28 results about "Single molecule localization" patented technology

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 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.

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.

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.

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 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.

The invention discloses a super-resolution imaging system and method based on single molecule quantum coherence, and belongs to the technical field of fluorescence imaging, the system comprises a femtosecond laser, a Michelson interference light path module, a microscopic module and a fluorescence detection module, the Michelson interference light path module splits femtosecond laser into a pump light path and a detection light path; the method comprises the following steps: determining zero delay positions of pump light and probe light; a spatial phase pattern is loaded through a spatial light modulator, a coherent superposition state with an adjustable delay phase is formed on a sample surface, and sparse random activation of single-molecule fluorescence is realized; and finally, collecting a plurality of frames of sparse fluorescence images, and reconstructing a super-resolution image through single molecule positioning and synthesis. According to the invention, the quantum coherence physical principle is utilized to regulate and control the single molecule luminescence state, the dependence on a complex chemical buffer solution is reduced, the sample preparation is simplified, and a new means is provided for the super-resolution imaging of a biological sample by combining the penetrability advantage of near-infrared femtosecond laser.

The application provides a single-element super-resolution ultrasonic imaging method based on acoustic metamaterials, which comprises the following steps: first, an acoustic field coding board is made of acoustic metamaterials to construct a single-element super-resolution ultrasonic imaging system; then, the system is calibrated in terms of acoustic field, and the system matrix in the imaging area is obtained by means of angular spectrum method and automatic convolution; secondly, the flowing microbubbles in the tissue are imaged by the single-element super-resolution ultrasonic imaging system to obtain a group of simulated original ultrasonic data, the acoustic coding board is rotated to different angles, the multi-angle data of the flowing microbubbles are collected, and multi-angle original ultrasonic echo data are obtained; then, based on the system matrix and the obtained original ultrasonic echo data, the ultrasonic microbubble image is reconstructed by means of linear inversion algorithm; finally, the center of the ultrasonic microbubble image is positioned by a single-molecule positioning method, the microbubble position is obtained, the microbubble positioning results of all frames are superimposed, and the single-element super-resolution ultrasonic imaging is realized.

A marker-free drift tracking systema and method, referred to as Adaptive localization Intersection based Drift correction (AID), for fast and precise drift correction for single-molecule localization microscopy (SMLM). The system and method utilize an adaptively adjusted position intersection map between two temporally adjacent sets of localized emitter coordinates from the imaging target to achieve robust and precise drift correction at a high computation speed.

The present application belongs to the technical field of fluorescent dyes, and particularly relates to a kind of fluorescent dye molecules capable of spontaneous blinking under physiological conditions and a synthesis method and application thereof.The spontaneous blinking fluorescent dye molecules of the present application take aniline squarylium cyanine as a mother nucleus;the spontaneous blinking fluorescent molecules can be prepared by Click reaction through intermediate squarylium fluorophore and azide targeting group;the spontaneously blinking fluorescent molecules designed and synthesized by the present application can occur spontaneous blinking behavior under physiological conditions, so that they are very suitable for single molecule localization microscopy, and can be used for cell super-resolution imaging.

The embodiment of the invention discloses a method and device for processing a single molecule positioning microscope image. According to the embodiment of the invention, band-pass filtering processing can be carried out on an original single-molecule positioning microscope image to obtain a band-pass filtering image; performing background homogenization processing on the band-pass filtering image to obtain a background homogenization image; performing contrast adjustment processing on the background-homogenized image to obtain a contrast-enhanced image; for each pixel point in the contrast enhanced image, performing brightness value comparison processing on the pixel point and an adjacent pixel point; when the brightness value of the pixel point is greater than the brightness value of the adjacent pixel point and the brightness value of the pixel point is greater than a preset brightness value, determining the pixel point as a candidate molecular position; and performing Gaussian point spread function fitting on the to-be-fitted pixel region corresponding to the candidate molecule position to obtain a single molecule coordinate. In the embodiment of the invention, the accuracy of single molecule positioning is improved.

The invention belongs to the technical field of data processing, and particularly relates to a multi-modal cell nucleus segmentation method and device, computer equipment and a storage medium. The method comprises the following steps: acquiring original image data generated by a single molecule tracing experiment and single molecule positioning point data corresponding to the original image data; based on the original image data and the single molecule positioning point data, constructing input features including at least two different modal information; and inputting the input features into a pre-trained neural network model to generate a segmentation result of the cell nucleus. According to the method, multi-modal input features are constructed by relying on original image data and single-molecule positioning point data, and accurate and stable cell nucleus segmentation results can be output in weak-texture and low-signal single-molecule tracing experiments in combination with the neural network model.

The invention discloses a system and a method for automatically searching an optimal inclined 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 reflector to enter an internal light path of an electric displacement table, then enters a dichroscope, is reflected by the dichroscope to enter a TIRF objective lens, is focused by the TIRF objective lens, and is irradiated on a sample carried by a cover glass; emitted light emitted by the sample is collected by the TIRF objective lens, transmitted by the dichroscope and reflected by the second reflector to enter the camera; the computer controls the electric displacement table to move to obtain a fluorescence image collected by the camera; and determining the position point with the maximum fluorescence signal intensity value as the optimal inclined layered light sheet illumination position. The method can automatically, quickly and accurately find the optimal inclined layered light sheet illumination position, avoids manual subjective judgment errors, has the advantages of automation, self-adaption and high accuracy, and remarkably improves the experimental efficiency and quality of single-molecule positioning super-resolution imaging.

The application discloses a kind of molecular level resolution microscopy MITI (Molecular-level Imaging based on Two-color Imaging) based on two-color single molecule positioning. The application uses the method of two-color imaging, and the positioning points of the originally indistinguishable fluorescent molecules are successfully distinguished by the two-color method, and the different positioning point sets are distributed into the corresponding fluorescent channels to be distinguished, and then the center position of the fluorescent molecule positioning point distribution and the positioning accuracy are improved again by fitting the distribution of the fluorescent molecule positioning points again, so as to improve the resolution. The application can achieve a spatial resolution of up to 2 nm, providing an important means for analyzing molecular-scale biological structures and interaction processes.

The invention discloses a multi-layer three-dimensional single-molecule positioning super-resolution imaging method and system based on a zoom lens, and the method comprises the steps: 1, carrying out the preparation before an experiment, completing the calibration of the zoom parameter of an electrically adjustable lens, and obtaining an astigmatism calibration curve, 2, fixing a sample, focusing, and starting an anti-drift function and a synchronous control program, 3, collecting a plurality of layers of three-dimensional single-molecule scintillation images in a frame-by-frame scanning mode; and 4, carrying out three-dimensional positioning and splicing reconstruction on all the collected single-molecule fluorescent scintillation images. The problems that the scanning speed is low and mechanical inertia exists when axial scanning is carried out by depending on mechanical movement of a microscope objective or an objective table in the prior art can be solved, and a brand new frame-by-frame scanning mode can be adopted due to the fact that ETL has the capacity of rapid zooming and accurate resetting. The problem that in a traditional layer-by-layer scanning mode, the laser irradiation time of a focal plane scanned later is longer than that of a focal plane scanned first is solved, and the influence of photobleaching on the imaging quality of different layers is reduced to the maximum extent.

Method for single molecule localization microscopy (SMLM) based on conventional fluorescent binder probes, including antibodies, that are continually transported at low concentration over a sample under continuous illumination and microscopic acquisition.