A slide with boundary identification and assisted focusing, apparatus and method

Abstract

The application provides a slide with boundary mark and auxiliary focusing, a device and a method. The slide comprises a slide base, a sample bearing area on a bearing surface of the slide base, removable positioning coordinates arranged in the sample bearing area, and at least two permanent boundary marks arranged at the periphery of the sample bearing area. The removable positioning coordinates are made of transient material which is dissolved in a pathological sample pre-treatment liquid and have a geometric configuration indicating a sample layout center and a direction. The at least two permanent boundary marks jointly define a scanning boundary of the sample bearing area on the bearing surface, and at least one of the at least two permanent boundary marks is different from the rest of the permanent boundary marks in geometric shape, for determining the spatial direction of the slide. The permanent boundary marks are used as focusing references for axial height measurement of a digital slice scanner to fit a reference focal plane, and as spatial position references for scanning image cutting and multi-piece alignment.

Description

Technical Field

[0001] This application relates to the field of specimen testing technology, and in particular to a glass slide, apparatus and method with boundary marking and auxiliary focusing. Background Technology

[0002] With the popularization of digital pathology and artificial intelligence analysis technologies, whole-field digital slides (WSI) have become the core data foundation for pathological diagnosis and multimodal AI model training. To obtain high-quality images, digital slide scanners need to identify sample regions and fit reference focal planes during the preview stage to achieve high-magnification automatic scanning, and register different stained serial sections of the same tissue block to a unified coordinate system in the construction of pathological AI.

[0003] In related technologies, low-contrast biological samples such as tumor organoids and microbiota biopsies are difficult to focus stably on using the sample itself, which easily leads to out-of-focus and missed scans. If focus marks are printed on the outside of the coverslip, the focal plane axis deviation will occur due to errors in the sealing medium and slide thickness. In addition, the lack of physical reference during manual slide retrieval leads to random deviations in the position and orientation of continuous slices on different slides, and the image size and boundaries output by the scanner are inconsistent, which significantly increases the difficulty of aligning and registering multimodal continuous slices. Summary of the Invention

[0004] In view of this, it is necessary to provide a glass slide, device and method with boundary marking and auxiliary focusing, which can overcome at least one of the above defects.

[0005] In a first aspect, embodiments of this application provide a glass slide with boundary markings and auxiliary focusing, the glass slide comprising:

[0006] The slide substrate, the sample carrying area located on the carrying surface of the slide substrate, the removable positioning coordinates set in the sample carrying area, and at least two permanent boundary markers set around the sample carrying area;

[0007] The removable positioning coordinates are made of transient material that fades in the pathological specimen pretreatment liquid and have a geometric configuration that indicates the center and orientation of the sample placement.

[0008] At least two of the permanent boundary markers are directly formed on the bearing surface. The permanent boundary markers include graphics with light-transmitting openings or surface microstructures. The imaging surface of the permanent boundary markers is within the same depth of focus as the imaging surface of the sample in the sample bearing area.

[0009] At least two of the permanent boundary markers jointly define the scanning boundary of the sample carrying area on the bearing surface, and at least one of the at least two permanent boundary markers is geometrically different from the other permanent boundary markers, in order to determine the spatial orientation of the slide;

[0010] Each of the permanent boundary markers serves as a focusing reference for the digital slice scanner to perform axial height measurement to fit the reference focal plane, and as a spatial position reference for cropping and aligning multiple slices of the scanned image.

[0011] In one embodiment, the removable positioning coordinates include mutually perpendicular horizontal and vertical reference lines, the intersection of the horizontal and vertical reference lines is located at the geometric center of the sample bearing area, and at least one of the horizontal or vertical reference lines is provided with an asymmetrical direction indication structure.

[0012] In one embodiment, the number of permanent boundary markers is four, and the four permanent boundary markers are respectively disposed on the four corners of the sample carrying area, and the geometric center or feature edge of the four permanent boundary markers correspond to the four vertices of the scanning boundary.

[0013] In one embodiment, the at least one permanent boundary marker that is geometrically different from the other permanent boundary markers includes:

[0014] It differs from the other permanent boundary markers in at least one of the following: graphic outline, opening orientation, size, or internal microstructure;

[0015] The morphological combination or coded arrangement of multiple permanent boundary markers is used to indicate the slice sequence number, sample batch, or staining type.

[0016] In one embodiment, the permanent boundary marker is formed on the bearing surface, and the protrusion height or recess depth of the permanent boundary marker relative to the bearing surface is 0.1 micrometers to 100 micrometers;

[0017] The slide also includes a coverslip covering the sample-bearing area and a sealing medium located between the slide substrate and the coverslip. The permanent boundary marker and the sample in the sample-bearing area are encapsulated together between the slide substrate and the coverslip.

[0018] In one embodiment, the transient material includes a water-soluble material, an alcohol-soluble material, or an organic solvent-soluble material;

[0019] The removable positioning coordinates are configured to be dissolved, peeled, or decolorized by the corresponding treatment reagents when the pathological specimen undergoes at least one step of dewaxing, rehydration, staining, or clearing.

[0020] In one embodiment, the permanent boundary marker includes a surface micro-etched structure, a laser refractive index modified structure, a metal oxide thin film pattern, or a solvent-resistant inorganic printed pattern formed on the bearing surface;

[0021] The outline width of the permanent boundary marker is 1 micrometer to 100 micrometers.

[0022] In one embodiment, a label area for recording sample information is provided at one end of the slide substrate;

[0023] A preset clamping clearance distance is maintained between the permanent boundary marker and the outer edge of the slide substrate, and the distance between each permanent boundary marker and the geometric center of the removable positioning coordinate is greater than the preset sample spreading distance.

[0024] Secondly, this application provides a digital slice scanning apparatus, the apparatus comprising:

[0025] An image acquisition mechanism is used to acquire microscopic images of the glass slide;

[0026] An axial focusing mechanism is used to drive the image acquisition mechanism or the glass slide to move along the optical axis; and

[0027] The control processing module is communicatively connected to the image acquisition mechanism and the axial focusing mechanism, and the control processing module is configured as follows:

[0028] The image acquisition mechanism is controlled to acquire a preview image of the slide, and the image coordinates of the at least two permanent boundary markers are identified in the preview image;

[0029] The spatial orientation of the slide is determined based on at least one permanent boundary marker that is geometrically different from the other permanent boundary markers;

[0030] The axial focusing mechanism is controlled to perform axial height measurement in the areas where each of the permanent boundary markers is located to obtain the corresponding optimal focal plane height, and a reference focal plane covering the sample bearing area is constructed by fitting each of the optimal focal plane heights.

[0031] Based on the reference focal plane, the image acquisition mechanism performs high-magnification digital scanning on the sample bearing area to obtain slice scan images; and

[0032] The sliced ​​scan image is standardized and cropped based on the scan boundary defined by the at least two permanent boundary markers.

[0033] Thirdly, embodiments of this application provide a digital slice scanning method, the method comprising:

[0034] Acquire a preview image of the slide, identify the image coordinates of the at least two permanent boundary markers in the preview image, and determine the spatial orientation of the slide based on the at least one permanent boundary marker that is geometrically different from the other permanent boundary markers;

[0035] Axial focusing height measurement is performed in the area where each of the permanent boundary markers is located to obtain the optimal focal plane height of each of the permanent boundary markers.

[0036] A reference focal plane covering the sample bearing area is generated by fitting the optimal focal plane height of each of the above-described focal planes.

[0037] A digital scan is performed on the sample within the sample-bearing area based on the reference focal plane to obtain an initial slice image; and

[0038] The initial slice image is sized and cropped based on the scan boundary defined by the at least two permanent boundary markers.

[0039] The embodiments of this application provide a glass slide, device, and method with boundary marking and auxiliary focusing. By constructing a dual benchmark collaborative system of "removable positioning coordinates during slide preparation" and "permanent boundary markings during scanning" on the glass slide bearing surface, the front-end and back-end physical connection between sample preparation and digital scanning is realized. On the one hand, the positioning coordinates can provide an intuitive physical center and orientation reference during the sample spreading or retrieval stage, and automatically fade away in subsequent preprocessing. This not only suppresses random tilting and eccentricity of the slides caused by manual retrieval from the source, but also avoids the obstruction and interference of markers on the final pathological image analysis. On the other hand, the permanent boundary markers are directly formed on the bearing surface and are in the same focal depth range as the sample. Structurally, this completely eliminates the axial focal plane deviation caused by uneven thickness of the sealing medium or coverslip due to the traditional marking on the outer surface of the coverslip. Combined with the light transmission configuration and heterogeneous error-proof design, it can provide the digital slide scanner with a precise and unique spatial orientation determination, a Z-axis reference focal plane fitting benchmark covering the entire field of view, and a standardized scanning boundary without affecting the imaging optical path. This effectively solves the technical defects such as defocusing, missed scanning, and inaccurate scanning range that are very likely to occur in automated scanning of low-contrast biological samples such as tumor organoids and micro-biopsy tissues. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the structure of a glass slide with boundary markings and auxiliary focusing provided in an embodiment of this application.

[0041] Figure 2This is a schematic diagram of the glass slide usage process provided in one embodiment of this application.

[0042] Figure 3 This is a schematic diagram of a permanent boundary marker combination method provided in an embodiment of this application.

[0043] Figure 4 This is a schematic diagram of multi-layer registration provided for an embodiment of this application.

[0044] Figure 5 This is a schematic diagram of the cross-sectional positional relationship provided in the embodiments of this application.

[0045] Figure 6 This is a schematic diagram of a digital slice scanning device provided in an embodiment of this application.

[0046] Figure 7 This is a schematic flowchart of a digital slice scanning method provided in an embodiment of this application. Detailed Implementation

[0047] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them.

[0048] It should be noted that, in the embodiments of this application, "at least one" refers to one or more, and "more than one" refers to two or more. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0049] It should be noted that in the embodiments of this application, the terms "first," "second," etc., are used only for descriptive purposes and should not be construed as indicating or implying relative importance, nor as indicating or implying order. Features specified as "first" or "second" may explicitly or implicitly include one or more of the stated features. In the description of the embodiments of this application, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or explanations. Any embodiment or design scheme described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design schemes. Specifically, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0050] Based on the embodiments described in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0051] With the popularization of digital pathology and artificial intelligence analysis technologies, whole-field digital slides (WSI) have become the core data foundation for pathological diagnosis and multimodal AI model training. To obtain high-quality images, digital slide scanners need to identify sample regions and fit reference focal planes during the preview stage to achieve high-magnification automatic scanning, and register different stained serial sections of the same tissue block to a unified coordinate system in the construction of pathological AI.

[0052] In related technologies, low-contrast biological samples such as tumor organoids and microbiota biopsies are difficult to focus stably on using the sample itself, which easily leads to out-of-focus and missed scans. If focus marks are printed on the outside of the coverslip, the focal plane axis deviation will occur due to errors in the sealing medium and slide thickness. In addition, the lack of physical reference during manual slide retrieval leads to random deviations in the position and orientation of continuous slices on different slides, and the image size and boundaries output by the scanner are inconsistent, which significantly increases the difficulty of aligning and registering multimodal continuous slices.

[0053] In view of this, the slide, apparatus and method with boundary marking and auxiliary focusing provided in this application realize the front-end physical connection between sample preparation and digital scanning by constructing a dual reference collaborative system of "removable positioning coordinates during slide preparation" and "permanent boundary markings during scanning" on the slide bearing surface. On the one hand, the positioning coordinates can provide an intuitive physical center and orientation reference during the sample spreading or retrieval stage, and automatically fade away in subsequent preprocessing. This not only suppresses random tilting and eccentricity of the slides caused by manual retrieval from the source, but also avoids the obstruction and interference of markers on the final pathological image analysis. On the other hand, the permanent boundary markers are directly formed on the bearing surface and are in the same focal depth range as the sample. Structurally, this completely eliminates the axial focal plane deviation caused by uneven thickness of the sealing medium or coverslip due to the traditional marking on the outer surface of the coverslip. Combined with the light transmission configuration and heterogeneous error-proof design, it can provide the digital slide scanner with a precise and unique spatial orientation determination, a Z-axis reference focal plane fitting benchmark covering the entire field of view, and a standardized scanning boundary without affecting the imaging optical path. This effectively solves the technical defects such as defocusing, missed scanning, and inaccurate scanning range that are very likely to occur in automated scanning of low-contrast biological samples such as tumor organoids and micro-biopsy tissues.

[0054] This application further integrates four-corner topological layout, microstructure morphology encoding, solvent-resistant micron-level molding process, and compatible scanning devices and methods to construct a standardized closed-loop process from pathological slide preparation and automatic focus-finding scanning to multimodal image registration. Morphological encoding of boundary markers automatically identifies the sequence and staining type of consecutive slides, preventing sample misordering and confusion. Local focus-finding based on a reference focal plane during scanning significantly reduces the computational and mechanical overhead of blind focus-finding throughout the low-contrast tissue region, improving the efficiency of high-throughput scanning. More importantly, using permanent boundary markers with consistent geometric topology on each slide as rigid geometric anchor points, the spatial transformation matrix across slides can be directly calculated. This allows multimodal consecutive slide images, such as those stained with hematoxylin-eosin (HE) and immunohistochemically (IHC) with different antibodies, to be directly normalized to a unified spatial coordinate system and output image. This significantly reduces the algorithmic complexity and computational overhead of multimodal consecutive slide image alignment and registration from the physical hardware source, providing a highly aligned standard dataset to support the accurate training of pathological artificial intelligence models and cross-stain annotation transfer.

[0055] Figure 1 This is a schematic diagram of a glass slide with boundary markings and auxiliary focusing provided in an embodiment of this application. For example... Figure 1 The slide 10 with boundary markings and auxiliary focusing shown includes at least the following parts: a slide substrate 1, a sample carrying area 3 located on the bearing surface of the slide substrate 1, removable positioning coordinates 4 set in the sample carrying area 3, and at least two permanent boundary markings 5 ​​set around the sample carrying area 3.

[0056] In this embodiment of the application, the slide with boundary markings and auxiliary focusing includes: a slide substrate 1, a sample carrying area 3 located on the carrying surface of the slide substrate 1, removable positioning coordinates 4 disposed within the sample carrying area 3, and at least two permanent boundary markings 5 ​​disposed around the sample carrying area 3.

[0057] Specifically, the removable positioning coordinate 4 is disposed inside the sample carrying area 3, made of transient material that fades in the pathological specimen pretreatment liquid, and has a geometric configuration that indicates the center and orientation of the sample placement. It provides a center and angle reference when the sample 6 is spread or retrieved, and fades in subsequent pretreatment to avoid obscuring the sample image. At least two permanent boundary markers 5 are directly formed on the carrying surface of the slide substrate 1. The permanent boundary marker 5 includes a graphic with a light-transmitting opening or surface microstructure. The imaging surface of the permanent boundary marker 5 is within the same depth of focus as the imaging surface of the sample 6 in the sample carrying area 3. At least two permanent boundary markers 5 (as shown in the attached figures 51, 52, 53, 54) jointly define the scanning boundary 13 of the sample carrying area 3 on the carrying surface, and at least one of the at least two permanent boundary markers 5 (as shown in the attached figures 51, 52, 53, 54) defines the scanning boundary 13 of the sample carrying area 3. Figure 1 The identifier 54 in the lower right corner is geometrically different from the other permanent boundary identifiers (such as...). Figure 1 The markings 51 (top left), 52 (top right), and 53 (bottom left) are used by the digital slice scanner to identify and determine the spatial orientation of the slide. During scanning and image processing, each of the permanent boundary markings 5 ​​serves as a focusing reference for the digital slice scanner to perform axial height measurement to fit a reference focal plane, and as a spatial position reference for cropping the scanned image and aligning multiple slides.

[0058] It is understandable that by coordinating the placement of removable positioning coordinates 4 and permanent boundary markers 5 on the bearing surface, a complete physical reference connection for the entire process of sample preparation and digital scanning analysis is achieved. The removable positioning coordinates 4, while constraining the initial position and orientation consistency of the slide or smear, completely eliminate visual interference to subsequent pathological images through fading during pretreatment; while the permanent boundary markers 5, directly formed on the bearing surface, are confocal with the sample, eliminating axial focal plane deviation caused by differences in the thickness of the coverslip or sealing medium in traditional structures. This not only provides high-precision Z-axis height measurement and global reference focal plane fitting for low-contrast samples to prevent defocusing and missed scans, but also provides a spatial registration reference for digital images under a unified coordinate system for unambiguous angular determination, standardized image cropping, and multi-slice cropping through morphological heterogeneity and the definition of the scanning boundary 13.

[0059] In this embodiment of the application, the geometric configuration and material fading mechanism of the removable positioning coordinate 4 are further explained. Figure 2 This is a schematic diagram of the glass slide usage process provided in one embodiment of this application.

[0060] Specifically, see Figure 1 and Figure 2The removable positioning coordinate 4 includes mutually perpendicular intersecting horizontal and vertical reference lines, the intersection of which is located at the geometric center of the sample-bearing area 3. At least one of the horizontal or vertical reference lines has an asymmetrical direction indicator structure (e.g., arrows, asymmetrical ends, specific notches, or short horizontal line markings) to indicate the head of the slide or a predetermined tissue anatomical direction. The removable positioning coordinate 4 is made of a transient material, including water-soluble polymers (such as polyvinyl alcohol, water-soluble cellulose), alcohol-soluble resins, or soluble waxes. In the microscopic specimen preparation process, such as... Figure 2 As shown in step S1, the operator aligns and spreads the sample 6 (such as a serial tissue section or organoid) along its center and long axis according to the removable positioning coordinates 4; upon entering... Figure 2 After the sample pretreatment process shown in step S2, the transient material is completely removed or faded by dissolution, peeling or chemical degradation when it comes into contact with conventional pathological reagents such as aqueous treatment solution, ethanol, xylene or clearing agent, so that the final specimen no longer has a visible image of the positioning coordinates when it enters step S3 for digital scanning.

[0061] Understandably, the intersecting horizontal and vertical geometric configuration with directional indicators provides a physical reference for manual slide retrieval or cell smear preparation, significantly reducing the translational and rotational dispersion of the same tissue block when spread on multiple slides. At the same time, the chemical dissolution properties of pathological pretreatment reagents and transient materials enable in-situ automatic removal without the need for additional cleaning procedures, completely eliminating the obstruction and interference of positioning markers on subsequent high-magnification tissue pathological morphology observation and image algorithm analysis.

[0062] In this embodiment of the application, the quantity distribution, boundary definition, heterogeneous error prevention, and hierarchical coding characteristics of the permanent boundary marker 5 are further explained. Figure 3 This is a schematic diagram of a permanent boundary marker combination method provided in an embodiment of this application. Figure 4 This is a schematic diagram of multi-layer registration provided for an embodiment of this application.

[0063] Specifically, see Figure 1 , Figure 3 and Figure 4 Preferably, four permanent boundary markers 5 are provided, and the four permanent boundary markers 5 are respectively arranged on the four corners of the sample carrying area 3, and the geometric centers or feature edges of the four permanent boundary markers 5 correspond to the four rectangular vertices of the scanning boundary 13. Figure 1 and Figure 3As shown in scheme C, among the four permanent boundary markers 5, at least one permanent boundary marker (such as the lower right marker 54) differs geometrically from the other permanent boundary markers. This difference manifests in at least one of the following: graphic outline (e.g., differences between rectangles, circles, triangles, L-shapes, or crosses), opening orientation, geometric dimensions, or internal microstructure, to form a globally unique angular mis-proofing reference. Furthermore, as... Figure 3 and Figure 4 As shown, the morphological combination or coded arrangement of multiple permanent boundary markers 5 are also used to characterize the sequential slice number 14, sample batch, or staining type (e.g., ...). Figure 4 Layer 1 was stained with HE, layer 2 with IHC-A, and layer 3 with IHC-B. Each layer corresponds to a specific combination of corner markers.

[0064] Understandably, the symmetrically distributed markers at the four corners directly define the physical geometric vertices of the rectangular scanning boundary 13, enabling the scanner to directly extract a standardized image field of view with a uniform aspect ratio based on the four vertices. The introduction of heterogeneous features completely eliminates the identification ambiguity caused by the rotation of the slide in the 0°, 90°, 180°, or 270° direction during automated loading or scanning acquisition. Meanwhile, morphological encoding enables multimodal continuous slices to automatically complete the binding of layer sequence and staining information during the scanning stage, preventing manual pairing errors and providing a reliable data organization basis for the subsequent image registration module to construct an image dataset in a unified coordinate system.

[0065] In this embodiment of the application, the micro-processing configuration, confocal height tolerance, and assembly and packaging structure of the permanent boundary marker 5 are further described. Figure 5 This is a schematic diagram of the cross-sectional positional relationship provided in the embodiments of this application.

[0066] Specifically, see Figure 5 The permanent boundary marker 5 is formed on the bearing surface of the slide substrate 1 to form a boundary marker layer 15. The permanent boundary marker 5 includes a surface micro-etched structure, a laser-induced refractive index modified structure, a metal oxide thin film pattern, or a solvent-resistant inorganic printed pattern, and its outline line width is preferably 1 micrometer to 100 micrometers. The protrusion height or recess depth of the permanent boundary marker 5 relative to the bearing surface is strictly controlled between 0.1 micrometers and 100 micrometers. The slide assembly also includes a coverslip 7 covering the sample bearing area 3 and a sealing medium 8 filling the space between the bearing surface of the slide substrate 1 and the coverslip 7. The permanent boundary marker 5 and the sample 6 in the sample layer 16 are jointly encapsulated between the slide substrate 1 and the coverslip 7, so that the imaging surface of the boundary marker layer 15 and the target imaging surface of the sample layer 16 are both within the same depth of field covered by the digital slice scanner focusing module 10, and a continuous reference focal plane 17 is generated by fitting.

[0067] Understandably, by directly and integrally fabricating the permanent boundary marker 5 onto the substrate bearing surface and controlling the height fluctuations to the micrometer level, the boundary marker and the sample are placed in the same physical medium layer. This coplanar and confocal design eliminates the axial focusing deviation caused by the thickness tolerance of the coverslip itself, tilted bonding, or uneven thickness of the sealing adhesive in traditional solutions. This ensures that the optimal focal height acquired by the scanner at the marker can directly and accurately reflect the spatial focal plane of the sample layer 16, providing a high-precision physical reference plane for rapid focusing of large-area, low-contrast biological samples.

[0068] In this embodiment, the global functional partitioning and anti-obstruction avoidance layout of the slide substrate 1 are further described.

[0069] Specifically, see Figure 1 The slide substrate 1 is a long strip-shaped structure. One end of its bearing surface is provided with a label area 2 for recording sample information (which can be used to affix paper labels, laser-mark QR codes, or write pathology numbers). The remaining area is divided into the sample bearing area 3. The four permanent boundary markers 5 (51, 52, 53, 54) are located near the edge of the sample bearing area 3, but with a preset clamping clearance distance from the outer periphery of the slide substrate 1. At the same time, the distance between each permanent boundary marker 5 and the geometric center of the removable positioning coordinate 4 located in the center of the sample bearing area 3 is greater than the preset sample spreading distance.

[0070] Understandably, setting a perimeter clamping clearance ensures that the boundary marker 5 is not obstructed by the mechanical structure when the slide is fixed and clamped by the slide loading mechanism, mechanical grippers, or scanning stage of the automatic slide scanner, thus guaranteeing complete exposure of the optical field of view. Meanwhile, maintaining sufficient sample spreading distance between the boundary marker and the center positioning coordinates ensures that after the tissue sample drifts or the organoid spreads, the sample edge still falls within the inner area surrounded by the boundary marker, preventing biological tissue from directly covering the boundary marker and affecting the scanner's high-precision extraction of the marker edge contour and axial height measurement.

[0071] Please refer to the following: Figure 6 , Figure 6 This is a schematic diagram of a digital slice scanning device provided in an embodiment of this application. Figure 6 As shown, the digital slice scanning device 20 includes: an image acquisition mechanism 21, an axial focusing mechanism 22, and a control processing module 23.

[0072] Specifically, the image acquisition mechanism 21 (e.g., including an objective lens, microscopic optical components, and an image sensor) is used to acquire microscopic images of the slide (including low-magnification preview images and high-magnification slice images); the axial focusing mechanism 22 (e.g., including a piezoelectric ceramic driver, a stepper motor, and a high-precision slide assembly) is used to drive the image acquisition mechanism 21 or the stage carrying the slide to move along the optical axis (Z-axis direction) to achieve continuous adjustment of the microscopic imaging focal length; the control processing module 23 is communicatively connected to the image acquisition mechanism 21 and the axial focusing mechanism 22, and the control processing module 23 is configured to perform the following control and processing operations:

[0073] First, the image acquisition mechanism 21 is controlled to acquire a low-magnification preview image of the slide, and the image coordinates of at least two permanent boundary markers 5 (such as the markers 51, 52, 53, and 54 at the four corners) are identified in the preview image using an image recognition algorithm (such as template matching, contour detection, or threshold segmentation). Second, the feature configuration and position of at least one permanent boundary marker (such as heterogeneous marker 54) that is geometrically different from the other permanent boundary markers are extracted and analyzed, thereby uniquely determining the current spatial orientation of the slide on the stage (such as 0°, 90°, 180°, or 270° direction) to achieve automatic rotation angle correction. Subsequently, the axial focusing mechanism 22 drives the system to perform axial height measurement scans in the areas where each of the permanent boundary markers 5 is located. The optimal focal plane height corresponding to each of the permanent boundary markers 5 is obtained through a sharpness evaluation algorithm. Based on the extracted optimal focal plane heights, a global reference focal plane 17 covering the entire sample carrying area 3 is constructed using a least squares plane fitting or surface interpolation algorithm. Next, based on the reference focal plane 17, the image acquisition mechanism 21 is controlled to perform high-magnification digital scanning within the sample carrying area 3 (high-magnification imaging and image stitching of sample 6 within a small axial offset range defined by the reference focal plane 17) to obtain high-definition slice scan images. Finally, based on the scanning boundary 13 jointly defined by at least two permanent boundary markers 5, the acquired and stitched slice scan images are standardized and the image size is normalized to output digital slices with a unified field of view reference.

[0074] Understandably, the digital slide scanning device 20, by combining the image acquisition mechanism 21, the axial focusing mechanism 22, and the control and processing module 23, fully utilizes the multi-dimensional physical properties of the permanent boundary markers 5 directly formed on the slide's bearing surface. By anchoring the focusing height reference to the high-contrast permanent boundary markers 5, a precise reference focal plane 17 can be constructed without relying on the contrast of the biological sample itself. This completely solves the problem of defocusing and missed scans that easily occur in traditional full-stroke focusing processes for low-contrast samples such as micro-biopsy tissues and tumor organoids. At the same time, the automatic orientation recognition and standard frame cropping achieved by the control and processing module 23 based on the heterogeneous markers and scanning boundaries 13 ensure a high degree of standardization in the spatial coordinates and image size of the output digital slides, significantly improving the operational efficiency and slide quality of automated batch scanning of pathological slides.

[0075] Please refer to the following: Figure 7 , Figure 7 This is a schematic flowchart of a digital slice scanning method provided in an embodiment of this application. Figure 7 The digital slicing scanning method shown includes at least the following steps: S100: acquiring a preview image of a glass slide, identifying the image coordinates of at least two permanent boundary markers in the preview image, and determining the spatial orientation of the glass slide based on at least one permanent boundary marker that is geometrically different from the other permanent boundary markers; S200: performing axial focusing and height measurement in the area where each permanent boundary marker is located to obtain the optimal focal plane height of each permanent boundary marker; S300: generating a reference focal plane covering the sample bearing area based on each optimal focal plane height; S400: performing digital scanning on the sample within the sample bearing area based on the reference focal plane to obtain an initial slice image; S500: performing size normalization cropping on the initial slice image based on the scanning boundary jointly defined by at least two permanent boundary markers.

[0076] In this embodiment, the digital slice scanning method is applied to the glass slide with boundary markings and auxiliary focusing described in the foregoing embodiments, and can be controlled and executed by the control and processing module 23 of the aforementioned digital slice scanning device 20, so as to achieve efficient focusing, standardized scanning and imaging of low-contrast biological samples 6 in the sample carrying area 3, and subsequent spatial alignment of multiple slices.

[0077] Specifically, the implementation process of each of the above steps is as follows:

[0078] In step S100, the control processing module 23 controls the image acquisition mechanism 21 to capture a low-magnification full-view preview image of the slide. In the preview image, template matching, edge contour extraction, or threshold segmentation algorithms are used to locate each permanent boundary marker 5 (such as the markers 51, 52, 53, and 54 at the four corners) and extract their pixel center coordinates. Furthermore, the geometric contour or notch direction of the heterogeneous permanent boundary marker 54 with asymmetric configuration is identified and extracted, and the rotation angle of the slide relative to the standard optical axis coordinate system is calculated accordingly (such as determining whether it is in the state of 0°, 90°, 180° or 270°), thus completing the initial orientation calibration and angle correction of the image.

[0079] In step S200, the control processing module 23 controls the axial focusing mechanism 22 to sequentially drive the optical system to move to the spatial region where each permanent boundary marker 5 is located, and performs axial scanning sampling in the optical axis direction (Z-axis direction). Using sharpness evaluation algorithms such as the Laplacian gradient function, frequency domain energy function, or gray-level variance function, the optimal focal plane height at which the image is sharpest at each permanent boundary marker 5 is calculated. (i.e., spatial focal coordinates) ).

[0080] In step S300, the control processing module 23 summarizes the spatial focus coordinates of each permanent boundary marker 5. The plane equation is calculated using the least squares plane fitting method. Alternatively, spline interpolation or polynomial surface fitting can be used to construct a continuous and smooth three-dimensional spatial surface, thereby fitting and generating a global reference focal plane 17 covering the entire sample bearing area 3.

[0081] In step S400, the control processing module 23 plans the scanning motion path under high magnification based on the generated reference focal plane 17. When the image acquisition mechanism 21 moves and scans along the sample carrying area 3 field by field, it sets the imaging focal length in real time according to the Z-axis height given by the reference focal plane 17, and within a small axial offset range defined by the reference focal plane 17 (e.g., ... Within the scope of the image, local fine-tuning and focusing can be performed or high-magnification image acquisition can be directly triggered. The acquired local microscopic fields of view are then stitched together to form a high-resolution initial slice image containing the complete sample 6.

[0082] In step S500, the control processing module 23 establishes a perspective or affine geometric transformation relationship from the actual scanning coordinate system to the preset standard output canvas based on the geometric scanning boundary 13 formed by the permanent boundary markers 5 determined in step S100. It performs translation, rotation correction and boundary clipping on the initial slice image and outputs a standardized digital slice image with a uniform image size, uniform aspect ratio and uniform starting origin.

[0083] Understandably, this digital slide scanning method transforms the traditional process of blindly searching for focus throughout the entire sample body into a process of accurately measuring the height at five permanent boundary markers with significant optical contrast and fitting a reference focal plane 17. This completely eradicates the problem of defocusing and missed scans caused by the inability to find a focal point during the scanning process of weak contrast specimens such as micro-biopsy and tumor organoids from the perspective of algorithm and control process. At the same time, through automatic direction determination of heterogeneous markers and size normalization and cropping based on the scanning boundary 13, the acquired digital slides have highly standardized physical and geometric benchmarks. This provides standardized data support for the accurate registration of multimodal continuous slide images in a unified spatial coordinate system and the training of AI pathological models, such as hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) staining with different antibodies.

[0084] It is understood that the glass slides, devices, and methods with boundary markings and auxiliary focusing provided in the embodiments of this application realize the front-end and back-end physical connection between sample preparation and digital scanning by constructing a dual benchmark collaborative system of "removable positioning coordinates during slide preparation" and "permanent boundary markings during scanning" on the glass slide bearing surface. On the one hand, the positioning coordinates can provide an intuitive physical center and orientation reference during the sample spreading or retrieval stage, and automatically fade away in subsequent preprocessing. This not only suppresses random tilting and eccentricity of the slides caused by manual retrieval from the source, but also avoids the obstruction and interference of markers on the final pathological image analysis. On the other hand, the permanent boundary markers are directly formed on the bearing surface and are in the same focal depth range as the sample. Structurally, this completely eliminates the axial focal plane deviation caused by uneven thickness of the sealing medium or coverslip due to the traditional marking on the outer surface of the coverslip. Combined with the light transmission configuration and heterogeneous error-proof design, it can provide the digital slide scanner with a precise and unique spatial orientation determination, a Z-axis reference focal plane fitting benchmark covering the entire field of view, and a standardized scanning boundary without affecting the imaging optical path. This effectively solves the technical defects such as defocusing, missed scanning, and inaccurate scanning range that are very likely to occur in automated scanning of low-contrast biological samples such as tumor organoids and micro-biopsy tissues.

[0085] Those skilled in the art should recognize that the above embodiments are only used to illustrate this application and are not intended to limit this application. Any appropriate changes and variations made to the above embodiments within the essential spirit and scope of this application fall within the scope of protection claimed in this application.

Claims

1. A glass slide with boundary markings and auxiliary focusing, characterized in that, The glass slide includes: The slide substrate, the sample carrying area located on the carrying surface of the slide substrate, the removable positioning coordinates set in the sample carrying area, and at least two permanent boundary markers set around the sample carrying area; The removable positioning coordinates are made of transient material that fades in the pathological specimen pretreatment liquid and have a geometric configuration that indicates the center and orientation of the sample placement. At least two of the permanent boundary markers are directly formed on the bearing surface. The permanent boundary markers include graphics with light-transmitting openings or surface microstructures. The imaging surface of the permanent boundary markers is within the same depth of focus as the imaging surface of the sample in the sample bearing area. At least two of the permanent boundary markers jointly define the scanning boundary of the sample carrying area on the bearing surface, and at least one of the at least two permanent boundary markers is geometrically different from the other permanent boundary markers, in order to determine the spatial orientation of the slide; Each of the permanent boundary markers serves as a focusing reference for the digital slice scanner to perform axial height measurement to fit the reference focal plane, and as a spatial position reference for cropping and aligning multiple slices of the scanned image.

2. The glass slide with boundary markings and auxiliary focusing according to claim 1, characterized in that, The removable positioning coordinates include mutually perpendicular horizontal and vertical reference lines. The intersection of the horizontal and vertical reference lines is located at the geometric center of the sample bearing area, and at least one of the horizontal or vertical reference lines is provided with an asymmetrical direction indication structure.

3. The glass slide with boundary markings and auxiliary focusing according to claim 1, characterized in that, The number of permanent boundary markers is four. The four permanent boundary markers are respectively set on the four corners of the sample carrying area, and the geometric center or feature edge of the four permanent boundary markers correspond to the four vertices of the scanning boundary.

4. The glass slide with boundary markings and auxiliary focusing according to claim 3, characterized in that, The at least one permanent boundary marker that is geometrically different from the other permanent boundary markers includes: It differs from the other permanent boundary markers in at least one of the following: graphic outline, opening orientation, size, or internal microstructure; The morphological combination or coded arrangement of multiple permanent boundary markers is used to indicate the slice sequence number, sample batch, or staining type.

5. The glass slide with boundary markings and auxiliary focusing according to any one of claims 1 to 4, characterized in that, The permanent boundary marker is formed on the bearing surface, and the protrusion height or recess depth of the permanent boundary marker relative to the bearing surface is 0.1 micrometers to 100 micrometers; The slide also includes a coverslip covering the sample-bearing area and a sealing medium located between the slide substrate and the coverslip. The permanent boundary marker and the sample in the sample-bearing area are encapsulated together between the slide substrate and the coverslip.

6. The glass slide with boundary markings and auxiliary focusing according to claim 1, characterized in that, The transient material includes water-soluble materials, alcohol-soluble materials, or materials soluble in organic solvents; The removable positioning coordinates are configured to be dissolved, peeled, or decolorized by the corresponding treatment reagents when the pathological specimen undergoes at least one step of dewaxing, rehydration, staining, or clearing.

7. The glass slide with boundary markings and auxiliary focusing according to claim 1, characterized in that, The permanent boundary markings include surface micro-etched structures, laser refractive index modified structures, metal oxide thin film patterns, or solvent-resistant inorganic printed patterns formed on the bearing surface; The outline width of the permanent boundary marker is 1 micrometer to 100 micrometers.

8. The glass slide with boundary markings and auxiliary focusing according to any one of claims 1 to 5, characterized in that, One end of the slide substrate is provided with a label area for recording sample information; A preset clamping clearance distance is maintained between the permanent boundary marker and the outer edge of the slide substrate, and the distance between each permanent boundary marker and the geometric center of the removable positioning coordinate is greater than the preset sample spreading distance.

9. A digital slice scanning device, characterized in that, The device includes: An image acquisition mechanism is used to acquire microscopic images of the glass slide; An axial focusing mechanism is used to drive the image acquisition mechanism or the glass slide to move along the optical axis; and The control processing module is communicatively connected to the image acquisition mechanism and the axial focusing mechanism, and the control processing module is configured as follows: The image acquisition mechanism is controlled to acquire a preview image of the slide, and the image coordinates of the at least two permanent boundary markers are identified in the preview image; The spatial orientation of the slide is determined based on at least one permanent boundary marker that is geometrically different from the other permanent boundary markers; The axial focusing mechanism is controlled to perform axial height measurement in the areas where each of the permanent boundary markers is located to obtain the corresponding optimal focal plane height, and a reference focal plane covering the sample bearing area is constructed by fitting each of the optimal focal plane heights. Based on the reference focal plane, the image acquisition mechanism performs high-magnification digital scanning on the sample bearing area to obtain slice scan images; and The sliced ​​scan image is standardized and cropped based on the scan boundary defined by the at least two permanent boundary markers.

10. A digital slice scanning method, characterized in that, The method includes: Acquire a preview image of the slide, identify the image coordinates of the at least two permanent boundary markers in the preview image, and determine the spatial orientation of the slide based on the at least one permanent boundary marker that is geometrically different from the other permanent boundary markers; Axial focusing height measurement is performed in the area where each of the permanent boundary markers is located to obtain the optimal focal plane height of each of the permanent boundary markers. A reference focal plane covering the sample bearing area is generated by fitting the optimal focal plane height of each of the above-described focal planes. A digital scan is performed on the sample within the sample-bearing area based on the reference focal plane to obtain an initial slice image; and The initial slice image is sized and cropped based on the scan boundary defined by the at least two permanent boundary markers.

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