A tissue multi-microscopic image registration method based on physical dot array markers

Abstract

The embodiment of the application discloses a tissue multi-microscopic image registration method based on a physical dot array marker. The method belongs to the technical field of tissue section image registration. The method comprises the following steps: obtaining a tissue section; forming a physical dot array on the surface of the tissue section by laser engraving; performing multi-staining on the tissue section with the physical dot array to obtain multi-staining results; collecting a plurality of microscopic images containing the physical dot array for the multi-staining results; and registering the plurality of microscopic images by taking the physical dot array as a registration reference. Based on the method, the spatial position alignment of the same tissue region in the plurality of microscopic images can be realized without cell nucleus re-staining, the interference of nuclear staining signals on marker coloration is avoided, and the problem that negative staining results cannot be accurately positioned and registered is solved.

Description

Technical Field

[0001] This invention relates to the field of tissue section image registration technology, and in particular to a method for multiple microscopic image registration of tissues based on physical dot matrix markers. Background Technology

[0002] Multiplex staining techniques (including multiplex immunohistochemistry, multiplex in situ hybridization, multiplex special staining, and the combined application of multiple staining techniques) enable the simultaneous in situ quantitative detection of multiple molecular indicators such as proteins, nucleic acids, carbohydrates, and lipids on a single tissue section. This is a core technique for analyzing tissue spatial heterogeneity and revealing the mechanisms of disease development. Image registration is a crucial step in the quantitative analysis of multiplex staining results, referring to matching and aligning each pixel in the multiple microscopic images generated by multiplex staining to the same planar coordinate position. Current mainstream methods for tissue microscopic image registration include: 1. Nuclear registration: This method is the most widely used. It requires a nuclear counterstaining step after each round of staining, followed by image registration using the nucleus as a spatial reference. However, nuclear staining signals are continuously superimposed on the tissue section, which can cause irreversible interference to the color development signals of subsequent markers.

[0003] 2. Registration based on intrinsic tissue features: This method uses vascular intersections, unique structural contours, and intrinsic pigment points within the tissue as references for image registration. However, multiple rounds of operation may damage or even eliminate these intrinsic features, potentially leading to registration failure.

[0004] 3. Registration using software tools: Although existing software tools can perform image registration of multiple staining results in tissues, it requires a digital slide scanner to acquire the stained tissue images. The specialized equipment and commercial software are expensive, making them unaffordable for primary healthcare and research institutions. Furthermore, for negatively stained images lacking identifiable spatial features, software tools often produce poor registration results.

[0005] Due to the aforementioned objective limitations of existing image registration methods, the clinical translation and large-scale application of tissue multiple staining techniques are constrained. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects of existing tissue microscopic image registration methods and provide a tissue multiple microscopic image registration method based on physical dot matrix labeling, which eliminates the dependence on cell nuclear counterstaining, significantly reduces detection costs and operational thresholds, and achieves accurate registration of negative staining results.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A tissue multiple microscopic image registration method based on physical dot matrix markers includes the following steps: S1: Prepare paraffin-embedded tissue sections with a thickness of 3-5 μm, bake the sections at 60-65 ℃ for 2-4 hours, and then allow them to cool naturally to room temperature; S2: Fix the tissue slice onto the platform of a non-contact labeling device, and form a permanent physical dot matrix mark on the surface of the effective analysis area of ​​the tissue slice by laser ablation. The physical dot matrix consists of multiple discrete mark points that penetrate the tissue layer and reach the surface of the glass slide. S3: Perform multiple rounds of multiple staining on tissue sections with the physical dot matrix markers formed, without the need for counterstaining of cell nuclei after each round of staining; S4: For each round of staining results, acquire a microscopic image containing at least 3 of the marked points, and all microscopic images contain the same physical dot matrix region; S5: Using the geometric center of the marker points of the permanent physical dot matrix as the coordinate anchor point, rigidly register multiple microscopic images to achieve spatial alignment of the same tissue regions.

[0008] Furthermore, the physical dot matrix is ​​a regularly arranged dot matrix, and the shape of the marker points can be any one of circles, ellipses, squares or triangles.

[0009] Furthermore, the physical dot matrix is ​​arranged in a rectangular array, with a center-to-center distance of 0.5-1.0 mm between adjacent markers and a diameter of 10-100 μm for each marker.

[0010] Furthermore, the physical dot matrix is ​​arranged in a rectangular array, with the area of ​​the rectangular region formed by four adjacent marker points being 0.25-1 mm. 2 .

[0011] Furthermore, the non-contact marking device mentioned in step S2 is a carbon dioxide laser engraving machine or an ultraviolet laser engraving machine, with a laser power of 1-4 W and a laser moving speed of 300-900 mm / min.

[0012] Further, the acquisition of a microscopic image containing at least three of the marked points in step S4 specifically involves: obtaining a full-section staining result image by scanning the tissue sections that have undergone each round of staining, and acquiring a microscopic image of the region defined by the physical dot matrix from the full-section staining result image; or acquiring a microscopic image of the region defined by the physical dot matrix in the tissue sections that have undergone each round of staining using a microscope.

[0013] Furthermore, the method also includes: sequentially inverting, converting grayscale, and performing pseudocolor processing on the registered multiple microscopic images, and then overlaying the layers to generate a pseudocolor overlay image for displaying the results of multiple staining.

[0014] Furthermore, the multiple staining includes at least one round of nuclear staining; the method further includes: S6: Extract the cell nucleus from the microscopic image corresponding to the cell nucleus staining, and divide it into single-cell regions using an image segmentation algorithm, which are then used as regions of interest; S7: Map the region of interest to each of the other registered microscopic images to obtain the signal intensity of each staining index within the region of interest in each of the other microscopic images; S8: For regions of interest, perform quantitative analysis at the single-cell level based on the signal intensity of each staining index.

[0015] Furthermore, the multiple staining includes any one or more of the following: multiple immunohistochemical staining, multiple immunofluorescence staining, multiple in situ hybridization staining, and multiple special chemical staining.

[0016] The present invention has at least the following beneficial effects: 1. Fundamentally eliminate nuclear signal interference: There is no need to perform nuclear counterstaining after each round of staining, which completely avoids the superimposed interference of nuclear staining signals on the color development of markers, and at the same time reduces experimental steps and effectively shortens the detection cycle.

[0017] 2. Completely solves the problem of registration for negative staining: A permanent physical matrix that penetrates the tissue layer is used as the registration reference. The marker is not affected by any staining, elution, or hydration operations. Even if the staining result is completely negative in a certain round, pixel-level accurate registration can still be achieved through the matrix.

[0018] 3. Extremely low economic and technical barriers: Only a regular desktop laser engraving machine and microscope are required, without the need for expensive multispectral imaging systems and commercial analysis software, reducing the detection cost from tens of thousands of yuan to thousands of yuan; the operation process is simple and easy to promote and apply in grassroots laboratories and clinical units.

[0019] 4. Excellent versatility and scalability: Applicable to all types of tissue sections and all mainstream multiple staining techniques, it can be seamlessly integrated with existing experimental procedures, while supporting standardized tissue region division and cross-sectional comparison of multiple batches of experiments, providing a unified technical platform for large-scale clinical research.

[0020] 5. This method can be widely applied in spatial omics and clinical pathology research scenarios such as tumor microenvironment analysis, neurodevelopment research, and immunohistochemical quantitative detection.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0022] Figure 1 This is a comparison diagram of the forming effect of physical dot matrix under different laser parameters in Embodiment 1 of the present invention.

[0023] Figure 2 This is an overall flowchart of the method for accurate registration of tissue multiple staining images using physical dot matrix markers as described in this invention.

[0024] Figure 3 This is a single-channel microscopic image of undifferentiated thyroid carcinoma tissue in Example 3 of the present invention, after hematoxylin staining of cell nuclei and analysis of eight immunohistochemical indicators.

[0025] Figure 4 This is a diagram illustrating the process of cell nucleus extraction and single-cell region division in Example 3 of the present invention.

[0026] Figure 5 This is a cumulative grayscale value distribution diagram of the eight indicators in Embodiment 3 of the present invention.

[0027] Figure 6 This is a distribution diagram of the number of positive cells for eight indicators in Example 3 of the present invention.

[0028] Figure 7 This is a histogram of single-cell area distribution in Embodiment 3 of the present invention.

[0029] Figure 8 This is a distribution diagram of the positive cell area for eight indicators in Example 3 of the present invention.

[0030] Figure 9 This is a spatial localization map of positive cells for eight indicators in Example 3 of the present invention.

[0031] Figure 10 This is a single-channel pseudo-color image of eight indicators in Embodiment 3 of the present invention.

[0032] Figure 11 This is a pseudo-color overlay image of eight-fold immunohistochemical staining in Example 3 of the present invention.

[0033] Figure 12 This is a map showing the location of the tissue regions collected from the thymoid thyroid carcinoma tissue in Example 4 of the present invention (numbers 1-3 are the tumor periphery regions, and numbers 4-6 are the tumor center regions).

[0034] Figure 13 The images shown are: (left) original microscopic image and manually assessed percentage of positive cells, (middle) pseudocolor and single-cell segmentation map, and (right) magnified view of the pseudocolor and single-cell segmentation map in the edge region of tumor No. 1 in Embodiment 4 of the present invention.

[0035] Figure 14 This is a pseudo-color overlay image of immunohistochemical staining of four indicators in the tumor margin and central regions in Example 4 of the present invention.

[0036] Figure 15This is a comparison diagram of the average cell area in the tumor margin region and the central region in Example 4 of the present invention.

[0037] Figure 16 This is a comparison of the average gray values ​​of POU2F3 cells in the tumor edge region and the central region in Example 4 of the present invention.

[0038] Figure 17 This is a comparison diagram of the proportion of Tuft and non-Tuft cells in the tumor edge region and center region in Example 4 of the present invention.

[0039] Figure 18 This is a comparison of the average gray values ​​of POU2F3 cells of Ki67+ / - in the tumor margin region and central region of Example 4 of the present invention. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0041] Example 1: Optimization of Physical Dot Laser Engraving Parameters In this embodiment, paraffin-embedded human tonsil tissue was serially sectioned to a thickness of 4 μm. After baking at 65°C for 2 hours, the sections were allowed to cool naturally to room temperature. A desktop CO2 laser engraving machine was used to engrave a physical lattice of different parameters on the tissue section surface. The theoretical diameter of the marker points was set to 50 μm, the spacing between the points was 0.707 mm, and the area of ​​the rectangular region enclosed by four points was 0.5 mm². 2 The markers are circular. After engraving, the sections are routinely dewaxed, stained with hematoxylin, and mounted with neutral resin. The sections are then scanned using a bright-field pathology slide scanner (SQS-12P, Shengqiang Technology) to observe the dot matrix formation effect.

[0042] Experimental results are as follows Figure 1 As shown: When the laser moving speed is 300 mm / min, some of the marked tissues were not completely ablated when the laser power is 1 W, and the diameter of the over-marked tissues increased significantly and the edges became irregular when the laser power is 4 W.

[0043] When the laser moving speed is 1200 mm / min, the marked point shows obvious trailing marks.

[0044] When the laser power is 2 W and the moving speed is 600 mm / min, the diameter of the marking point is about 50 μm, the edge is neat and clear, and it penetrates the tissue layer to reach the surface of the glass slide.

[0045] Based on the above results, the optimal laser engraving parameters for this invention are determined to be: laser power 2 W and laser moving speed 600 mm / min.

[0046] Example 2: Overall process of registration method This embodiment details the complete operation process of the method described in this invention, such as... Figure 2 As shown: Tissue section preparation: The paraffin-embedded tissue block was cut into continuous sections with a thickness of 4 μm, attached to a glass slide to prevent detachment, baked at 65℃ for 2 hours and then cooled naturally.

[0047] Physical dot matrix engraving: Fix the glass slide on the platform of the laser engraving machine, adjust the laser focal length to the tissue surface, import the preset square array dot matrix pattern, set the laser power to 2 W and the moving speed to 600 mm / min, start the engraving program, and form a permanent physical dot matrix in the effective analysis area of ​​the tissue.

[0048] Multiple staining procedure: Perform multiple staining on the slides according to the standard experimental protocol. After each round of staining, directly acquire the image without counterstaining the cell nuclei. If single-cell quantitative analysis is required, a cell nuclei staining can be added before the first round of staining or after the last round of staining.

[0049] Microscopic image acquisition: After each round of staining, a microscopic image containing at least 3 markers is acquired using a microscope or whole-section scanner, ensuring that all images contain the same dot matrix area.

[0050] Image rigid registration: Using the Register Virtual Stack Slices plugin of the open-source image analysis software FIJI, multiple images are automatically rigidly registered with the geometric center of the physical dot matrix markers as the coordinate anchor point to achieve spatial alignment.

[0051] Pseudocolor overlay generation: After registration, each channel image is sequentially inverted, converted to grayscale, and processed with pseudocolor. Photoshop software is then used to overlay the layers, generating a multi-index pseudocolor overlay image.

[0052] Single-cell quantitative analysis: Based on cell nuclear staining images, the StarDist plugin is used to automatically extract cell nuclei, and the single-cell region is segmented and a region of interest (ROI) is generated using the watershed algorithm; the ROI is mapped to each registered staining image, the staining signal intensity of each single cell is extracted, and subsequent statistical analysis is performed.

[0053] It should be noted that this method is not only applicable to multiple immunohistochemical staining, but also to multiple immunofluorescence staining, multiple in situ hybridization staining, multiple special chemical staining, and combined staining of two or more of the above staining techniques. All of the above staining types involve multiple rounds of staining operations and corresponding image registration requirements.

[0054] Example 3: Eight-fold immunohistochemical staining analysis of undifferentiated thyroid carcinoma tissue In this embodiment, a paraffin section of undifferentiated thyroid carcinoma was subjected to physical dot matrix labeling, hematoxylin nuclear staining, and eight-fold immunohistochemical staining (CD56, CK, CD4, TG, CD3, CD8, MC, Ki67), and image registration and quantitative analysis were performed according to the procedure described in Example 2.

[0055] Experimental results are as follows Figures 3 to 11 As shown: Figure 3 Single-channel microscopic images of hematoxylin nuclear staining and eight immunohistochemical markers are shown. All images clearly show physical dot matrix markers in completely consistent positions.

[0056] Figure 4 The process of extracting cell nuclei and segmenting single cells based on hematoxylin nuclear staining results was demonstrated, and the segmentation results were highly consistent with the actual cell distribution.

[0057] Figures 5-8 The cumulative gray value, number of positive cells, single cell area, and distribution of positive cell area of ​​eight indicators were displayed respectively. The quantitative results objectively reflect the expression differences of each indicator.

[0058] Figure 9 The spatial localization of positive cells for each indicator is shown.

[0059] Figure 10 and Figure 11 The images show single-channel pseudocolor and pseudocolor overlay images of eight indicators, clearly and intuitively presenting the spatial distribution and co-expression patterns of different cell subpopulations.

[0060] The results of this implementation show that precise registration of multiple microscopic images of tissues was achieved through physical dot arrays.

[0061] Example 4: Quadruple immunohistochemical staining analysis of thymoid thyroid carcinoma tissue In this embodiment, paraffin-embedded tissue sections from a case of thymoid thyroid carcinoma were subjected to physical dot matrix labeling, hematoxylin nuclear staining, and quadruple immunohistochemical staining (CK, POU2F3, CD117, Ki67), with a focus on analyzing cellular heterogeneity between the tumor center and periphery. Cells co-positive for CD117 and POU2F3 were defined as Tuft cells.

[0062] Experimental results are as follows Figures 12 to 18 As shown: Figure 12 This shows the locations of six rectangular tissue regions used for subsequent image analysis, with regions 1-3 representing the tumor periphery and regions 4-6 representing the tumor center.

[0063] Figure 13 The images show the original microscopic image and manually assessed percentage of positive cells under immunohistochemical staining for four indicators in tissue region 1 (left), pseudocolor and single-cell segmentation map (middle), and magnified local view of pseudocolor and single-cell segmentation map (right).

[0064] Figure 14 The pseudo-color overlay image of immunohistochemical staining of four indicators in the tumor margin and central regions visually demonstrates the spatial distribution characteristics of each indicator in different regions of the tumor, showing differences between the tumor margin and central regions.

[0065] Figure 15 The study showed no significant difference in the average cell area between the tumor periphery and the central region.

[0066] Figure 16 The average gray value of POU2F3 showed a significant difference between the tumor margin and central regions.

[0067] Figure 17 The results showed a significant difference in the proportion of Tuft cells between the tumor periphery and the central region.

[0068] Figure 18 The analysis of the difference in average gray values ​​of POU2F3 between Ki67+ and Ki67- cells in the tumor periphery and central regions showed that the average gray value of POU2F3 in Ki67+ cells in the tumor periphery region was significantly higher than that in Ki67- cells; in addition, the expression level of POU2F3 in Ki67- cells in the tumor central region was significantly higher than that in Ki67- cells in the tumor periphery region.

[0069] The above results suggest that Tuft cells are enriched at the forefront of tumor invasion. The results of this study demonstrate that precise registration of multiple microscopic images of tissues was achieved through a physical lattice.

[0070] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be readily implemented by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for registering multiple microscopic images of tissue based on physical lattice markers, comprising the steps of acquiring tissue sections, performing multiple staining on the tissue sections, acquiring multiple microscopic images and registering them, characterized in that, The method specifically includes the following steps: S1: Prepare paraffin-embedded tissue sections with a thickness of 3-5 μm, bake the sections at 60-65 ℃ for 2-4 hours, and then allow them to cool naturally to room temperature; S2: Fix the tissue slice onto the platform of a non-contact labeling device, and form a permanent physical dot matrix mark on the surface of the effective analysis area of ​​the tissue slice by laser ablation. The physical dot matrix consists of multiple discrete mark points that penetrate the tissue layer and reach the surface of the glass slide. S3: Perform multiple rounds of multiple staining on tissue sections with the permanent physical dot matrix markers formed, without the need for counterstaining of cell nuclei after each round of staining; S4: For each round of staining results, acquire a microscopic image containing at least 3 of the marked points, and all microscopic images contain the same physical dot matrix region; S5: Using the geometric center of the marker points of the permanent physical dot matrix as the coordinate anchor point, rigidly register multiple microscopic images to achieve spatial alignment of the same tissue regions.

2. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 1, characterized in that, The physical dot matrix is ​​a regularly arranged dot matrix, and the shape of the marker points can be any one of circles, ellipses, squares or triangles.

3. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 2, characterized in that, The physical dot matrix is ​​arranged in a rectangular array, with a center-to-center distance of 0.5-1.0 mm between adjacent markers and a diameter of 10-100 μm for each marker.

4. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 3, characterized in that, The physical dot matrix is ​​arranged in a rectangular array, with the area of ​​a rectangular region formed by four adjacent markers being 0.25-1 mm. 2 .

5. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 1, characterized in that, The non-contact marking device mentioned in step S2 is a carbon dioxide laser engraving machine or an ultraviolet laser engraving machine, with a laser power of 1-4 W and a laser moving speed of 300-900 mm / min.

6. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 1, characterized in that, The acquisition of a microscopic image containing at least three of the marker points in step S4 specifically involves: obtaining a full-section staining result image by scanning tissue sections that have undergone each round of staining, and acquiring a microscopic image of the region defined by the physical dot matrix from the full-section staining result image; Alternatively, a microscope can be used to acquire microscopic images of the regions defined by the physical dot matrix in tissue sections that have undergone each round of staining.

7. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 1, characterized in that, The method further includes: sequentially inverting, converting grayscale, and processing pseudocolor on multiple registered microscopic images, and then overlaying the layers to generate a pseudocolor overlay image for displaying the results of multiple staining.

8. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 1, characterized in that, The multiple staining includes at least one round of nuclear staining; the method further includes: S6: Extract the cell nucleus from the microscopic image corresponding to the cell nucleus staining, and divide it into single-cell regions using an image segmentation algorithm, which are then used as regions of interest; S7: Map the region of interest to each of the other registered microscopic images to obtain the signal intensity of each staining index within the region of interest in each of the other microscopic images; S8: For regions of interest, perform quantitative analysis at the single-cell level based on the signal intensity of each staining index.

9. The tissue multiple microscopic image registration method based on physical lattice markers according to claim 1, characterized in that, The multiple staining includes any one or more of the following: multiple immunohistochemical staining, multiple immunofluorescence staining, multiple in situ hybridization staining, and multiple special chemical staining.

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