An ultramicroscopic imaging system and a method for detecting biomarkers

Abstract

The application provides a kind of super-resolution imaging system and biomarker detection method, comprising: using super-resolution imaging system to carry out single molecule fluorescence imaging to specific biomarker in biological sample, obtains multiple frames original microfluorograph;Multiple frames original microfluorograph is handled and analyzed to extract the super-resolution distribution characteristic information of specific biomarker on the surface of biological sample, and its content distribution and other quantitative characteristics in biological sample.The application obtains positioning table data by processing original microfluorograph;The positioning point in positioning table data is rendered to generate super-resolution image;Super-resolution image is carried out convolution operation, open operation and convolution filtering, region segmentation to calculate the area of cell and the number and area of single cluster of cluster, and the density of cluster and the area proportion of cluster are determined using the two, and the nearest distance between clusters is calculated using cluster center point, which greatly improves the accuracy of biomarker detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microscopic optical imaging and image processing, and in particular, to an ultraprecision imaging system and a biomarker detection method. BACKGROUND

[0002] Alzheimer's disease (AD) is the most common form of dementia, which is an irreversible neurodegenerative disease. "Early diagnosis" and "early intervention" are the key to preventing AD. However, the commonly used techniques are expensive (Positron Emission Tomography, PET) or have certain invasiveness (cerebrospinal fluid biomarker detection), which are not easily accepted by patients.

[0003] In recent years, the detection of blood biomarkers for AD has made rapid progress. Current 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, which has high affinity with albumin, apolipoprotein, α2-macroglobulin and other proteins in plasma. Although the detection technology of Aβ protein in plasma based on mass spectrometry (such as immunoprecipitation coupled mass spectrometry, antibody-free liquid chromatography mass spectrometry, etc.) and single molecule array with ultra-high sensitivity can improve the detection limit of Aβ protein, although there have been a large number of studies on multiple variables before plasma detection and standardization processing guidelines before plasma analysis have been developed, the plasma matrix is relatively complex, and individual 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 pass through the blood-brain barrier, flow into the blood with cerebrospinal fluid, and easily enrich on the surface of red blood cells in peripheral blood. However, the concentration of biomarkers in blood is much smaller than that in cerebrospinal fluid, so high-sensitivity technology is needed for accurate measurement. Currently, the detection methods of AD biomarkers in blood have certain limitations, such as limited sensitivity of immunofluorescence assay and ELISA assay; although atomic force microscopy has ultra-high resolution, its detection results may be affected by other similar proteins. There are the following defects:

[0005] (1) Sample processing procedure is complex: The existing AD early diagnosis method based on blood biomarkers mainly detects the concentration of AD biomarkers in plasma. A small number of studies detect AD biomarkers on the surface of red blood cells by enzyme-linked immunosorbent assay (ELISA) and europium immunoassay. That is, peripheral 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 the problems of multiple steps, complex operation, long time consumption, and high technical requirements. Studies have shown that the same blood plasma, different laboratories use the same or different methods to detect (detection methods: enzyme-linked immunosorbent assay (ELISA), single molecule array (Single molecule array), IP-LC-MS (Immunoprecipitation coupled to liquid chromatography mass spectrometry)), the results will have certain differences. In other words, the accuracy of plasma-based AD biomarker detection is easily affected by the detection process (personnel operation error, blood storage conditions, etc.).

[0006] (2) Lack of biological specificity: Some studies have used atomic force microscopy to study the morphology (size, structure, and texture, etc.) of Aβ protein aggregates on the surface of peripheral red blood cells of AD patients, and compared and analyzed the clinical data to explore the feasibility of using spatial distribution characteristics of protein aggregates as AD diagnostic criteria. However, the detection of red blood cell surface biomarkers based on atomic force microscopy still has some problems in accuracy or specificity: the concentration measurement method based on immunosorbent 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 have used single molecule localization imaging technology to detect the density of characteristic proteins of multiple myeloma cells, the number of cells counted is small, and the detection parameter only uses the density of characteristic proteins as an indicator to judge the progress of the disease. However, in single molecule localization imaging, the quenching of target proteins 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 number of fluorescent molecules collected by the camera will gradually decrease. That is, when the content of target proteins is low, the number of fluorescent molecules collected may not be sufficient to support the acquisition of super-resolution data or affect the quality of super-resolution data, which will affect the accuracy of the subsequent algorithm for density detection. In view of this, an ultra-resolution imaging system and a biomarker detection method are urgently needed to solve the problems existing in the prior art. SUMMARY

[0007] In view of this, the present application provides an ultraprecision imaging system and a biomarker detection method, aiming to solve the problems of complex blood biomarker sample processing flow, great influence of background fluorescence on image acquisition, narrow imaging field of view, and low accuracy of biomarker detection results in the prior art.

[0008] In one aspect, the present application provides an ultraprecision imaging system, comprising: a laser beam combining unit, a laser illumination unit, and a fluorescent 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 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 laser emitted by the first laser source; the multimode fiber transmitter is arranged between the exit end of the first laser homogenization and collimation unit and the laser illumination unit, and is used to combine, transmit, and collimate the output of the two lasers emitted by the first laser source and the second laser source, and to vibrationally isolate the laser beam combining unit and the laser illumination unit; the multimode fiber transmitter has a small-core-diameter multimode fiber, and the entrance end of the small-core-diameter multimode fiber is provided with a first objective lens, so as to improve the efficiency of coupling the combined laser into the multimode fiber; the laser illumination unit is located at the exit end of the multimode fiber transmitter, and is used to focus the combined laser onto a biological sample; and the fluorescent signal detection unit is located at the exit end of the laser illumination unit, and is used to collect the fluorescent signal emitted by the fluorescent molecules on the biological sample.

[0009] Further, in the above ultraprecision imaging system, the first laser homogenization and collimation unit comprises: a first lens, an engineered diffuser, and a second lens; wherein the first lens and the second lens are arranged between the exit end of the first laser source and the entrance end of the multimode fiber transmitter, and are used to collimate the laser emitted by the first laser source; and the engineered diffuser is arranged between the focal planes of the first lens and the second lens, and is used to homogenize the laser emitted by the first laser source.

[0010] Further, in the super-resolution imaging system, the laser beam combining unit further comprises a first mirror, a second mirror, a third mirror, a fourth mirror, a first set of dichroic mirrors and a second set of dichroic mirrors; the first mirror and the second mirror are both arranged at a first preset angle between the exit end of the first laser source and the entrance end of the first collimation and homogenization unit, and the first mirror and the second mirror are parallel to each other; the first set of dichroic mirrors is arranged between the exit end of the first collimation and homogenization unit and the entrance end of the multimode fiber transmitter; and the first set of dichroic mirrors is arranged opposite to the second mirror; the third mirror and the fourth mirror are both arranged at a second preset angle at the exit end of the second laser source, and the third mirror and the fourth mirror are parallel to each other; and the second set of dichroic mirrors is arranged at the exit end of the fourth mirror and opposite to the fourth mirror.

[0011] Further, in the super-resolution imaging system, the multimode fiber transmitter further comprises a second objective lens; wherein,

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

[0013] Further, in the super-resolution imaging system, the laser illumination unit comprises a laser beam expanding unit and a focusing assembly; the laser beam expanding unit is arranged at the output end of the laser beam combining unit to expand the collimated laser beam; the focusing assembly is arranged between the output end of the laser beam expanding unit and the input end of the fluorescence signal detection unit to focus the expanded laser beam onto the back focal plane of the objective lens of the fluorescence signal detection unit; and the fifth mirror and the sixth mirror are arranged between the laser beam expanding unit and the exit end of the second objective lens.

[0014] Further, in the super-resolution imaging system, a focus stabilization control system is further included; the focus stabilization control system is arranged on one side of the fluorescence signal detection unit to maintain the stability of the focal point during imaging.

[0015] The present application can further reduce the coherence of the illuminating laser by utilizing the aliasing effect of different modes in the multimode optical fiber transmitter, thereby achieving uniform illumination. Meanwhile, the multimode optical fiber transmitter can transmit higher power laser and couple more laser spot modes, which can focus two lasers on the back focal plane of the objective lens in the fluorescence signal detection unit, meet the total internal reflection (TIR) illumination condition, 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, and then transmitted by the multimode optical fiber transmitter and expanded and focused by the illumination unit, the laser can be accurately irradiated to the biological sample labeled by the fluorescence probe, and the evanescent wave is generated by total internal reflection at the sample and slide interface to illuminate the sample and excite the sample to generate fluorescence signals. The fluorescence signals are collected by the fluorescence signal detection unit for imaging, which helps to increase the field of view of super-resolution localization imaging, realizes large field of view and uniform illumination imaging effect, and greatly improves the processing throughput of biological sample images.

[0016] In another aspect, the present application also provides a biomarker detection method, comprising the following steps:

[0017] 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;

[0018] Analyzing and processing 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.

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

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

[0021] Obtaining localization table data by processing the multiple frames of original microscopic fluorescence images;

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

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

[0024] Performing image opening operation and convolution filtering on the super-resolution image that has not been down-sampled to remove small noise points;

[0025] The image after the opening operation is subjected to connected domain analysis, the biomarkers in the image are segmented into isolated clusters, and all the segmented clusters are counted to obtain the number, area and center point of each cluster of all biomarker clusters;

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

[0027] Further, in the biomarker detection method, the biological sample is blood cells, and the fluorescent labeling of the biological sample with specific biomarkers comprises the following steps:

[0028] The blood cells with specific biomarkers are extracted and diluted, and then added to a treated confocal culture dish, and left for a period of time to allow the blood cells to adhere to the wall;

[0029] After adding a fixing solution to the confocal culture dish to fix the blood cells, a blocking solution is used to block the blood cells;

[0030] A primary antibody is used to incubate the blood cells to specifically recognize and bind to the specific biomarkers on the blood cells;

[0031] A secondary antibody labeled with fluorescent dye is used to incubate the blood cells incubated with the primary antibody to complete the fluorescent labeling of the biological sample with specific biomarkers.

[0032] The present application obtains multiple frames of original microscopic fluorescence images by collecting data from the biological sample with specific biomarkers that has been fluorescently labeled, and analyzes and processes the multiple frames of original microscopic fluorescence images to obtain the localization table information of the specific biomarkers on the surface of the biological sample and the super-resolution image. Finally, the super-resolution distribution characteristic information of the specific molecules on the surface of the biological sample is obtained by algorithm, thereby greatly improving the accuracy of the detection results of the biomarkers. BRIEF DESCRIPTION OF DRAWINGS

[0033] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are for purposes of illustration only and are not intended to limit the present application thereto. Moreover, like reference numerals in the Figures are intended to represent the same parts throughout the Figures. In the Figures:

[0034] Figure 1 The schematic diagram of the super-resolution localization imaging principle in the embodiments of the present application;

[0035] Figure 2 The optical path diagram of the super-resolution positioning imaging system provided by the embodiment of the present application;

[0036] Figure 3 The imaging field diagram based on the total internal reflection illumination of the small core diameter multi-mode optical fiber in the super-resolution positioning imaging system provided by the embodiment of the present application;

[0037] Figure 4 The normalized light intensity diagram at the dashed line in the corresponding Figure 3

[0038] Figure 5 The flowchart of the red blood cell surface AD biomarker detection method provided by the embodiment of the present application;

[0039] Figure 6 The flowchart of the clustering analysis of the multiple frames of original microscopic fluorescence images in the embodiment of the present application;

[0040] Figure 7 The principle diagram of the miscellaneous signal filtering in the embodiment of the present application;

[0041] Figure 8 The super-resolution image of the single red blood cell surface Aβ protein in the embodiment of the present application;

[0042] Figure 9 The diagram of dividing the cell boundary to remove the extracellular signal and obtaining the cell area in the embodiment of the present application;

[0043] Figure 10 The images before and after the image opening operation on the super-resolution image which has not been down-sampled in the embodiment of the present application;

[0044] Figure 11 The clustering effect diagram of the super-resolution data of the single red blood cell surface Aβ protein after the algorithm processing in the embodiment of the present application;

[0045] Figure 12 The nearest neighbor distance distribution diagram of the single red blood cell membrane surface Aβ protein in the embodiment of the present application;

[0046] Figure 13 The density statistical result of the gradient experiment of the multiple red blood cell membrane surface Aβ proteins in the embodiment of the present application;

[0047] Figure 14 The nearest neighbor distance statistical result of the gradient experiment of the multiple red blood cell membrane surface Aβ proteins in the embodiment of the present application;

[0048] Figure 15 The protein cluster area ratio statistical result of the gradient experiment of the multiple red blood cell membrane surface Aβ proteins in the embodiment of the present application. DETAILED DESCRIPTION ​

[0049] Exemplary embodiments of the present disclosure will be described in greater detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it is understood that the present disclosure can be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure can be more thoroughly understood, and so that the scope of the present disclosure can be completely conveyed to those skilled in the art. It is noted that the embodiments in the present disclosure and the features in the embodiments can be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0050] Immunofluorescence (IF) is a labeling technique widely used in fluorescence microscopic imaging, which specifically recognizes and labels target proteins in cells by antibodies, thereby realizing in situ imaging. Single molecule localization microscopy (SMLM) is a technique that uses laser to excite fluorescently labeled target proteins, and realizes random blinking of single fluorescent molecules of target proteins with the assistance of special imaging buffer. Single fluorescent molecules are captured by a high-precision microscope imaging system, and tens of thousands of original pictures of point-like blinking are collected to reduce overlapping effects, thereby improving the resolution of imaging. Finally, the collected pictures are reconstructed by algorithm to obtain super-resolution images and corresponding localization table; therefore, the spatial resolution of SMLM can reach 20-30 nm, which is much higher than that of ordinary fluorescence microscope. In combination with immunofluorescence technology, the distribution of biomarkers on the cell surface can be clearly revealed, thereby realizing accurate density detection.

[0051] Referring to Figure 1 , the basic principle of single molecule localization super-resolution microscopy technology is as follows:

[0052] (1) Use light-activated fluorescent probe labeled samples. Light-activated fluorescent molecules are an important prerequisite for realizing super-resolution localization imaging. Light-activated fluorescent molecules can be converted between bright state and dark state, which is called light switching phenomenon. This means that each fluorescent molecule can be imaged in time, showing spatially isolated and non-overlapping PSF (point spread function), avoiding overlapping of fluorescent molecule signals in the diffraction limit region.

[0053] (2) Activate fluorescent probes labeled biological structures. A specific wavelength laser is used to activate fluorescent molecules, so that only a small number of randomly distributed fluorescent molecules are converted to bright state each time. Thereafter, another wavelength laser is used to excite fluorescent molecules to emit fluorescence. The fluorescent molecules switch between bright state and dark state, resulting in a "blinking" phenomenon of the fluorescent molecules.

[0054] (3) Detection and localization of single-molecule spatial positions. A detector collects the fluorescence signals emitted by fluorescent molecules in their bright state. Only a small number of single-molecule fluorescence signals are collected per frame because 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 a high signal-to-noise ratio. Currently, electron-multiplying charge-coupled devices (EMCCDs) and scientific-grade complementary metal-oxide-semiconductor (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 image, the Gaussian intensity spectrum of the molecule's horizontal and vertical positions can be obtained, and the peak position is the centroid of the fluorescent molecule. It should be noted that the imaging throughput of SMLMs does not solely depend on the detector; the photophysical properties of the fluorophore (scintillation rate) are also another important factor.

[0055] (4) Reconstruct the super-resolution image. Repeat the above process extensively to obtain thousands or even tens of thousands of single-molecule images. Using the sufficient 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 that, through the cooperation of various optical elements, achieves a large field of view and uniform illumination imaging effect, improves sample processing throughput, and thus improves the efficiency and accuracy of biomarker detection.

[0057] Single-molecule localization imaging techniques based on wide-field fluorescence illumination are prone to strong background fluorescence when imaging AD biomarkers on the surface of erythrocytes, which is detrimental to obtaining high-quality images and thus reduces detection capability. Single-molecule localization imaging based on total internal reflection (TIR) ​​illumination can suppress defocused background fluorescence during the imaging process, making it very suitable for studying the super-resolution distribution characteristics of AD biomarkers on the surface of erythrocytes. However, existing single-molecule localization imaging schemes based on TIR illumination are limited by complex optical paths (free-space laser transmission) or small illumination fields (the single-mode fiber used in conventional TIR illumination cannot transmit high-power lasers, limiting the illumination field), which is not conducive to the collection and statistical analysis of a large number of erythrocytes. To address this deficiency, this invention proposes a large field-of-view single-molecule localization imaging system based on fiber-optic TIR.

[0058] System Implementation Example:

[0059] See Figure 2The single-molecule localization imaging system of this invention includes: a laser beam combining unit 100, a laser illumination unit, and a fluorescence signal detection unit 200; wherein, the laser beam combining unit 100 includes: a first laser source I1, a second laser source I2, a first laser homogenization and collimation unit 102, and a multimode fiber optic 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 emission 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 fiber optic transmitter 101 is disposed in the first laser homogenization unit 102. The collimation unit 102, located between its output end and the laser illumination unit, 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 provide vibration isolation between the laser combining unit 100 and the laser illumination unit. The multimode fiber transmitter has a small-core multimode fiber, and a first objective lens is provided at the entrance end of the small-core multimode fiber to improve the efficiency of coupling the combined laser beam into the multimode fiber. The laser illumination unit is located at the output end of the multimode fiber transmitter 101 to focus the combined laser beam onto the biological sample. The fluorescence signal detection unit 200 is located at the output end of the laser illumination unit to collect the fluorescence signal emitted by fluorescent molecules on the biological sample.

[0060] Specifically, the first laser source I1 emits a laser 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 fluorescent molecules are easily quenched when the target protein is small, 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 refers to the laser beam resulting from the combination of the laser emitted by the first laser source and the laser emitted by the second laser source.

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

[0064] In this embodiment, the laser beam is coupled into the small-core-diameter multimode 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 10μm, which is smaller than the core diameter of the multimode fiber (core diameter is 50μm), 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] To ensure that the beam output from the multimode fiber optic transmitter can be well collected, a second objective lens OBJ2 (10X objective lens) with a numerical aperture of NA0.25 is selected for collimation, and its collimation effect is better than that of other lenses.

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

[0067] Furthermore, the multimode fiber transmitter 101 is positioned between the laser beam combining unit 100 and the laser illumination unit, which is beneficial for placing the laser beam combining unit 100 and the laser illumination unit on two different optical platforms. The vibration generated during laser use will not affect the imaging optical path, thus not affecting single-molecule fluorescence imaging.

[0068] Existing technologies using single-mode fiber (SMF) for TIR illumination suffer from limitations. Due to the small core diameter, the transmitted laser power is limited to the hundreds of milliwatts, and the laser spot shape is highly demanding, hindering the achievement of large field-of-view single-molecule localization imaging. In contrast, multimode fiber (MMF) has a larger core diameter (typically 30-400 μm), enabling the transmission of higher-power lasers and coupling more laser spot modes. Research shows that focusing laser light from small-core-diameter (less than 100 μm) MMF onto the back focal plane of the objective lens satisfies TIR illumination requirements, achieving an imaging field of view up to 200 μm, covering dozens of blood cells and increasing imaging throughput. Therefore, replacing the single-mode fiber (SMF) in conventional single-molecule localization imaging systems with small-core-diameter MMF to build a large field-of-view single-molecule localization imaging system based on TIR illumination can significantly improve the throughput of blood cell biomarker detection and shorten detection time.

[0069] The laser illumination unit includes a laser beam expander and a focusing component L5. The laser beam expander is located at the output end of the laser beam combiner 100 and is used to expand the aligned laser beam. The focusing component L5 is located between the output end of the laser beam expander and the input end of the fluorescence signal detection unit 200, and is used to focus the expanded laser beam onto the back focal plane of the objective lens of the fluorescence signal detection unit 200. A fifth reflecting mirror M5 and a sixth reflecting mirror M6 are disposed between the laser beam expander 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 expanding unit. The laser beam expanding unit includes an excitation filter EX, a third lens L3, and a fourth lens L4; wherein the excitation filter EX is disposed between the third lens L3 and the fourth lens L4 to purify the combined laser beam in the optical path; and an aperture stop is provided at the exit end of the fourth lens L4 to eliminate stray light.

[0071] More specifically, the excitation filter EX is model FF01-390 / 482 / 532 / 640-25, Semrock; the third lens L3 is model AC254-060-A, Thorlabs; and the fourth lens L4 is model AC508-150-A, Thorlabs. An aperture stop Diaphragm (ID50, Thorlabs) is placed behind the fourth lens L4 to eliminate stray light, suppress background, and improve the image's signal-to-noise ratio. In this embodiment, a seventh reflecting mirror M7 is also provided at the exit 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 dichroic mirror DM3, a notch filter NF, an emission filter EM, a tube lens TL, and a camera C; wherein the fourth dichroic mirror DM3, the notch filter NF, and the emission filter EM are disposed 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 third objective lens can be a UPL APO 60X or Olympus. The fourth dichroic filter DM3 is model ZT405 / 488 / 532 / 640rpc-XT, the chroma notch filter NF is model NF03-405 / 488 / 532 / 635E-25 (Semrock), and the emission filter EM is model ET705 / 100 (Chroma). The camera C is preferably a high-sensitivity camera; in this embodiment, an sCMOS camera (Dhyana 400BSI V2, Tucsen Photonics) is used.

[0074] It is evident from the above that: the embodiments of the present invention utilize the aliasing effect of different modes in the multimode fiber transmitter to further reduce the coherence of the illumination laser, thereby achieving uniform illumination; simultaneously, since the multimode fiber transmitter can transmit higher power lasers and couple more laser spot modes, it can focus two laser beams onto the back focal plane of the objective lens in the fluorescence signal detection unit, satisfying the total internal reflection TIR illumination condition, which can significantly increase 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 fiber transmitter and expanded and focused by the illumination unit, so that the laser can illuminate the biological sample labeled by the fluorescent probe at an accurate angle, and generate evanescent wave illumination of the sample at the interface between the sample and the slide through total internal reflection, exciting the sample to generate a fluorescence signal, and then collecting the fluorescence signal for imaging by the fluorescence signal detection unit, which helps to increase the field of view of super-resolution localization imaging, achieve a large field of view and uniform illumination imaging effect, and greatly improve the processing throughput of biological sample images.

[0075] In the above embodiments, 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 disposed between the emitting end of the first laser source I1 and the incident end of the multimode fiber transmitter 101, for collimating the laser emitted by the first laser source I2; the engineered diffuser ED1 is disposed between the focal planes of the first lens L1 and the second lens L2, for homogenizing the laser emitted by the first laser source I1.

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

[0077] In this embodiment, because the laser is projected onto the sample end, the coherence of the laser will produce random interference pattern laser speckle, which is detrimental to single-molecule fluorescence imaging. By placing an engineered diffuser ED1 between the focal planes of the first lens L1 and the second lens L2, the transmission angle of different rays in the beam can be effectively changed, altering the incident beam direction and disrupting the laser coherence. By providing high transmittance, the divergence angle, spatial distribution of light, and intensity profile of diffused light can be controlled, improving the uniformity of the field of view and providing better conditions for subsequent quantitative analysis. However, the engineered diffuser ED1 has the disadvantage of high divergence. Therefore, this problem is solved by placing it on the focal plane of the first lens L1 (focusing lens) and the second lens L2 (collimating lens), thereby improving the laser transmission efficiency. In this embodiment, the engineered diffuser ED1 and the multimode fiber transmitter can eliminate the random speckle generated by laser illumination, achieving 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 dichroic mirror DM3, a notch filter NF, an emission filter EM, a tube lens TL, and a camera C; wherein the fourth dichroic mirror DM3, the notch filter NF, and the emission filter EM are disposed 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 third objective lens OBJ3 is a UPL APO 60X, Olympus; the fourth dichroic filter DM3 is a ZT405 / 488 / 532 / 640rpc-XT; the notch filter NF is a NF03-405 / 488 / 532 / 635E-25, Semrock; the emission filter EM is an ET705 / 100, Chroma; the camera C is preferably a high-sensitivity camera, and in this embodiment, an sCMOS camera (Dhyana 400BSI V2, Tucsen Photonics) is used.

[0080] In the above embodiments, 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 set of dichroic mirrors DM2, and a second set of dichroic mirrors DM1; wherein, the first reflector M1 and the second reflector M2 are both inclined at a first preset angle between the emitting 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 set of dichroic mirrors DM2 is disposed between the emitting end of the second reflector M2 and the incident end of the multimode fiber transmitter 101, and the first set of dichroic mirrors DM2 is disposed opposite to the second reflector M2;

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

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

[0083] In long-duration single-molecule localization imaging, to prevent focus shift caused by factors such as sample drift and temperature changes (focus shift leads to image blurring and resolution degradation), this embodiment may further include a focus stabilization control system 300. The focus stabilization control system 300 is disposed on one side of the fluorescence signal detection unit 200 and is used to maintain focus stability during imaging, thereby preventing focus shift caused by factors such as sample drift and temperature changes. This is particularly important for long-duration super-resolution imaging, as focus shift leads to image blurring and resolution degradation.

[0084] See Figure 2 In this embodiment, the quadrant detector QD, the prism reflector Prism, the bandpass filter F1, the 850nm infrared light, the ninth reflector M9, the tenth reflector M10, and the third dichroic mirror DM4 constitute the focus stabilization control system 300 to ensure that the focus is always in the optimal position.

[0085] Specifically, the QD has a data refresh rate of 100Hz and can support a focus stabilization frequency of at least 50Hz.

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

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

[0088] The fourth dichroic mirror DM3 can guide the beam of 850nm infrared light combined from the first laser source I1, the second laser source I2 and the focusing control system 300 into the third objective lens OBJ3 of the fluorescence signal detection unit 200; the third dichroic mirror DM4 can guide the 850nm infrared light beam 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 DM4, and the fourth dichroic mirror DM3.

[0090] In practice, the laser beam enters the optical path of the imaging system through the third dichroic mirror DM4, and then is transmitted to the biological sample through the fourth dichroic mirror DM3 and the third objective lens OBJ3. The light reflected back from the biological sample then passes through the third objective lens OBJ3, the fourth dichroic mirror DM3, and the third dichroic mirror DM4 in sequence to return to the focus stabilization control system 300. The focus stabilization control system 300 determines the focal plane by the change in the position of the light beam, and then makes adjustments according to the change in the focal plane to ensure the stability of the focus.

[0091] In this embodiment, an illumination mode switching unit is provided in the optical path between the fourth lens L4 and the focusing component L5 to switch between total internal reflection illumination (TIRF) and epi-illumination (EPI) modes and two fluorescence imaging modes to obtain more comprehensive information about the biological sample. The illumination mode switching unit is a Stage module (capable of switching between TIRF and epi-illumination fluorescence modules). The illumination mode switching unit includes an eighth reflecting mirror M8 and a focusing lens L5. In this embodiment, the fifth reflecting mirror M5 and the sixth reflecting mirror M6 are set at a third preset angle, and the seventh reflecting mirror M7 and the eighth reflecting mirror M8 are set at a fourth preset angle. Both the third and fourth preset angles can be 45°.

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

[0093] First, the transmission direction of the 640nm laser emitted by the first laser source I1 is adjusted using the first reflecting mirror M1 and the second reflecting mirror M2. L1 and L2 are achromatic cemented doublet lenses with a focal length of 30mm. The transmission direction of the 405nm laser emitted by the second laser source I2 is adjusted using the third reflecting mirror M3, the fourth reflecting mirror M4, and the second set of dichroic mirrors DM1. 405nm is the activation light, and the spot quality requirement is not high, so there is no need to use an engineering diffuser for spot homogenization. The 405nm laser is reflected by the first set of dichroic mirrors DM2, while the 640nm laser is transmitted.

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

[0095] After collimation, the two laser beams are coupled into a multimode fiber optic transmitter via the first objective lens OBJ1. To ensure good beam collection from the multimode fiber optic transmitter, a second objective lens OBJ2 with a larger NA (aperture area) is used for collimation after the beam exits the transmitter. The combined laser beam, after collimation by the second objective lens OBJ2, is then expanded and purified by the third lens L3, excitation filter EX, and fourth lens L4 of the laser beam expander unit. To suppress 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 expanded laser beam is focused onto the back focal plane of the third objective lens OBJ3 in the fluorescence signal detection unit via focusing assembly L5, the third dichroic mirror DM4, and the fourth dichroic mirror DM3. Finally, the laser beam illuminates the fluorescently labeled biological sample through the third objective lens OBJ3 in the fluorescence signal detection unit, exciting fluorescent molecules and collecting the fluorescence signal. The excitation light is then filtered out by a notch filter NF, and the dye fluorescence is purified by an emission filter EM to ensure high fluorescence transmittance. Finally, 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 this embodiment of the invention was tested, and the power density of the 640nm laser at the sample end was found to be 6.06kW / cm². 2 It fully meets the power density requirements of single-molecule localization imaging for the sample end; and it can still provide sufficiently high illumination intensity and good illumination uniformity when the imaging field diameter is as high as 100μm.

[0098] In summary, the large field-of-view single-molecule localization imaging system based on fiber-optic total internal reflection illumination (TIRF) constructed in this invention features a large field of view, low background, and high signal-to-noise ratio. The large field-of-view TIRF illumination facilitates the acquisition of a large amount of biological sample information in a single shot, increasing the single-shot imaging throughput. This allows for the acquisition of more super-resolution imaging data of blood cell biomarkers in a shorter time, providing better technical support for analyzing the distribution characteristics of biomarkers in large numbers of blood cells (100 or more red blood cells).

[0099] Method Implementation Examples:

[0100] See Figures 5-15 The present invention also provides a method for processing images of biological samples with specific biomarkers, comprising the following steps:

[0101] Step S1: Fluorescently label specific biomarkers in the biological sample and perform single-molecule fluorescence imaging on the biomarkers to obtain multiple frames of raw microscopic fluorescence images.

[0102] Specifically, in this embodiment, 100,000 frames of raw microscopic fluorescence images are obtained. The biological sample is blood cells, such as red blood cells, white blood cells, and platelets. Compared to existing detection methods using mixed solutions like serum and plasma, the blood cell-based detection method reduces interference from signal-free samples, thereby improving detection sensitivity. Furthermore, using a super-resolution imaging system based on a large field-of-view single-molecule localization method to detect multiple (100 or more) blood cells from the same batch of blood samples effectively eliminates the impact of individual cell differences on the detection results and simplifies the sample processing procedure.

[0103] In this embodiment, the biological sample containing a specific biomarker is preferably a blood cell sample. The fluorescence signal of the blood cell sample enriched with the biomarker is captured by a detector to generate a single-frame image. This process is repeated to generate a raw microscopic fluorescence image containing multiple images. The fluorescent molecule signals in each raw microscopic fluorescence image are extracted and processed to generate entries in the localization table data.

[0104] Combined again Figure 5 Fluorescent labeling of biological samples with specific biomarkers includes the following steps:

[0105] Step S11: Extract blood cells with specific biomarkers, dilute them, and then add them to a pretreated confocal culture dish. Let them stand for a period of time to allow the blood cells to adhere to the dish.

[0106] This step, when implemented in practice, may include:

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

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

[0109] In practice, 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 h to allow the Aβ1-42 powder to dissolve completely.

[0111] b. Blow nitrogen for about 10 minutes to allow HFIP to evaporate and obtain an Aβ1-42 film. Then, dissolve the Aβ1-42 film with a small amount of dimethyl sulfoxide (DMSO).

[0112] c. Add hydroxyethylpiperazine ethanethioic acid (HEPES) to adjust to the required concentration. If a small amount of crystals are present, they can be dissolved by ultrasound. After placing in a 4°C refrigerator for 24 hours, dispense the product and store it in a -80°C refrigerator.

[0113] (2) Extract blood cells and incubate blood cells with biomarker solutions of different concentrations for a period of time to enrich the biomarkers on the surface of the blood cells. In this embodiment, red blood cells are preferred.

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

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

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

[0117] b. Simulated red blood cells from AD patients: Red blood cells were incubated with Aβ protein solutions of different concentrations for about 24 hours to enrich Aβ protein on the surface of the red blood cells. The supernatant was then removed to obtain incubated red blood cells. The cells were then washed twice with an incubation solution without Aβ protein and centrifuged to remove unbound Aβ protein. The resulting concentrated red blood cell solution was the simulated red blood cells from AD patients.

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

[0119] In practice, confocal culture dishes with good light transmittance (Mat Tek, P35G-1.5-14-C) were treated with 0.1% poly-L-lysine to facilitate subsequent experiments. Red blood cells simulating AD patients were diluted to the required concentration with DMEM basal medium, and 400 μL of the diluted red blood cell suspension was added to the confocal culture dish. The dish was then incubated for 30 min to allow the red blood cells to adhere to the wall, which can achieve better labeling results.

[0120] Because sample offset occurs during imaging, and the offset of the fluorescent microspheres matches the offset of the target protein on the cell, the fluorescent microspheres do not flicker during imaging, while the target protein on the cell flickers as a single molecule. The cell offset can be corrected using a positioning table of the fluorescent microspheres. In this embodiment, fluorescent microspheres are added to the confocal culture dish before this step. The diameter of the fluorescent microspheres can be 100 nm.

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

[0122] Step S12: After fixing the blood cells in the confocal culture dish with fixative, block the blood cells with blocking solution.

[0123] In practice, remove excess incubation solution from the confocal culture dish, add fixative and fix for 15 min, then wash with PBS phosphate buffer to remove excess fixative. After washing, add blocking solution to block the red blood cells.

[0124] Step S13: Incubate the above-mentioned red blood cells with a primary antibody to specifically recognize and bind to the Aβ protein on the blood cells.

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

[0126] Step S14: Incubate blood cells that have been incubated with primary antibody using a secondary antibody labeled with a fluorescent dye to complete the fluorescent labeling of biological samples with specific biomarkers.

[0127] In practice, the secondary antibody is diluted with 3% BSA, 200 μL of the secondary antibody is added to the well of the dish and incubated in the dark for about 40 minutes. Then, it is washed with PBS phosphate buffer to remove excess secondary antibody.

[0128] In this embodiment, the primary antibody is incubated to allow it to locate the target protein. The secondary antibody binds to the primary antibody, which amplifies the fluorescence signal to a certain extent. After fluorescence 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 steps S11-S14 above are used for the preparation and fluorescent labeling of model blood cell samples. For fluorescent labeling of real blood samples from patients, only steps (3)-S14 are required.

[0130] It can be seen that this invention only requires the characteristic measurement and analysis of biomarkers on the surface of blood cells, and completes the detection of biomarkers on the surface of blood cells with high sensitivity, high accuracy and high specificity, thereby reducing the impact of differences in experimental operation procedures on the measurement results and greatly reducing the error pressure on experimental personnel; it requires a small sample volume, only a few microliters of blood are needed to meet the experimental requirements; other substances in the blood have little impact on the results of this experiment.

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

[0132] Specifically, the super-resolution distribution characteristics of specific biomarkers on the surface of the biological sample include: the density of specific biomarker clusters, the area ratio of specific biomarkers on the surface of the biological sample, and the average and standard deviation of the nearest neighbor distances of specific biomarker clusters. That is, the detection results are no longer presented as concentration, but rather as the super-resolution distribution characteristics of biomarkers detected on the surface of disease-related blood cells (the density of specific biomarker clusters, the area ratio of specific biomarkers on the surface of the biological sample, and the average and standard deviation of the nearest neighbor distances of specific biomarker clusters), and the features of the super-resolution distribution characteristics can be quantified. In this embodiment, the biomarker features include: the density of specific biomarker clusters, the area ratio of specific biomarkers on the surface of the biological sample, and the average and standard deviation of the nearest neighbor distances of specific biomarker clusters.

[0133] A series of raw microscopic fluorescence images are obtained by repeatedly exposing biological samples with specific biomarkers using a super-resolution imaging system. Each exposure activates only a small number of fluorescent molecules, ensuring 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 and related information of each fluorescent molecule are determined to obtain the localization table data.

[0136] Specifically, the original image is processed using a single-molecule localization algorithm to obtain localization table data. The localization table includes 12 columns of information (as shown in Table 1), which, in addition to coordinate information, also include the peak signal intensity (PI) and signal-to-noise ratio (SNR) of the localization point.

[0137] In one specific implementation of this embodiment, the localization algorithm used is QC-STORM (QC is an abbreviation for Quality Control), which is a GPU-accelerated method for image preprocessing and molecular recognition, maximum likelihood estimation localization, super-resolution image rendering, and statistical information analysis. Its function is to calculate information such as the position of signal points and the full width at half maximum (FWHM) in the localization microscopic image to form a localization table.

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

[0139] Table 1

[0140]

[0141]

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

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

[0144] In practice, a Gaussian model is used to render the localized points to obtain a super-resolution image with a pixel size of 5nm. The pixel size of the generated super-resolution image is 5nm, meaning that each pixel represents a 5nm × 5nm region.

[0145] (3) Combination Figure 9 The super-resolution image is convolved to obtain a downsampled image to delineate the boundaries of the biological samples, and the total area of ​​the biological samples is calculated.

[0146] In practice, the super-resolution image obtained in the previous step is convolved with a Gaussian kernel of size 100 pixels to obtain a downsampled image, which is used to delineate cell boundaries. Extracellular signals are then removed based on the cell boundaries, and the cell area is calculated. The pixel size of the image is selected based on the density of biomarker clusters on the cell.

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

[0148] In practice, a disk structure of 5 pixels (determined according to the cluster density) is used to perform 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] By using a disk structure of 5 pixels to perform opening operations, small specks with a diameter of less than 5 pixels in the image can be effectively removed.

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

[0151] (5) Combination Figure 11 The image after the opening operation is subjected to connected component analysis to segment the biomarkers in the image into isolated clusters. The number, area and center point of each cluster of all biomarkers are obtained by statistical analysis of all the segmented clusters.

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

[0153] (6) Combination Figures 11-12 The density and area ratio of biomarker clusters in the biological sample are determined by using the total area of ​​the biological sample and the number and area of ​​all biomarker clusters, and the mean and standard deviation of the nearest neighbor distance of the biomarker cluster are calculated by using the center point of each cluster.

[0154] Existing clustering algorithms developed for single-molecule localization microscopy, such as DBSCAN, ClusterViSu, and FACAM, are based on point cloud data from calibration tables. They only utilize the positional coordinates of localization points, neglecting information such as the intensity of these points. However, cell surface biomarkers are distributed in isolated clusters, and their super-resolution imaging results also correspond to this isolated cluster distribution, meaning cluster classification can be performed based on this image information. Furthermore, the rendering and reconstruction process of super-resolution images utilizes multi-dimensional information such as coordinates, photon counts, and localization accuracy from the localization table. Therefore, compared to clustering methods that only utilize localization point coordinates, clustering methods based on super-resolution images can analyze higher-density data.

[0155] See Figure 5 , Figures 13-15 In the control group, healthy red blood cells were directly incubated with DMEM basal medium. In the experimental group, the prepared Aβ protein solution was diluted to different concentrations with DMEM basal medium and then incubated with healthy red blood cells at 37°C for approximately 24 hours to enrich Aβ protein on the red blood cell surface. The incubation solution was then discarded, and the incubated red blood cells were used for immunofluorescence labeling experiments. Super-resolution imaging was then performed to obtain super-resolution data. Finally, the super-resolution distribution characteristics of Aβ protein on the red blood cell surface were analyzed using an algorithm. For each sample, super-resolution data from 100 red blood cells were randomly selected. The clustering algorithm used in this embodiment was employed to calculate the size, density, nearest neighbor distance, and area percentage of Aβ protein clusters on the surface of each red blood cell. The statistical results of the obtained protein cluster density, nearest neighbor distance, and area percentage are shown below. Figures 13-15 As shown in the figure, when red blood cells are incubated with Aβ protein solutions of different concentrations, the density and area ratio of Aβ protein clusters on the surface of red blood cells increase with the increase of incubation concentration, while the nearest neighbor distance of Aβ protein clusters on the surface of red blood cells decreases to a certain extent. This proves that the detection method provided in this embodiment of the invention can effectively and accurately detect the density, area ratio, and mean and standard deviation of the nearest neighbor distance of Aβ protein clusters on the surface of red blood cells.

[0156] As can be seen, the clustering algorithm of this invention can process super-resolution images with high-density biomarkers, providing more valuable detection parameters and quantifying 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 the imaging process, thereby significantly improving the accuracy of the detection results.

[0157] In this embodiment, the process for 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. Then, the cell segmentation module extracts the cells to be analyzed from the image. The isolated signal clusters are then separated by the image filtering and segmentation module. Finally, the data statistics module is used to statistically analyze the area and nearest neighbor distance of the signal clusters to obtain the clustering results (i.e., the super-resolution distribution characteristics of the target protein).

[0159] It is evident from the above that this embodiment acquires multiple frames of raw microscopic fluorescence images by collecting data from biological samples that have been fluorescently labeled with specific biomarkers, and analyzes and processes these multiple frames of raw microscopic fluorescence images to obtain the localization table information and super-resolution images of specific biomarkers on the surface of the biological samples. Finally, an algorithm is used to obtain the super-resolution distribution feature information of specific molecules on the surface of the biological samples, thereby greatly improving the accuracy of biomarker detection results.

[0160] The relevant parts of the method embodiments and the system embodiments described above can be referred to each other, and will not be repeated here.

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

[0162] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A super-resolution imaging system, characterized in that, include: The system includes a laser beam combining unit, a laser illumination unit, and a fluorescence signal detection unit; among which, The laser beam combining unit includes: a first laser source, a second laser source, a first laser homogenization and collimation unit, and a multimode fiber optic 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 emission end of the first laser source and is used to homogenize and collimate the laser emitted by the first laser source. The multimode fiber optic transmitter is disposed 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, as well as to isolate the laser beam combining unit and the laser illumination unit from vibration. The multimode fiber transmitter has a small-core multimode fiber, and the entrance end of the small-core multimode fiber is provided with a first objective lens to improve the efficiency of coupling the combined laser beam into the multimode fiber. The first laser homogenization and collimation unit includes: a first lens, an engineered diffuser, and a second lens; wherein the first lens and the second lens are disposed between the emitting end of the first laser source and the incident end of the multimode fiber optic transmitter to collimate the laser emitted by the first laser source; the engineered diffuser is disposed between the focal planes of the first lens and the second lens to homogenize the laser emitted by the first laser source. 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 set of dichroic mirrors, and a second set of dichroic mirrors; wherein, The first reflector and the second reflector are both inclined at a first preset angle between the emitting end of the first laser source and the incident end of the first laser homogenization and collimation unit, and the first reflector and the second reflector are parallel to each other. The first set of dichroic mirrors is disposed between the emitting end of the first laser homogenization and collimation unit and the incident end of the multimode fiber optic transmitter; and the first set of dichroic mirrors is disposed opposite to the second reflector. The third and fourth reflectors are both inclined at the emission end of the second laser source along a second preset angle, and the third and fourth reflectors are parallel to each other; the second set of dichroic mirrors is disposed at the emission end of the fourth reflector and is disposed opposite to the fourth reflector. The laser illumination unit is located at the output end of the multimode 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 fluorescence signals emitted by fluorescent molecules on biological samples.

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

3. The super-resolution imaging system according to claim 2, characterized in that, The laser illumination unit includes: a laser beam expander and a focusing assembly; wherein... The laser beam expanding unit is located at the output end of the laser beam combining unit and is used to expand the beam of the aligned combined laser. The focusing component is disposed between the output end of the laser beam expander and the input end of the fluorescence signal detection unit, and is used to focus the expanded laser beam onto the back focal plane of the objective lens of the fluorescence signal detection unit. A fifth and a sixth reflecting mirror are provided between the laser beam expander unit and the exit end of the second objective lens.

4. The super-resolution imaging system according to claim 3, characterized in that, Also includes: Focus stabilization control system; among which, The focus stabilization control system is located on one side of the fluorescence signal detection unit and is used to maintain the stability of the focus during the imaging process.

Images