Super-resolution imaging system and biomarker detection method

Abstract

The invention provides a super-resolution imaging system and a biomarker detection method.The method comprises the steps that the super-resolution imaging system is used for conducting single-molecule fluorescence imaging on a specific biomarker in a biological sample, and multiple frames of original microscopic fluorescence images are obtained; and processing and analyzing the multiple frames of original microscopic fluorescence images to extract super-resolution distribution characteristic information of specific biomarkers on the surface of the biological sample, and content distribution and other quantitative characteristics of the specific biomarkers in the biological sample. The method comprises the following steps: processing an original microscopic fluorescence image to obtain positioning table data; rendering positioning points in the positioning table data to generate a super-resolution image; performing convolution operation, opening operation, convolution filtering and region segmentation on the super-resolution image to calculate the area of cells, the number of clusters and the area of a single cluster, determining the density of the clusters and the area ratio of the clusters by using the two, and calculating the closest distance between the clusters by using the center points of the clusters; and the accuracy of a biomarker detection result is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microscopic optical imaging and image processing, and in particular to a super-resolution imaging system and a biomarker detection method. Background Art

[0002] Alzheimer's disease (AD) is the most common form of dementia and an irreversible neurodegenerative disease. Early diagnosis and early intervention are the key to preventing AD. However, commonly used technologies are expensive (Positron Emission Tomography, PET) or invasive (cerebrospinal fluid biomarker testing), which is not easy for patients to accept.

[0003] In recent years, the detection of blood biomarkers for AD has made rapid progress. At present, the research on blood detection methods for AD mainly includes plasma detection and red blood cell detection. Amyloid β-protein (Aβ) is one of the core biomarkers of Alzheimer's disease. It has a high affinity with albumin, apolipoprotein, α2-macroglobulin, etc. in plasma. Although single molecule arrays with ultra-high sensitivity and mass spectrometry-based detection technologies for Aβ protein in plasma (such as immunoprecipitation-coupled mass spectrometry, antibody-free liquid chromatography-mass spectrometry, etc.) can improve the detection limit of Aβ protein, and although a large number of studies have been conducted on multiple variables before plasma detection and standardized processing guidelines have been formulated before plasma analysis, the plasma matrix is ​​relatively complex and personal operation details are difficult to standardize. These differences will affect the accuracy of the detection results of AD biomarkers in plasma, thereby affecting the clinical applicability of biomarkers.

[0004] Studies have shown that Aβ protein in cerebrospinal fluid can cross the blood-brain barrier, flow into the blood with the cerebrospinal fluid, and easily enrich on the surface of red blood cells in peripheral blood. However, the concentration of biomarkers in the blood is much lower than that in the cerebrospinal fluid, so high-sensitivity technology is required for accurate measurement. At present, the detection methods of AD biomarkers in the blood have certain limitations, such as the limited sensitivity of immunofluorescence assays and ELISA assays; although atomic force microscopy has ultra-high resolution, its detection results may be affected by other similar proteins. The specific defects are as follows:

[0005] (1) Complex sample processing procedures: Existing blood biomarker-based AD early diagnosis methods mainly detect the concentration of AD biomarkers in plasma; a small number of studies have also used enzyme-linked immunosorbent assay (ELISA) and europium immunoassay to detect AD biomarkers on the surface of red blood cells. That is, peripheral blood red blood cells can be considered as an additional source for screening and detecting AD biomarkers. However, due to the complex blood environment, the detection of target proteins has many steps, complex operations, long time consumption, and high technical requirements. Studies have shown that for the same plasma, different laboratories use the same or different methods to detect (detection methods include: enzyme-linked immunosorbent assay (ELISA), single molecule array (Single molecule array), IP-LC-MS (Immunoprecipitation coupled toliquid chromatography mass spectrometry), and the results will be different. In other words, the accuracy of plasma-based AD biomarker detection is easily affected by the detection process (human operation errors, blood storage conditions, etc.).

[0006] (2) Lack of biological specificity: Some studies have used atomic force microscopy to study the morphology (size, structure, texture, etc.) of Aβ protein aggregates on the surface of peripheral blood red blood cells of AD patients, and compared and analyzed them with clinical data to explore the feasibility of using the spatial distribution characteristics of protein aggregates as a diagnostic standard for AD. However, the detection of red blood cell surface biomarkers based on atomic force microscopy still has certain problems in terms of accuracy or specificity: the concentration measurement method based on immunoadsorption is easily affected by the sample preparation process, and the morphology measurement using atomic force microscopy lacks biological specificity. On the other hand, although some people currently use single-molecule localization imaging technology to detect the characteristic protein density of multiple myeloma cells, the number of cells counted is small, and the detection parameters only use the characteristic protein density as an indicator to judge the progress of the disease. However, in single-molecule localization imaging, the quenching of the target protein during the experiment will affect the accuracy of the final density detection. In short, as the single-molecule localization imaging process continues, the fluorescent molecules in the bright state will be gradually bleached, and the fluorescent molecules collected by the camera will gradually decrease. That is, when the target protein content is low, the number of fluorescent molecules finally collected may not be enough to support the acquisition of super-resolution data or affect the quality of super-resolution data, which will affect the accuracy of subsequent algorithm density detection. In view of this, a super-resolution imaging system and biomarker detection method are urgently needed to solve the problems existing in the existing technology. Summary of the invention

[0007] In view of this, the present invention proposes a super-resolution imaging system and a biomarker detection method, aiming to solve the problems in the prior art that the blood biomarker sample processing process is complicated, is greatly affected by background fluorescence during image acquisition, has a narrow imaging field of view, and has low accuracy of biomarker detection results.

[0008] In one aspect, the present invention proposes a super-resolution imaging system, comprising: a laser beam combining unit, a laser illumination unit and a fluorescence signal detection unit; wherein the laser beam combining unit comprises: a first laser source, a second laser source, a first laser homogenization and collimation unit and a multimode optical fiber transmitter; the first laser source and the second laser source are arranged in parallel, the first laser source is used to excite fluorescent molecules, and the second laser source is used to activate fluorescent molecules; the first laser homogenization and collimation unit is located at the exit end of the first laser source, and is used to homogenize and collimate the light field of the laser emitted by the first laser source; the multimode optical fiber transmitter is arranged at the exit end of the first laser homogenization and collimation unit and the laser illumination unit, for combining, transmitting and collimating the two laser beams emitted by the first laser source and the second laser source, and for vibration isolation between the laser combining unit and the laser illumination unit; the multimode optical fiber transmitter has a small-core multimode optical fiber, and a first objective lens is provided at the inlet end of the small-core multimode optical fiber, for improving the efficiency of coupling the combined laser into the multimode optical fiber; the laser illumination unit is located at the exit end of the multimode optical fiber transmitter, for focusing the combined laser beam onto the biological sample; the fluorescence signal detection unit is located at the exit end of the laser illumination unit, for collecting the fluorescence signal emitted by the fluorescent molecules on the biological sample.

[0009] Furthermore, in the above-mentioned super-resolution imaging system, the first laser homogenization and collimation unit includes: a first lens, an engineering diffuser, and a second lens; wherein the first lens and the second lens are arranged between the output end of the first laser source and the incident end of the multi-mode optical fiber transmitter to collimate the laser emitted by the first laser source; and the engineering diffuser is arranged between the focal planes of the first lens and the second lens to homogenize the laser emitted by the first laser source.

[0010] Furthermore, in the above-mentioned super-resolution imaging system, the laser beam combining unit also includes: a first reflector, a second reflector, a third reflector, a fourth reflector, a first group of dichroic mirrors and a second group of dichroic mirrors; wherein, the first reflector and the second reflector are both tilted along a first preset angle and arranged between the output end of the first laser source and the input end of the first collimation and homogenization unit, and the first reflector and the second reflector are parallel to each other, the first group of dichroic mirrors are arranged between the output end of the first collimation and homogenization unit and the input end of the multi-mode optical fiber transmitter; and the first group of dichroic mirrors are arranged opposite to the second reflector; the third reflector and the fourth reflector are both tilted along a second preset angle and arranged at the output end of the second laser source, and the third reflector and the fourth reflector are parallel to each other; the second group of dichroic mirrors are arranged at the output end of the fourth reflector and opposite to the fourth reflector.

[0011] Furthermore, in the above-mentioned super-resolution imaging system, the multimode optical fiber transmitter further comprises: a second objective lens; wherein,

[0012] The second objective lens is arranged at the output end of the small-core multimode optical fiber to collimate the combined laser beam.

[0013] Furthermore, in the above-mentioned super-resolution imaging system, the laser illumination unit includes: a laser beam expansion unit and a focusing assembly; wherein the laser beam expansion unit is arranged at the output end of the laser beam combining unit to expand the collimated laser; the focusing assembly is arranged between the output end of the laser beam expansion unit and the input end of the fluorescence signal detection unit to focus the expanded laser onto the rear focal plane of the objective lens of the fluorescence signal detection unit; a fifth reflector and a sixth reflector are arranged between the laser beam expansion unit and the output end of the second objective lens.

[0014] Furthermore, the above-mentioned super-resolution imaging system also includes: a focus stabilization control system; wherein the focus stabilization control system is arranged on one side of the fluorescence signal detection unit, and is used to maintain the stability of the focus during the imaging process.

[0015] The present invention can further reduce the coherence of the illumination laser by utilizing the aliasing effect of different modes in a multimode optical fiber transmitter, thereby achieving uniform illumination; at the same time, because the multimode optical fiber transmitter can transmit higher-power lasers and can couple more laser spot modes, two laser beams can be focused on the rear focal plane of the objective lens in the fluorescence signal detection unit, which can meet the total internal reflection TIR illumination condition, can greatly improve the flux of biomarker detection in biological samples, and shorten the detection time; after the laser is homogenized and collimated by a first laser homogenization and collimation unit, it is then transmitted by the multimode optical fiber transmitter and expanded and focused by the illumination unit, so that the laser can irradiate the biological sample marked by the fluorescent probe at an accurate angle, and perform total internal reflection at the interface between the sample and the slide to generate an evanescent wave to illuminate the sample, excite the sample to generate a fluorescence signal, and then collect the fluorescence signal through the fluorescence signal detection unit for imaging, which is helpful to increase the field of view of super-resolution positioning imaging, achieve an imaging effect of a large field of view and uniform illumination, and greatly improve the processing throughput of biological sample images.

[0016] On the other hand, the present invention also proposes a biomarker detection method, comprising the following steps:

[0017] Fluorescently labeling specific biomarkers in the biological sample, and performing single-molecule fluorescence imaging on the biomarkers to obtain multiple frames of original microscopic fluorescence images;

[0018] The multiple frames of original microscopic fluorescence images are analyzed and processed to extract super-resolution distribution characteristic information of specific biomarkers on the surface of the biological sample.

[0019] Furthermore, in the above-mentioned biomarker detection method, the super-resolution distribution characteristic information of specific biomarkers on the surface of the biological sample includes: the density of the specific biomarker cluster, the area ratio of the specific biomarker on the surface of the biological sample, and the average value and distribution standard deviation of the nearest neighbor distance of the specific biomarker cluster.

[0020] Furthermore, in the above biomarker detection method, analyzing and processing the multiple frames of original microscopic fluorescence images includes:

[0021] By processing multiple frames of original microscopic fluorescence images, localization table data is obtained;

[0022] Rendering the positioning points in the positioning table data to generate a super-resolution image;

[0023] Performing a convolution operation on the super-resolution image to obtain a down-sampled image to demarcate the boundary of the biological sample, and calculating the total area of ​​the biological sample;

[0024] Perform image opening and convolution filtering on the super-resolution image that has not been downsampled to remove small noise;

[0025] Performing connected domain analysis on the image after the opening operation, segmenting the biomarkers in the image into isolated clusters, and performing statistics on all the segmented clusters to obtain the number and area of ​​all biomarker clusters and the center point of each cluster;

[0026] The density and area proportion of the biomarker clusters in the biological sample are determined using the total area of ​​the biological sample, the number of all biomarker clusters, and the area of ​​a single cluster, and the average value and distribution standard deviation of the nearest neighbor distance of the biomarker cluster are calculated using the center point of each cluster.

[0027] Furthermore, in the above biomarker detection method, the biological sample is a blood cell, and fluorescent labeling of the biological sample having a specific biomarker includes the following steps:

[0028] Extracting blood cells with specific biomarkers, diluting them, and then adding them to the treated confocal culture dish, and letting them stand for a period of time to allow the blood cells to adhere to the wall;

[0029] After adding a fixative solution to the confocal culture dish to fix the blood cells, the blood cells are blocked with a blocking solution;

[0030] Incubating the blood cells with a primary antibody to specifically recognize and bind to a specific biomarker of the blood cells;

[0031] The blood cells incubated with the primary antibody are incubated with a secondary antibody labeled with a fluorescent dye to complete the fluorescent labeling of the biological sample with a specific biomarker.

[0032] The present invention acquires multiple frames of original microscopic fluorescence images by collecting data from fluorescently labeled biological samples with specific biomarkers, and analyzes and processes the multiple frames of original microscopic fluorescence images to obtain localization table information and super-resolution images of specific biomarkers on the surface of the biological samples. Finally, the super-resolution distribution feature information of specific molecules on the surface of the biological samples is acquired through an algorithm, thereby greatly improving the accuracy of the detection results of the biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0034] Figure 1 Schematic diagram of the principle of super-resolution positioning imaging in an embodiment of the present invention;

[0035] Figure 2 An optical path diagram of a super-resolution positioning imaging system provided by an embodiment of the present invention;

[0036] Figure 3 An imaging field diagram based on total internal reflection illumination of a small-core multimode optical fiber in a super-resolution positioning imaging system provided in an embodiment of the present invention;

[0037] Figure 4 For the corresponding Figure 3 Schematic diagram of normalized light intensity at the middle dashed line;

[0038] Figure 5 A schematic diagram of a process for detecting AD biomarkers on the surface of red blood cells provided by an embodiment of the present invention;

[0039] Figure 6 It is a flow chart of clustering analysis of multiple frames of original microscopic fluorescence images in an embodiment of the present invention;

[0040] Figure 7 This is a schematic diagram of the noise signal filtering principle in an embodiment of the present invention;

[0041] Figure 8 This is a super-resolution image of Aβ protein on the surface of a single red blood cell in an embodiment of the present invention;

[0042] Fig. 9 A schematic diagram of dividing cell boundaries, removing extracellular signals, and obtaining cell areas in an embodiment of the present invention;

[0043] Fig.10 In the embodiment of the present invention, an image opening operation is performed on the super-resolution image that has not been downsampled, and the image before and after the small noise is removed;

[0044] Fig.11 This is a clustering effect diagram of the super-resolution data of Aβ protein on the surface of a single red blood cell after being processed by an algorithm in an embodiment of the present invention;

[0045] Fig.12 This is a nearest neighbor distance distribution diagram of Aβ protein on the surface of a single red blood cell membrane in an embodiment of the present invention;

[0046] Fig.13 The density statistics results of the gradient experiments of Aβ proteins on the surface of multiple red blood cell membranes in the embodiments of the present invention;

[0047] Fig.14 The nearest neighbor distance statistics result of the gradient experiment of Aβ protein on the surface of multiple red blood cell membranes in the embodiment of the present invention;

[0048] Fig.15 These are statistical results of protein cluster area percentages from multiple gradient experiments of Aβ protein on the surface of erythrocyte membranes in the embodiments of the present invention. DETAILED DESCRIPTION

[0049] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0050] Immunofluorescence (IF) is a labeling technology widely used in fluorescence microscopy. It uses antibodies to specifically identify and label target proteins in cells, thereby achieving in situ imaging. Single Molecule Localization Microscopy (SMLM) uses lasers to excite fluorescently labeled target proteins, and with the assistance of special imaging buffers, single fluorescent molecules of target proteins are randomly flashed. Single fluorescent molecules are captured by a high-precision microscope imaging system, and tens of thousands of original images of point flashes are collected to reduce the overlapping effect, thereby improving the resolution of imaging. Finally, the collected images are reconstructed through algorithms to obtain super-resolution images and corresponding localization tables; therefore, the spatial resolution of SMLM can reach 20 to 30 nm, which is much higher than that of ordinary fluorescence microscopes. Combined with immunofluorescence technology, it can clearly reveal the distribution of biomarkers on the cell surface, thereby achieving accurate density detection.

[0051] See also Figure 1 The basic principles of single-molecule localization super-resolution microscopy are as follows:

[0052] (1) Samples labeled with photoactivated fluorescent probes. Photoactivated fluorescent molecules are an important prerequisite for achieving super-resolution localization imaging. Photoactivated fluorescent molecules can switch between bright and dark states, which is called the photo-switching phenomenon. This means that each fluorescent molecule can be imaged in a time-sharing manner, showing spatially isolated, non-overlapping PSFs (point spread functions), avoiding the overlap of fluorescent molecule signals in the diffraction-limited region.

[0053] (2) Activate the fluorescent probe that marks the biological structure. Use a laser of a specific wavelength to activate the fluorescent molecules, so that only a small number of randomly sparsely distributed fluorescent molecules are converted to the bright state each time. Thereafter, use a laser of another wavelength to excite the fluorescent molecules to emit fluorescence. The fluorescent molecules switch between the bright state and the dark state, causing the fluorescent molecules to "blink".

[0054] (3) Detecting and locating the spatial position of single molecules. The detector is used to collect the fluorescence signal emitted by the fluorescent molecules in the bright state. Only a small number of single-molecule fluorescence signals are collected in each frame because the bright-state fluorescent molecules are randomly distributed in space. Since the single-molecule fluorescence signal is relatively weak and the imaging spot covers multiple pixels, the detector must have high sensitivity and high signal-to-noise ratio. At present, electron multiplying charge-coupled devices (EMCCD) and scientific-grade complementary metal oxide semiconductors (sCMOS) are commonly used for single-molecule fluorescence imaging. The PSF of the fluorescent molecule collected by the detector can be approximated as a two-dimensional Gaussian distribution. By fitting, the Gaussian distribution intensity spectrum of the horizontal and vertical positions of the molecule can be obtained, and its peak position is the center of mass position of the fluorescent molecule. It should be noted that the imaging throughput of SMLM does not only depend on the detector. The photophysical properties of the fluorophore (blinking rate) are also another important factor.

[0055] (4) Reconstruct super-resolution images. Repeat the above process in large numbers to obtain thousands or even tens of thousands of single-molecule images. Using enough single-molecule localization information in these single-molecule images for reconstruction, the final super-resolution image and localization table information can be obtained.

[0056] Based on this, the present invention proposes a super-resolution imaging system, which achieves an imaging effect with a large field of view and uniform illumination through the cooperation of various optical elements, improves the processing throughput of samples, and further improves the efficiency of biomarker detection and the accuracy of detection results.

[0057] Since the single-molecule localization imaging technology based on wide-field fluorescence illumination is easily accompanied by strong background fluorescence when imaging AD markers on the surface of red blood cells, it is not conducive to obtaining high-quality images, thereby reducing the detection capability. Single-molecule localization imaging based on total internal reflection (TIR) ​​illumination can suppress the out-of-focus background fluorescence during the imaging process, which is very suitable for studying the super-resolution distribution characteristics of AD biomarkers on the surface of red blood cells. However, the existing single-molecule localization imaging scheme based on TIR illumination is limited by the complex optical path (free-space laser transmission) or the small illumination field of view (the single-mode optical fiber used in conventional TIR illumination cannot transmit high-power lasers, which limits the illumination field of view), which is not conducive to the collection and statistical analysis of a large number of red blood cells. In view of this shortcoming, the present invention proposes a large-field single-molecule localization imaging system based on fiber-optic TIR.

[0058] System Example:

[0059] See also Figure 2The single-molecule localization imaging system of the embodiment of the present invention comprises: a laser beam combining unit 100, a laser illumination unit and a fluorescence signal detection unit 200; wherein the laser beam combining unit 100 comprises: a first laser source I1, a second laser source I2, a first laser homogenization and collimation unit 102 and a multimode optical fiber transmitter 101; wherein the first laser source I1 and the second laser source I2 are arranged in parallel, the first laser source I1 is used to excite fluorescent molecules, and the second laser source I2 is used to activate fluorescent molecules; the first laser collimation unit 102 is located at the exit end of the first laser source I1, and is used to homogenize and collimate the laser emitted by the first laser source I1; the multimode optical fiber transmitter 101 is arranged at the first laser homogenization and collimation unit 102; Between the output end of the collimation unit 102 and the laser illumination unit, it is used to combine, transmit and collimate the two laser beams emitted by the first laser source I1 and the second laser source I2, and to perform vibration isolation between the laser combining unit 100 and the laser illumination unit; the multimode optical fiber transmitter has a small-core multimode optical fiber, and the inlet end of the small-core multimode optical fiber is provided with a first objective lens to improve the efficiency of coupling the combined laser into the multimode optical fiber; the laser illumination unit is located at the output end of the multimode optical fiber transmitter 101, and is used to focus the combined laser onto the biological sample; the fluorescence signal detection unit 200 is located at the output end of the laser illumination unit, and is used to collect the fluorescence signal emitted by the fluorescent molecules on the biological sample.

[0060] Specifically, the first laser source I1 emits laser light with a wavelength of 640 nm to excite fluorescent molecules.

[0061] The second laser source I2 emits a laser with a wavelength of 405 nm. Since the fluorescent molecules are easily quenched when the first laser source I1 collects the target protein when there is less target protein, the laser emitted by the second laser source I2 can be used to activate the quenched fluorescent molecules.

[0062] In this embodiment, the combined laser beam refers to the laser beam obtained by combining the laser beam emitted by the first laser source and the laser beam emitted by the second laser source.

[0063] The multimode optical fiber transmitter 101 comprises: a first objective lens OBJ1, a small-core multimode optical fiber MMF and a second objective lens OBJ2; wherein the first objective lens OBJ1 is located at the incident end of the small-core multimode optical fiber MMF to improve the efficiency of coupling the combined laser into the small-core multimode optical fiber MMF; the second objective lens OBJ2 is arranged at the exit end of the small-core multimode optical fiber MMF to collimate the combined laser.

[0064] In this embodiment, the laser beam is coupled into the small-core multimode optical fiber MMF through the first objective lens OBJ1 (10X objective lens). Since the diameter of the focused spot after passing through the first objective lens OBJ1 is about 10 μm, which is smaller than the core diameter of the multimode optical fiber (the core diameter is 50 μm), the laser transmission efficiency at the illumination end can be improved, and the excitation light power density requirements for single-molecule localization imaging can be better met.

[0065] In order to ensure that the light beam output from the multimode optical fiber transmitter can be well collected, the second objective lens OBJ2 (10X objective lens) with a numerical aperture of NA0.25 is selected to collimate it, and the collimation effect is better than other lenses.

[0066] In this embodiment, the core diameter of the small-core multimode optical fiber (MMF) in the multimode optical fiber transmitter 101 is preferably 30-100 μm. For example, a multimode optical fiber (MMF) with a core diameter of 50 μm can be selected to achieve total internal reflection (TIR) ​​illumination, thereby suppressing off-focus fluorescence background and improving the quality of microscopic images. In addition, the use of the first objective lens OBJ1 in the multimode optical fiber transmitter 101 can greatly improve the laser transmission efficiency (the full-process transmission efficiency can reach more than 40%) and the laser illumination power, thereby achieving large-field illumination.

[0067] In addition, the multimode optical fiber transmitter 101 is arranged between the laser beam combining unit 100 and the laser illumination unit, which is conducive to placing the laser beam combining unit 100 and the laser illumination unit on two different optical platforms. The vibration generated when the laser is used will not affect the imaging optical path, thereby not affecting the single-molecule fluorescence imaging.

[0068] In the prior art, TIR illumination using single-mode fiber (SMF) is not conducive to achieving large-field single-molecule localization imaging because of its small core diameter, the laser power that can be transmitted is in the order of hundreds of milliwatts, and the spot shape of the laser is very demanding. Compared with single-mode fiber (SMF), multimode fiber (MMF) has a larger core diameter (generally 30-400μm), can transmit higher-power lasers, and can couple more laser spot modes. Studies have shown that focusing the laser from a small-core-diameter (less than 100μm) multimode fiber (MMF) on the back focal plane of the objective lens can meet the TIR illumination conditions, and the imaging field of view under this configuration can be as high as 200μm, thereby covering dozens of blood cells and improving the imaging flux. Therefore, replacing the single-mode fiber (SMF) in the conventional single-molecule localization imaging system with a small-core-diameter multimode fiber (MMF) to build a large-field single-molecule localization imaging system based on TIR illumination can greatly improve the flux of blood cell biomarker detection and shorten the detection time.

[0069] The laser illumination unit includes: a laser beam expansion unit and a focusing component L5; wherein the laser beam expansion unit is arranged at the output end of the laser beam combining unit 100, for expanding the laser after being collimated; the focusing component L5 is arranged between the output end of the laser beam expansion unit and the input end of the fluorescent signal detection unit 200, for focusing the expanded laser onto the rear focal plane of the objective lens of the fluorescent signal detection unit 200; a fifth reflector M5 and a sixth reflector M6 are arranged between the laser beam expansion unit and the second objective lens OBJ2. The focusing component L5 can be a focusing lens.

[0070] Specifically, the combined laser beam after being collimated by the second objective lens OBJ2 is expanded by a laser beam expansion unit. The laser beam expansion unit includes: an excitation filter EX, a third lens L3 and a fourth lens L4; wherein an excitation filter EX is arranged between the third lens L3 and the fourth lens L4 to purify the combined laser beam in the optical path; an aperture stop is arranged at the exit end of the fourth lens L4 to eliminate stray light.

[0071] More specifically, the excitation filter EX model is FF01-390 / 482 / 532 / 640-25, Semrock; the third lens L3 model is AC254-060-A, Thorlabs; the fourth lens L4 model is AC508-150-A, Thorlabs. An aperture diaphragm (ID50, Thorlabs) is placed after the fourth lens L4 to eliminate stray light, suppress the background, and improve the signal-to-noise ratio of the image. In this embodiment, a seventh reflector M7 is also provided at the output end of the fourth lens L4.

[0072] In this embodiment, the fluorescence signal detection unit 200 may include: a third objective lens OBJ3, a fourth group of dichroic mirrors DM3, a notch filter NF, an emission filter EM, a tube lens TL and a camera C; wherein the fourth group of dichroic mirrors DM3, the notch filter NF and the emission filter EM are arranged between the exit end of the third objective lens OBJ3 and the lens of the camera C.

[0073] More specifically, the camera C is used in conjunction with the third objective lens OBJ3 to capture and record the fluorescence signal. The model of the third objective lens may be UPL APO 60X, Olympus. The model of the fourth dichroic mirror DM3 is ZT405 / 488 / 532 / 640rpc-XT, the model of the Chroma notch filter NF is NF03-405 / 488 / 532 / 635E-25, Semrock; the model of the emission filter EM is ET705 / 100, Chroma; the camera C is preferably a camera with higher sensitivity, and in this embodiment, a sCMOS camera (Dhyana 400BSI V2, Tucsen Photonics) is selected.

[0074] It is obvious from the above that: the embodiment of the present invention utilizes the aliasing effect of different modes in the multimode optical fiber transmitter to further reduce the coherence of the illumination laser, thereby achieving uniform illumination; at the same time, because the multimode optical fiber transmitter can transmit higher power lasers and can couple more laser spot modes, two laser beams can be focused on the rear focal plane of the objective lens in the fluorescence signal detection unit, which can meet the total internal reflection TIR illumination condition, and can greatly improve the throughput of biomarker detection in biological samples and shorten the detection time; after the laser is homogenized and collimated by the first laser homogenization and collimation unit, it is then transmitted by the multimode optical fiber transmitter and expanded and focused by the illumination unit, so that the laser can irradiate the biological sample marked with the fluorescent probe at an accurate angle, and perform total internal reflection at the interface between the sample and the slide to generate an evanescent wave to illuminate the sample, excite the sample to generate a fluorescence signal, and then collect the fluorescence signal through the fluorescence signal detection unit for imaging, which helps to increase the field of view of super-resolution positioning imaging, achieve an imaging effect of a large field of view and uniform illumination, and greatly improve the processing throughput of biological sample images.

[0075] In the above embodiment, the first laser homogenization and collimation unit 102 includes: a first lens L1, an engineered diffuser ED1 and a second lens L2; ​​wherein the first lens L1 and the second lens L2 are arranged between the output end of the first laser source I1 and the incident end of the multimode optical fiber transmitter 101, so as to collimate the laser emitted by the first laser source I2; the engineered diffuser ED1 is arranged between the focal planes of the first lens L1 and the second lens L2, so as to homogenize the laser emitted by the first laser source I1.

[0076] Specifically, the first lens L1 and the second lens L2 may be a pair of achromatic doublet lenses with a focal length of 30 mm.

[0077] In this embodiment, since the laser is projected onto the sample end, the coherence of the laser will generate random interference laser speckle, which is not conducive to single-molecule fluorescence imaging. By providing an engineered diffuser ED1 between the focal planes of the first lens L1 and the second lens L2, the transmission angle of different light rays in the light beam can be effectively changed to change the direction of the incident light beam and destroy the coherence of the laser; by providing high transmittance, controlling the divergence angle, the spatial distribution of light and the intensity profile of the diffused light, the uniformity of the field of view is improved to provide better conditions for subsequent quantitative analysis. However, the engineered diffuser ED1 has the disadvantage of high divergence, so this problem is solved by placing it on the focal planes of the first lens L1 (focusing lens) and the second lens L2 (collimating lens), thereby improving the transmission efficiency of the laser. In this embodiment, the random speckle generated by laser illumination can be eliminated by using the engineered diffuser ED1 and the multimode optical fiber transmitter, so as to achieve the purpose of homogenizing the illumination field of view.

[0078] In this embodiment, the fluorescence signal detection unit 200 may include: a third objective lens OBJ3, a fourth group of dichroic mirrors DM3, a notch filter NF, an emission filter EM, a tube lens TL and a camera C; wherein the fourth group of dichroic mirrors DM3, the notch filter NF and the emission filter EM are arranged between the exit end of the third objective lens OBJ3 and the lens of the camera C.

[0079] More specifically, the camera C is used in conjunction with the third objective lens OBJ3 to capture and record the fluorescence signal. The model of the third objective lens OBJ3 is UPL APO 60X, Olympus; the model of the fourth dichroic mirror DM3 is ZT405 / 488 / 532 / 640rpc-XT; the model of the notch filter NF is NF03-405 / 488 / 532 / 635E-25, Semrock; the model of the emission filter EM is ET705 / 100, Chroma; the camera C is preferably a camera with higher sensitivity, and in this embodiment, a sCMOS camera (Dhyana 400BSI V2, Tucsen Photonics) is selected.

[0080] In the above embodiment, the laser beam combining unit 100 further includes: a first reflector M1, a second reflector M2, a third reflector M3, a fourth reflector M4, a first group of dichroic mirrors DM2, and a second group of dichroic mirrors DM1; wherein the first reflector M1 and the second reflector M2 are both tilted along a first preset angle and arranged between the output end of the first laser source I1 and the incident end of the first collimation and homogenization unit 102, and the first reflector M1 and the second reflector M2 are parallel to each other, the first group of dichroic mirrors DM2 is arranged between the output end of the second reflector M2 and the incident end of the multimode optical fiber transmitter 101, and the first group of dichroic mirrors DM2 and the second reflector M2 are arranged opposite to each other;

[0081] The third reflector M3 and the fourth reflector M4 are both arranged at the exit end of the second laser source I2 at an inclined angle along a second preset angle; the third reflector M3 and the fourth reflector M4 are parallel to each other, and the second group of dichroic mirrors DM1 is arranged at the exit end of the fourth reflector M4 and opposite to the fourth reflector M4.

[0082] Specifically, the first group of dichroic mirrors DM2 is tilted in a direction opposite to the first preset angle, and the second group of dichroic mirrors DM1 is tilted in a direction opposite to the second preset angle. The first preset angle and the second preset angle may both be 45°.

[0083] In long-term single-molecule localization imaging, in order to prevent focus shift due to factors such as sample drift and temperature change (focus shift will cause image blur and resolution reduction), this embodiment may also include: a focus control system 300; wherein the focus control system 300 is arranged on one side of the fluorescence signal detection unit 200, and is used to maintain the stability of the focus during the imaging process, thereby preventing focus shift due to factors such as sample drift and temperature change. This is particularly important for long-term super-resolution imaging, because focus shift will cause image blur and resolution reduction.

[0084] See also Figure 2 In this embodiment, the quadrant detector QD (Quadrant Detector), Prism is a right-angle prism reflector, the bandpass filter F1, 850nm infrared light, the ninth reflector M9, the tenth reflector M10 and the third group of dichroic mirrors DM4 constitute a focus control system 300 to ensure that the focus is always at the optimal position.

[0085] Specifically, QD's data refresh rate reaches 100HZ and can support a stable focus frequency of at least 50HZ.

[0086] In the above embodiments, the third group of dichroic mirrors DM4 is arranged at the exit end of the focusing assembly L5, and the fourth group of dichroic mirrors DM3 is arranged on the optical path between the third group of dichroic mirrors DM4 and the third objective lens OBJ3.

[0087] Specifically, the model of the third dichroic mirror DM4 can be ZT405 / 488 / 532 / 640rpc-XT, Chroma.

[0088] The fourth group of dichroic mirrors DM3 can guide the light beam after the first laser source I1, the second laser source I2 and the 850nm infrared light in the focus control system 300 into the third objective lens OBJ3 of the fluorescence signal detection unit 200; the third group of dichroic mirrors DM4 can combine the 850nm infrared light into the system optical path.

[0089] The laser beam is focused onto the back focal plane BFP of the third objective lens OBJ3 through the focusing assembly L5, the third dichroic mirror group DM4 and the fourth dichroic mirror group DM3.

[0090] In specific implementation, the laser beam passes through the third group of dichroic mirrors DM4 to enter the optical path of the imaging system, and then passes through the fourth group of dichroic mirrors DM3 and the third objective lens OBJ3 to be transmitted to the biological sample. The light reflected from the biological sample then passes through the third objective lens OBJ3, the fourth group of dichroic mirrors DM3 and the third group of dichroic mirrors DM4 in sequence to return to the focus control system 300. The focus control system 300 determines the focal plane by the change in the position of the light, and then makes adjustments based on the change in the focal plane to ensure the stability of the focus.

[0091] In this embodiment, an illumination mode switching unit is provided on the optical path between the fourth lens L4 and the focusing assembly L5, which is used to switch between total internal reflection illumination (TIRF) and epi-illumination (EPI) and two fluorescence imaging modes, so as to obtain more comprehensive information of biological samples. The illumination mode switching unit is a Stage module (total internal reflection fluorescence and epi-fluorescence modules can be switched). An eighth reflector M8 and a focusing lens L5 are provided in the illumination mode switching unit. In this embodiment, the fifth reflector M5 and the sixth reflector M6 are arranged at a third preset angle, and the seventh reflector M7 and the eighth reflector M8 are arranged at a fourth preset angle. The third preset angle and the fourth preset angle can both be 45°.

[0092] The optical path flow of the imaging system provided by the embodiment of the present invention is described in detail below:

[0093] First, use the first reflector M1 and the second reflector M2 to adjust the transmission direction of the 640nm laser emitted by the first laser source I1. L1 and L2 are achromatic doublet lenses with a focal length of 30mm. Use the third reflector M3, the fourth reflector M4 and the second dichroic mirror DM1 to adjust the transmission direction of the 405nm laser emitted by the second laser source I2. 405nm is the activation light, which does not require high spot quality, and does not require the use of an engineered diffuser for spot homogenization. The laser with a wavelength of 405nm is reflected by the first dichroic mirror DM2, and the laser with a wavelength of 640nm is transmitted.

[0094] Multimode fiber optic transmitters are prone to laser speckle. The embodiment of the present invention uses an engineered diffuser ED1 to improve the uniformity of the field of view and ensure better quantitative analysis. The engineered diffuser ED1 has the disadvantage of high divergence, which can be solved by placing it on the focal plane of the first lens L1 (focusing lens) and the second lens L2 (collimating lens) to improve the laser coupling efficiency.

[0095] After the two laser beams are collimated, they are coupled into the multimode fiber optic transmitter through the first objective lens OBJ1. In order to ensure that the light beam output from the multimode fiber optic transmitter is well collected, the second objective lens OBJ2 with a larger NA is selected for collimation after the light beam is output from the multimode fiber optic transmitter. The combined laser beam after collimation by the second objective lens OBJ2 is expanded and purified by the third lens L3, the excitation filter EX and the fourth lens L4 of the laser beam expansion unit. In order to suppress the fluorescence background and improve the signal-to-noise ratio of the image, an aperture stop Iris3 (ID50, Thorlabs) is placed after the fourth lens L4 to eliminate stray light.

[0096] The beam after expansion is focused onto the back focal plane of the third objective lens OBJ3 in the fluorescence signal detection unit through the focusing assembly L5, the third dichroic mirror DM4 and the fourth dichroic mirror DM3. Finally, the laser beam is irradiated onto the fluorescently labeled biological sample through the third objective lens OBJ3 in the fluorescence signal detection unit, excites the fluorescent molecules and collects the fluorescence signal, and then the notch filter NF filters out the excitation light, and the emission filter EM purifies the dye fluorescence to ensure the high transmittance of the fluorescence, and the fluorescence signal is focused onto the sCMOS camera C through the tube lens TL.

[0097] Combination Figure 3 and Figure 4 The super-resolution imaging system based on the single-molecule localization method in the embodiment of the present invention was tested and it was found that the power density of the 640nm laser at the sample end was 6.06kW / cm 2 , fully meeting the requirements of single-molecule localization imaging for sample-side power density; and, when the imaging field of view diameter is as high as 100μm, it can still provide sufficiently high illumination intensity and good illumination uniformity.

[0098] In summary, the large-field single-molecule localization imaging system based on fiber-optic total internal reflection illumination constructed by the present invention has the characteristics of large field of view, low background and high signal-to-noise ratio. Large-field TIRF illumination is conducive to obtaining a large amount of biological sample information at a single time, improving the single imaging flux, thereby obtaining more super-resolution imaging data of blood cell biomarkers in a shorter time, and providing better technical support for analyzing the biomarker distribution characteristics of a large number of blood cells (100 red blood cells and more).

[0099] Method Example:

[0100] See also Figure 5-Figure 15 The present invention also provides a method for processing an image of a biological sample having a specific biomarker, comprising the following steps:

[0101] Step S1, fluorescently labeling specific biomarkers in a biological sample, and performing single-molecule fluorescence imaging on the biomarkers to obtain multiple frames of original microscopic fluorescence images.

[0102] Specifically, in this embodiment, 100,000 frames of original microscopic fluorescence images are obtained. The biological sample is a blood cell. For example, it can be red blood cells, white blood cells and platelets. Compared with the detection method in the prior art in mixed solutions such as serum and plasma, the detection method based on blood cells can reduce the interference of samples without signals, thereby improving the detection sensitivity. At the same time, the super-resolution imaging system based on the large-field single-molecule localization method is used to detect multiple (100 or more) blood cells of the same batch of blood samples, which can effectively eliminate the influence of individual cell differences on the detection results, and simplify the sample processing process.

[0103] In this embodiment, the biological sample with a specific biomarker is preferably a blood cell sample. The detector captures the fluorescence signal of the blood cell sample enriched with the marker to generate a single frame image, and the above steps are repeated to generate an original microscopic fluorescence image containing multiple images. The fluorescent molecule signal in each original microscopic fluorescence image is extracted and processed to generate an entry in the positioning table data.

[0104] Recombination Figure 5 , fluorescent labeling of biological samples with specific biomarkers includes the following steps:

[0105] Step S11, extracting blood cells with specific biomarkers, diluting them, and then adding them to the pre-treated confocal culture dish, and letting them stand for a period of time to allow the blood cells to adhere to the wall.

[0106] The specific implementation of this step may include:

[0107] (1) Prepare biomarker solutions of different concentrations.

[0108] Among them, the biomarker is preferably Aβ protein, which has good biological specificity.

[0109] In specific implementation, this step includes the following sub-steps:

[0110] a. Dissolve Aβ1-42 powder in pre-cooled hexafluoroisopropanol (HFIP) solution to a final concentration of 1 mM and incubate at room temperature for about 1 hour to allow the Aβ1-42 powder to fully dissolve.

[0111] b. Blow nitrogen for about 10 minutes to volatilize HFIP to obtain Aβ1-42 film, and then use a small amount of dimethylsulfoxide (DMSO) to dissolve the Aβ1-42 film.

[0112] c. Add hydroxyethylpiperazine ethanesulfonic acid (HEPES) to adjust to the required concentration. A small amount of crystals can be dissolved by ultrasound. Place in a 4°C refrigerator for 24 hours and then divide into smaller portions and store in a -80°C refrigerator.

[0113] (2) Extracting blood cells, incubating the blood cells with biomarker solutions of different concentrations for a period of time, so that the biomarkers are enriched on the surface of the blood cells. In this embodiment, the blood cells are preferably red blood cells.

[0114] In specific implementation, this step includes the following sub-steps:

[0115] a. Extraction of red blood cells: Take 4 μL of fresh whole blood from the fingertips and mix it with 6 mL of PBS-GB solution. Remove impurities in the blood by centrifugation and remove the supernatant to obtain red blood cell concentrate.

[0116] Specifically, this step uses peripheral blood from healthy donors to simulate red blood cell samples from AD patients.

[0117] b. Simulating red blood cells from AD patients: Incubate red blood cells with Aβ protein solutions of different concentrations for about 24 hours to allow Aβ protein to accumulate on the surface of red blood cells. Then remove the supernatant to obtain the incubated red blood cells. Wash them twice with incubation solution without Aβ protein, and centrifuge to remove unbound Aβ protein. The concentrated red blood cell solution obtained after incubation is the red blood cells simulating AD patients.

[0118] (3) After the confocal culture dish is treated, the blood cells with specific biomarkers are diluted and added to the confocal culture dish, and left to stand for a period of time to allow the red blood cells to adhere to the wall.

[0119] In the specific implementation, a confocal culture dish with good light transmittance (Mat Tek, P35G-1.5-14-C) was treated with 0.1% poly-lysine to facilitate subsequent experiments; the red blood cells of simulated AD patients were diluted to the required concentration with DMEM basal culture medium, and 400 μL of the diluted red blood cell suspension was added to the confocal culture dish, and incubated for 30 minutes to allow the red blood cells to adhere to the wall, which can achieve a better labeling effect.

[0120] Since the sample will produce an offset during the imaging process, the offset of the fluorescent microsphere is consistent with the offset of the target protein on the cell. The fluorescent microsphere will not flash in the imaging state, while the target protein on the cell flashes as a single molecule. The offset of the cell can be corrected by positioning the fluorescent microsphere. In this embodiment, before this step, fluorescent microspheres are added to the confocal culture dish. The diameter of the fluorescent microsphere can be 100nm.

[0121] The specific implementation steps are as follows: add the diluted fluorescent microsphere solution to the confocal culture dish treated with poly-lysine, dry it in an oven to make the fluorescent microspheres adhere to the confocal culture dish, and then add the biological sample for incubation and immunofluorescence labeling.

[0122] Step S12, after adding a fixative solution into the confocal culture dish for fixation, the blood cells are blocked with a blocking solution.

[0123] In the specific implementation, the excess incubation solution in the confocal culture dish is removed, a fixative solution is added for 15 minutes, and then washed with PBS phosphate buffer to remove the excess fixative solution. After washing, a blocking solution is added to block the red blood cells.

[0124] Step S13, incubating the red blood cells with a primary antibody to specifically recognize and bind to the Aβ protein on the blood cells.

[0125] In specific implementation, the primary antibody was diluted with 3% BSA, 200 μL of the primary antibody was added to the wells of the dish and incubated for 2 hours, and then washed with PBS phosphate buffer to remove excess primary antibody.

[0126] Step S14, incubate the blood cells incubated with the primary antibody with a secondary antibody labeled with a fluorescent dye to complete the fluorescent labeling of the biological sample with a specific biomarker.

[0127] In specific implementation, the secondary antibody was diluted with 3% BSA, 200 μL of the secondary antibody was added to the wells of the dish and incubated for about 40 minutes in the dark, and then washed with PBS phosphate buffer to remove excess secondary antibody.

[0128] In this embodiment, the primary antibody is incubated to locate the target protein, and the secondary antibody binds to the primary antibody to a certain extent to amplify the fluorescence signal. After fluorescent labeling, the sample in the confocal culture dish can be directly added to the imaging buffer for single-molecule localization imaging, or it can be temporarily stored in a 4°C refrigerator.

[0129] The above steps S11-S14 are used for the preparation and fluorescence labeling of the model blood cell sample. To perform fluorescence labeling on a real blood sample from a patient, only steps (3)-S14 are required.

[0130] It can be seen that the present invention only needs to perform characteristic measurement and analysis on the biomarkers on the surface of blood cells to complete high-sensitivity, high-accuracy and high-specificity detection of biomarkers on the surface of blood cells, thereby reducing the impact of differences in experimental operation processes on the measurement results and greatly reducing the error pressure of experimenters; the sample volume requirement is small, and only a few microliters of blood are needed to meet the experimental needs; other substances in the blood have little effect on the results of this experiment.

[0131] Step S2, analyzing and processing the multiple frames of original microscopic fluorescence images, so as to extract super-resolution distribution characteristic information of specific biomarkers on the surface of the biological sample.

[0132] Specifically, the super-resolution distribution characteristics of the specific biomarkers on the surface of the biological sample include: the density of the specific biomarker cluster, the area ratio of the specific biomarker on the surface of the biological sample, and the average value and distribution standard deviation of the nearest neighbor distance of the specific biomarker cluster. That is, the test results are no longer presented in the form of concentration, but present the super-resolution distribution characteristics of the biomarkers on the surface of the blood cells related to the detection of the disease (the density of the specific biomarker cluster, the area ratio of the specific biomarker on the surface of the biological sample, and the average value and distribution standard deviation of the nearest neighbor distance of the specific biomarker cluster), and the characteristics of the super-resolution distribution characteristics can be quantified. The biomarker features in this embodiment include: the density of the specific biomarker cluster, the area ratio of the specific biomarker on the surface of the biological sample, and the average value and distribution standard deviation of the nearest neighbor distance of the specific biomarker cluster.

[0133] A series of original microscopic fluorescence images are obtained by multiple exposures of biological samples with specific biomarkers through a super-resolution imaging system. Only a small number of fluorescent molecules are activated each time to ensure that the fluorescence signal is sparsely distributed.

[0134] Combination Figure 6 In this embodiment, the analysis and processing of the multiple frames of original microscopic images includes:

[0135] (1) By processing multiple frames of original microscopic fluorescence images, the position of each fluorescent molecule and related information are determined to obtain positioning table data.

[0136] Specifically, the single-molecule localization algorithm is used to process the original image to obtain the localization table data; the localization table includes 12 columns of information (such as Table 1), which includes the peak signal intensity (Peak Intensity, PI) and signal-to-noise ratio (Signal-Nosie Ratio, SNR) of the localization point in addition to the coordinate information.

[0137] In a specific implementation of this embodiment, the positioning algorithm used is QC-STORM (QC is the abbreviation of Qualitycontrol), which is based on GPU-accelerated image preprocessing and molecular recognition, maximum likelihood estimation positioning, super-resolution image rendering and statistical information analysis method. Its function is to calculate the signal point position, half-peak width and other information in the positioning microscopic image to form a positioning table.

[0138] See also Figure 7 In this embodiment, the coordinate relationship between the positioning points in the positioning table can be used to delete the noise signal. If no other coordinate point is found within rnm (in this experiment, the r value range is 2.5-20nm, preferably 10nm, and the r value can be adjusted according to the molecular point density) near a positioning point, the signal point is deleted.

[0139] Table 1

[0140]

[0141]

[0142] (2) Combination Figure 8 , rendering the positioning points in the positioning table data to generate a super-resolution image.

[0143] In this embodiment, the positioning point refers to the one obtained by the single-molecule positioning algorithm, and each positioning point contains information such as the precise position (x, y) and intensity of the fluorescent molecule.

[0144] In the specific implementation, the positioning points are rendered using the Gaussian model to obtain a super-resolution image with a pixel size of 5nm. The pixel size of the generated super-resolution image is 5nm, which means that each pixel represents an area of ​​5nm×5nm.

[0145] (3) Combination Fig. 9 , performing a convolution operation on the super-resolution image to obtain a down-sampled image to demarcate the boundary of the biological sample, and calculating the total area of ​​the biological sample.

[0146] In specific implementation, the super-resolution image obtained in the previous step is convolved with a Gaussian kernel of size 100 pixels to obtain a downsampled image to demarcate the cell boundaries; the extracellular signals are deleted according to the cell boundaries, and the cell area is calculated. The pixel size of the image is selected according to the density of the biomarker clusters on the cells.

[0147] (4) Combination Fig.10 , image opening and convolution filtering are performed on the super-resolution image that has not been downsampled to remove small noise.

[0148] In specific implementation, a disk structure with a size of 5 pixels (determined according to the size of the cluster density) is used to perform an image opening operation on the super-resolution image that has not been downsampled, so as to further remove noise points smaller than 5 pixels in the image.

[0149] Using a 5-pixel disk structure for opening operation can effectively remove small noise points with a diameter less than 5 pixels in the image.

[0150] Downsampling: Through convolution operation, the resolution of the image is reduced, but the main structural features are retained, which facilitates the subsequent cell boundary division.

[0151] (5) Combination Fig.11 , performing connected domain analysis on the image after the opening operation, segmenting the biomarkers in the image into isolated clusters, and performing statistics on all the segmented clusters to obtain the number, area and center point of all biomarker clusters.

[0152] Specifically, the cluster here may be a protein cluster, the biological sample may be a cell, and the protein cluster density is calculated according to protein cluster density = number of protein clusters / cell area.

[0153] (6) Combination Figure 11 to Figure 12 The total area of ​​the biological sample and the number and area of ​​all biomarker clusters are used to determine the density and area proportion of the biomarker clusters in the biological sample, and the center point of each cluster is used to calculate the average value and distribution standard deviation of the nearest neighbor distance of the biomarker cluster.

[0154] Existing clustering algorithms developed for single-molecule localization microscopy, such as DBSCAN, ClusterViSu, and FACAM, are all based on point cloud data in the calibration table. They only use the position coordinate information of the positioning points, and do not consider information such as the intensity of the positioning points. However, biomarkers on the cell surface are distributed in isolated clusters, so their super-resolution imaging results also correspond to isolated cluster distributions, and clusters can be classified based on this image information. In addition, the rendering and reconstruction process of super-resolution images uses multi-dimensional information such as coordinates, number of photons, and positioning accuracy in the positioning table. Therefore, compared with clustering methods that only use positioning point coordinate information, clustering methods based on super-resolution images can analyze higher density data.

[0155] See also Figure 5 , Figures 13 to 15 The blank group was directly incubated with healthy red blood cells using DMEM basal culture medium. The experimental group was diluted with the prepared Aβ protein solution to different concentrations using DMEM basal culture medium, and then incubated with healthy red blood cells at 37°C for about 24 hours to enrich the Aβ protein on the surface of the red blood cells. The incubation solution was then discarded, and the incubated red blood cells were taken for immunofluorescence labeling experiments. Super-resolution imaging was then performed to obtain super-resolution data. Finally, the super-resolution distribution characteristics of the Aβ protein on the surface of the red blood cells were analyzed by an algorithm. Super-resolution data of 100 red blood cells were randomly selected from each group of samples, and the size, density, nearest neighbor distance and area proportion of the protein cluster of the Aβ protein on the surface of each red blood cell were calculated using the clustering algorithm in this embodiment. The statistical results of the density, nearest neighbor distance and area proportion of the obtained protein clusters are shown in FIG. Figures 13 to 15 As shown in the figure, it can be seen that when the red blood cells are incubated with Aβ protein solutions of different concentrations, as the incubation concentration increases, the density and area ratio of the Aβ protein clusters on the red blood cell surface also increase, and the nearest neighbor distance of the Aβ protein clusters on the red blood cell surface also decreases to a certain extent, proving that the detection method provided in the embodiment of the present invention can effectively and accurately detect the density of the Aβ protein clusters on the red blood cell surface, the area ratio of the Aβ protein clusters, and the average value and distribution standard deviation of the nearest neighbor distance of the Aβ protein clusters.

[0156] It can be seen that the clustering algorithm of the present invention can process super-resolution images with high-density biomarkers, provide detection parameters with better value and quantify image features: in addition to detecting the cluster density of biomarkers on disease-related cells, it also adds statistical analysis of the nearest neighbor distance and area ratio of biomarkers. These quantitative parameters greatly reduce the impact of target protein quenching on density detection during imaging, thereby greatly improving the accuracy of the detection results.

[0157] In this embodiment, the process of extracting biomarker features from super-resolution images is as follows:

[0158] The super-resolution data is rendered into a super-resolution image through the data rendering module, and then the cells to be analyzed in the image are extracted using the cell segmentation module. The isolated signal clusters are then separated through the image filtering and segmentation module. Finally, the area and the nearest neighbor distance of the signal cluster are counted using the data statistics module to obtain the clustering result (i.e., the super-resolution distribution characteristic information of the target protein).

[0159] It can be clearly concluded from the above that this embodiment acquires data of a biological sample that has been fluorescently labeled with a specific biomarker to obtain multiple frames of original microscopic fluorescence images, and analyzes and processes the multiple frames of original microscopic fluorescence images, thereby obtaining the localization table information and super-resolution images of the specific biomarkers on the surface of the biological sample, and finally obtains the super-resolution distribution feature information of the specific molecules on the surface of the biological sample through an algorithm, thereby greatly improving the accuracy of the biomarker detection results.

[0160] The related parts between the method embodiment and the above-mentioned system embodiment can be referred to each other and will not be described in detail here.

[0161] See again Figure 5 The following is a detailed description of the process of collecting and analyzing super-resolution data in the present invention using a specific embodiment: (1) Turn on the super-resolution positioning imaging system and the focus control system, then adjust the system's lighting mode to the total internal reflection (TIR) ​​state, and drop lens oil on the objective lens to select a folder. (2) Place the prepared biological sample on the stage, and ensure that the placement position is within the range that the objective lens can move to; fix the sample and do not move it at will during the process. (3) Immerse the biological sample in the imaging buffer, find the focal plane of the sample in the bright field or low laser power state, and select the ROI area (the ROI area should contain target cells and fluorescent microspheres). The ROI area refers to the area in the image that requires special attention and processing, containing structures or features of interest. (4) Adjust the focus system and imaging system, increase the laser power, make the target protein flash randomly with the assistance of the imaging buffer, and collect tens of thousands of single-molecule fluorescence images. (5) Perform positioning processing on the collected single-molecule fluorescence images to obtain positioning table data (such as coordinate position, positioning accuracy and intensity, etc.), and then generate a grayscale image through image rendering. (6) The lateral offset generated during cell imaging is corrected using the location information of the fluorescent microspheres to obtain the corrected location table data. (7) Finally, the corrected location table data is analyzed using a clustering algorithm to obtain the super-resolution distribution characteristics of Aβ protein on the surface of red blood cells (protein cluster size, density, nearest neighbor distance and area ratio).

[0162] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A super-resolution imaging system, characterized in that: include: Laser beam combining unit, laser illumination unit and fluorescence signal detection unit; wherein, The laser beam combining unit comprises: a first laser source, a second laser source, a first laser homogenization and collimation unit and a multimode optical fiber transmitter; The first laser source and the second laser source are arranged in parallel, the first laser source is used to excite fluorescent molecules, and the second laser source is used to activate fluorescent molecules; the first laser homogenization and collimation unit is located at the output end of the first laser source, and is used to homogenize and collimate the light field of the laser emitted by the first laser source; The multimode optical fiber transmitter is arranged between the output end of the first laser homogenization and collimation unit and the laser illumination unit, and is used to combine, transmit and collimate the two laser beams emitted by the first laser source and the second laser source, and to perform vibration isolation between the laser combining unit and the laser illumination unit; The multimode optical fiber transmitter has a small core diameter multimode optical fiber, and a first objective lens is provided at the inlet end of the small core diameter multimode optical fiber to improve the efficiency of coupling the combined laser into the multimode optical fiber; The laser illumination unit is located at the output end of the multimode optical fiber transmitter and is used to focus the combined laser beam onto the biological sample; The fluorescence signal detection unit is located at the output end of the laser illumination unit and is used to collect the fluorescence signals emitted by the fluorescent molecules on the biological sample.

2. The super-resolution imaging system according to claim 1, characterized in that: The first laser homogenization and collimation unit comprises: a first lens, an engineering diffuser, and a second lens; wherein, The first lens and the second lens are arranged between the output end of the first laser source and the input end of the multimode optical fiber transmitter to collimate the laser emitted by the first laser source; the engineering diffuser is arranged between the focal planes of the first lens and the second lens to homogenize the laser emitted by the first laser source.

3. The super-resolution imaging system according to claim 1, characterized in that: The laser beam combining unit further includes: a first reflecting mirror, a second reflecting mirror, a third reflecting mirror, a fourth reflecting mirror, a first group of dichroic mirrors and a second group of dichroic mirrors; wherein, The first reflector and the second reflector are both tilted along a first preset angle and arranged between the output end of the first laser source and the input end of the first collimating and homogenizing unit, and the first reflector and the second reflector are parallel to each other, the first group of dichroic mirrors are arranged between the output end of the first collimating and homogenizing unit and the input end of the multimode optical fiber transmitter; and the first group of dichroic mirrors are arranged opposite to the second reflector; The third reflector and the fourth reflector are both arranged at the exit end of the second laser source at an inclined angle along a second preset angle, and the third reflector and the fourth reflector are parallel to each other; the second group of dichroic mirrors is arranged at the exit end of the fourth reflector and is arranged opposite to the fourth reflector.

4. The super-resolution imaging system according to claim 1, characterized in that: The multimode optical fiber transmitter further includes: a second objective lens; wherein, The second objective lens is arranged at the output end of the small-core multimode optical fiber to collimate the combined laser beam.

5. The super-resolution imaging system according to claim 4, characterized in that: The laser illumination unit comprises: a laser beam expansion unit and a focusing assembly; wherein, The laser beam expansion unit is arranged at the output end of the laser beam combining unit, and is used to expand the collimated combined laser beam; The focusing assembly is arranged between the output end of the laser beam expansion unit and the input end of the fluorescence signal detection unit, and is used to focus the expanded laser onto the rear focal plane of the objective lens of the fluorescence signal detection unit; A fifth reflecting mirror and a sixth reflecting mirror are arranged between the laser beam expansion unit and the output end of the second objective lens.

6. The super-resolution imaging system according to claim 5, characterized in that: Also includes: A focus control system; wherein: The focus stabilization control system is arranged on one side of the fluorescence signal detection unit, and is used to maintain the stability of the focus during the imaging process.

7. A biomarker detection method, characterized in that: The following steps are involved: Fluorescently labeling specific biomarkers in the biological sample, and performing single-molecule fluorescence imaging on the biomarkers to obtain multiple frames of original microscopic fluorescence images; The multiple frames of original microscopic fluorescence images are analyzed and processed to extract super-resolution distribution characteristic information of specific biomarkers on the surface of the biological sample.

8. The method for detecting a biomarker according to claim 7, characterized in that: The super-resolution distribution characteristic information of the specific biomarkers on the biological sample surface includes: the density of the specific biomarker cluster, the area ratio of the specific biomarker on the biological sample surface, and the average value and distribution standard deviation of the nearest neighbor distance of the specific biomarker cluster.

9. The method for detecting a biomarker according to claim 8, characterized in that: Analyzing and processing the multiple frames of original microscopic fluorescence images includes: By processing multiple frames of original microscopic fluorescence images, localization table data is obtained; Rendering the positioning points in the positioning table data to generate a super-resolution image; Performing a convolution operation on the super-resolution image to obtain a down-sampled image to demarcate the boundary of the biological sample, and calculating the total area of ​​the biological sample; Perform image opening and convolution filtering on the super-resolution image that has not been downsampled to remove small noise; Performing connected domain analysis on the image after the opening operation, segmenting the biomarkers in the image into isolated clusters, and performing statistics on all the segmented clusters to obtain the number and area of ​​all biomarker clusters and the center point of each cluster; The total area of ​​the biological sample, the number of clusters of all biomarkers, and the area of ​​a single cluster are used to determine the density and area proportion of the biomarker clusters in the biological sample, and the center point of each cluster is used to calculate the average value and distribution standard deviation of the nearest neighbor distance of the biomarker cluster and the area proportion of the biomarker on the biological sample.

10. The method for detecting a biomarker according to claim 7, characterized in that: The biological sample is a blood cell, and fluorescent labeling of the biological sample with a specific biomarker includes the following steps: Extracting blood cells with specific biomarkers, diluting them, and then adding them to the treated confocal culture dish, and letting them stand for a period of time to allow the blood cells to adhere to the wall; After adding a fixative solution to the confocal culture dish to fix the blood cells, the blood cells are blocked with a blocking solution; Incubating the blood cells with a primary antibody to specifically recognize and bind to a specific biomarker of the blood cells; The blood cells incubated with the primary antibody are incubated with a secondary antibody labeled with a fluorescent dye to complete the fluorescent labeling of the biological sample with a specific biomarker.

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