructing a conversion matrix, and mapping the positions of the single molecules of two different channels; measuring the fluorescence intensity of the single molecules in the two channels, calculating polarization values, and grouping the positioning of the single molecules according to the difference of the polarization values; calculation of a single molecule positioning distribution center and MIPI positioning precision is carried out on grouped single molecule positioning; and carrying out super-resolution image reconstruction to obtain a molecular scale resolution image. The method utilizes polarization measurement to distinguish adjacent single fluorescent molecules, and is suitable for high-density fluorescence labeled sample imaging.

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.

The application discloses a single-molecule image positioning and processing design platform based on PyQt, which comprises an image preprocessing module, an image positioning module, an image post-processing module and an interactive interface layer built based on PyQt, and the modules are connected with each other and work cooperatively, the target single-molecule image from the interactive interface layer is preprocessed by the image preprocessing module to obtain a processed image, the pre-trained single-molecule positioning deep learning network model is called by the image positioning module to perform forward inference on the processed image, single-molecule fluorescent point positioning and super-resolution reconstruction are completed, and a positioning image is obtained; the target single-molecule image is scaled to the same size as the positioning image by the image post-processing module, and then the positioning result of the single-molecule fluorescent point corresponding to the positioning image is superimposed on the scaled target single-molecule image to obtain a positioning comparison image, and the integration of single-molecule image reading, preprocessing, deep learning positioning, post-processing and result visualization is realized.

The application discloses a system and method for automatically searching for an optimal oblique layered light sheet illumination position, and belongs to the technical field of image data processing. The method comprises the following steps: exciting light is collimated and expanded, is adjusted by a first mirror to be incident to an internal light path of an electric displacement table, is reflected by a dichroic mirror to enter a TIRF objective, is focused by the TIRF objective, and is irradiated on a sample carried by a cover glass; the emitted light of the sample is collected by the TIRF objective, is transmitted by the dichroic mirror, is reflected by a second mirror, and enters a camera; a computer controls the electric displacement table to move, and acquires a fluorescence image collected by the camera; and a position point with the maximum fluorescence signal intensity value is determined as the optimal oblique layered light sheet illumination position. The application can automatically, quickly and accurately find the optimal oblique layered light sheet illumination position, avoids artificial subjective judgment errors, has the advantages of automation, self-adaptation and high accuracy, and significantly improves the experimental efficiency and quality of single-molecule localization super-resolution imaging.

The invention relates to optical microscopic imaging, and discloses a three-dimensional single-molecule positioning microscope which comprises a microscope body and an operation assembly, the microscope body comprises a light path transmission device, an objective lens and a camera, and the light path transmission device enables incident exciting light to penetrate through the objective lens to irradiate a sample and generate a total internal reflection evanescent field; the objective lens acquires a fluorescence signal and transmits the fluorescence signal to the camera, the camera acquires a sample data set and displays an image, and the data set comprises a positioning point plane coordinate and a photon number representing fluorescence signal intensity; the operation assembly is used for obtaining a positioning point cloud cluster according to the data set, z-axis coordinates of positioning points are obtained through calculation of a double-index model and fluorescence signal intensity, and the double-index model comprises addition of angle radiation intensity and angle refraction intensity; the operation assembly also groups the positioning point cloud clusters according to the sample prior information, carries out re-positioning according to groups, carries out calculation to obtain three-dimensional information with higher resolution, carries out the reconstruction and display of a super-resolution image according to the three-dimensional information, and further enhances the resolution.

The invention discloses a single-molecule super-resolution microscopic imaging method based on depth spatio-temporal information integration, and belongs to the field of biological super-resolution microscopic imaging. The implementation method comprises the following steps: establishing an initial network model for positioning the ultrahigh-density fluorescent molecules; training parameters and network hyper-parameters are set, and a flicker mechanism and a time window are fused to obtain training data; training the network by using the training data until an iteration round reaches a training parameter setting round, and model effect evaluation is close to stability between the training rounds and does not continue to be obviously improved; converting to-be-measured data into photon data through camera parameters, inputting the photon data into the network, and outputting a probability matrix and positioning information; fluorescent molecule position information of each frame is obtained according to a set probability threshold value, and a super-resolution positioning map of the fluorescent molecules is obtained through reconstruction. The method is combined with a single-molecule positioning microtechnique, the positioning precision of ultrahigh-density super-resolution imaging is improved, and the method is suitable for fluorescence microscopic imaging of fixed cells under different imaging conditions.

This invention discloses a multimodal single-molecule localization method and system based on in-site diffusion function and fringe illumination, aiming to solve the problems of low photon utilization and decreased accuracy of defocused location positioning in existing single-molecule localization algorithms. This invention acquires multiphase-shifted single-molecule image sequences using spatially adjustable three-dimensional fringe light; reassembles data using a time-sliding window strategy to reuse scintillation signals; then, a cubic spline interpolation model of the in-site diffusion function is constructed for preliminary localization, and precise three-dimensional fringe parameters are extracted based on a two-stage direction vector estimation algorithm; finally, a maximum likelihood multi-parameter joint estimation model is constructed to output high-precision three-dimensional molecular coordinates. This method is perfectly compatible with modulated illumination at arbitrary tilt angles and distorted point diffusion functions of arbitrary shapes, not only significantly compensating for defocused positioning errors but also achieving a significant improvement of more than 2 times in lateral positioning accuracy and more than 2.5 times in axial positioning accuracy.

The invention discloses a near-infrared self-flickering fluorescent dye as well as a preparation method and application thereof. The near-infrared self-flickering fluorescent dye has one of structures as shown in a formula I and a formula II. Azetidine is introduced to inhibit a twisted charge transfer effect in molecules, so that silicon rhodamine has the characteristics of high light stability, high brightness, cell permeability and the like. Based on thermodynamic equilibrium of intramolecular spiralization of a benzyl alcohol structure in the dye, the fluorescent dye can realize single-molecule localization super-resolution fluorescence imaging of various subcellular structures in living cells, and realizes visualization of structures including lysosomes, mitochondria, cell nucleuses, microfilaments and the like on a nano scale. Besides, the absorption wavelength of the dye is 650nm, the fluorescence emission wavelength reaches 666nm and reaches a near-infrared region, damage of exciting light to cells is reduced in the super-resolution imaging process, and the dynamic life process of the cells under normal physiological conditions can be visualized;

The application discloses an interference single-molecule positioning microscopic imaging device and imaging method based on mirror reflection and single objective. The device comprises a light source system, an illumination and modulation system, a fluorescent imaging system and a signal control and processing system. After the excitation light passes through the objective, the light is reflected by the mirror located above the objective, and a standing wave interference field is formed at the sample to excite fluorescent molecules. The axial position of the mirror is accurately adjusted to modulate the illumination phase; based on the change of the fluorescent intensity in the collected single-molecule image with the interference fringes, the phase of the single-molecule image relative to the interference fringes is calculated, and the axial relative position information is combined for analysis, so that the nanoscale axial positioning precision is realized. The application adopts the optical path structure of single objective combined with mirror reflection, avoids the high-precision pairing and complex alignment of the traditional double objective, and realizes the axial resolution close to the double-objective 4Pi single-molecule positioning microscope. Meanwhile, the system complexity, assembly and adjustment difficulty and manufacturing cost are significantly reduced.