Method and device for acquiring fluorescent region image in biological section

Abstract

The invention discloses a method and a device for acquiring a fluorescent area image in a biological section, and the method comprises the steps: integrating a low-power fluorescent objective lens and a high-power fluorescent objective lens on an electric objective lens converter, and enabling the two fluorescent objective lenses to be flexibly switched in the same light path through the electric objective lens converter, the method comprises the following steps of: scanning (image acquisition) a whole biological section under the illumination of a fluorescence excitation light source by adopting a low-power fluorescence objective lens to splice a complete tissue area image of the biological section, calculating to obtain X and Y coordinate values of an image boundary of a fluorescent tissue area in the biological section, and determining an area needing to be scanned; and the high-power fluorescent objective lens is used for accurately scanning the fluorescent tissue area in the biological section and splicing the image, so that the complete image of the fluorescent area in the biological section is obtained, the scanning area is reduced, and the working efficiency is improved.

Description

Technical Field

[0001] The present invention relates to a method and a device for acquiring biological slice images, and in particular to a method and a device for acquiring fluorescent area images in biological slices. Background Art

[0002] The existing method of obtaining the image of the fluorescent area in the biological slice is to use an ordinary fluorescent slice scanner for scanning. Since the fluorescent objective lens in the fluorescent slice scanner is a high-magnification fluorescent objective lens of 20 times or 40 times, it is necessary to first use a marker pen to mark the tissue area on the biological slice, and then use the fluorescent slice scanner to collect the biological slice to obtain the image including the marked tissue area, and then perform edge detection on the image to obtain the edge contour of the marked tissue area, and then perform connected area detection on the edge contour to obtain a set composed of several connected areas; perform convex hull detection on each connected area, and finally identify the tissue area marked by the marker pen. After obtaining the tissue area, the fluorescent objective lens of the fluorescent slice scanner is used to perform subsequent scanning and detection on the tissue area of ​​the biological slice, so as to obtain the image of the fluorescent area in the biological slice.

[0003] The existing method requires the user to mark the biological slices to be scanned with a marker in advance. When multiple biological slices need to be scanned, this step increases the workload. The tissue on the biological slice needs to be illuminated by a light source of a specific wavelength to emit fluorescence so that the slice scanner can collect images. When the user uses a marker to mark the area (under natural light), it can only roughly mark the location of the tissue area, which increases the scanning range of the subsequent slice scanning work. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method and device for obtaining fluorescent area images in biological slices, which can accurately obtain the pixel position of biological slice tissue and accurately locate the subsequent tissue scanning range without the need for a fluorescent marker.

[0005] The technical solution adopted by the present invention to solve the above technical problem is: a method for obtaining a fluorescent area image in a biological slice, comprising the following steps: Step 1: Place the biological slice to be scanned on a detection platform that can move in the horizontal plane along the mutually perpendicular X and Y directions, and turn on the fluorescent excitation light source to excite the biological slice to produce fluorescence; Step 2: Insert a low-magnification fluorescent objective lens into the optical path, move the detection platform to place the biological slice to be scanned within the imaging field of view of the low-magnification fluorescent objective lens, and focus to obtain a clear biological slice image; Step 3: The mobile detection platform scans the tissue area of ​​the complete biological slice and collects all rectangular images in the tissue area of ​​the biological slice through an imaging device; Step 4: In the tissue region of the biological slice, all the acquired rectangular images are stitched together to obtain a complete tissue region image of the biological slice; Step 5: Based on the complete biological slice image, calculate the X and Y coordinate values ​​of the image boundary of the tissue area where fluorescence occurs in the biological slice; Step 6: Replace the low-power fluorescence objective lens with a high-power fluorescence objective lens and focus; Step 7: According to the X, Y coordinate values ​​of the image boundary of the fluorescent tissue area obtained in step 5, the mobile detection platform scans the entire fluorescent tissue area and collects all rectangular images in the fluorescent tissue area through an imaging device, and the size of the rectangular image is the same as that in step 4; Step 8: In the tissue area where fluorescence occurs, all the collected rectangular images are stitched together to obtain an image of the fluorescent area in the complete biological slice.

[0006] Compared with the prior art, the advantage of the present invention is that a low-power fluorescent objective lens is used to scan (capture images) the entire biological slice under the illumination of a fluorescent excitation light source to splice a complete tissue area image of the biological slice, and the X, Y coordinate values ​​of the image boundary of the tissue area where fluorescence occurs in the biological slice are calculated, thereby determining the area to be scanned, and then a high-power fluorescent objective lens is used to accurately scan the tissue area where fluorescence occurs in the biological slice and splice the images to obtain an image of the fluorescent area in the complete biological slice, thereby reducing the scanning area and improving work efficiency.

[0007] Preferably, the low-power fluorescence objective lens is a 1x or 2x fluorescence objective lens, and the high-power fluorescence objective lens is a 20x or 40x fluorescence objective lens.

[0008] Preferably, the specific method for collecting all rectangular images in the tissue area is: ① Move the detection platform to ensure that the upper left corner of the tissue area is within the observable imaging field of view; ② Move the detection platform backward along the Y axis according to the predetermined Y axis step distance to get closer to the operator, and collect a rectangular image with each step. When the lower edge of the tissue area appears in the imaging field of view, stop moving the detection platform; ③ Move the detection platform to the left along the X-axis. When the lower right corner of the tissue area appears in the imaging field of view, stop moving the detection platform. ④ Move the detection platform forward along the Y-axis away from the operator according to the preset Y-axis step distance, and collect a rectangular image with each step. When the upper edge of the tissue area appears in the imaging field of view, stop moving the detection platform to complete the rectangular image collection of all tissue areas.

[0009] Preferably, the length of the long side of the rectangular image is greater than or equal to the distance the detection platform moves along the X-axis.

[0010] Preferably, the specific method for stitching all the collected rectangular images is: ① Read the rectangular images from left to right and from front to back. Read the first rectangular image first. If the rectangular image read subsequently is the first row or the first column, splice it according to the default method, that is, the rectangular image of the first row is spliced ​​with the offset in the left direction, and the rectangular image of the first column is spliced ​​with the offset in the back direction. At the same time, for each rectangular image, according to its splicing path relative to the first rectangular image, record the cumulative registration degree and cumulative offset coordinates on the path. ② The rectangular images that are not in the first row and the first column are judged from the registration degree of the back direction and the left direction, and the direction with the larger registration degree is used to stitch the rectangular images; when the registration degree of the left direction and the back direction is not greater than 0, if the standard registration degree of the left direction is 0, the back direction is used for stitching; in other cases, the left direction is selected for stitching; then, according to the stitching path of the current rectangular image relative to the first rectangular image, the cumulative registration degree and the cumulative offset coordinates on the path are recorded; ③ Determine the position of the current rectangular image relative to the first rectangular image by accumulating the offset coordinates, and gradually process each rectangular image to complete the stitching of all rectangular images.

[0011] Preferably, the specific method for splicing two adjacent rectangular images is: Select overlapping areas in adjacent rectangular images. The specific stitching method is: ①According to the prior information, the overlapping areas of adjacent visual field images are intercepted and converted into grayscale images; ②Use SURF algorithm to find feature points and calculate descriptors; ③ Obtain matching pairs through Brute-Force matching method; ④ Set the threshold by finding the minimum distance, and get a good match pair through threshold filtering; ⑤ Use the random sampling consistency algorithm to screen for better matching pairs and obtain the final offset and alignment.

[0012] The present invention implements a device for the method of obtaining a fluorescent area image in a biological slice, comprising a fluorescent excitation light source for exciting the biological slice to generate fluorescence, a detection platform for placing a slice clamp tray, an imaging device for collecting tissue area images, and a fluorescent objective lens assembly, wherein the fluorescent objective lens assembly is composed of an electric objective lens converter and at least one low-power fluorescent objective lens and at least one high-power fluorescent objective lens arranged on the electric objective lens converter, the electric objective lens converter is used to switch the low-power fluorescent objective lens or the high-power fluorescent objective lens into the optical path, and the detection platform can move up and down in the Z-axis direction, move forward and backward in the Y-axis direction, and move left and right in the X-axis direction.

[0013] The above-mentioned device integrates a low-magnification fluorescent objective lens and a high-magnification fluorescent objective lens on a motorized objective lens converter. The motorized objective lens converter can flexibly switch between the two fluorescent objective lenses in the same optical path. After determining the area to be scanned by the low-magnification fluorescent objective lens, the high-magnification fluorescent objective lens can be used to accurately scan the fluorescent tissue area in the biological slice and splice the image.

[0014] Preferably, the slice holder tray is placed on the detection platform by magnetic attraction, and the slice holder tray can be pulled out when in use.

[0015] Preferably, the slice clamp tray is provided with five biological sample placement cavities.

[0016] Preferably, the low-power fluorescence objective lens is a 1x or 2x fluorescence objective lens, and the high-power fluorescence objective lens is a 20x or 40x fluorescence objective lens. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the structure of a device for acquiring a fluorescent area image in a biological slice in an embodiment of the present invention; The numbers in the figure are: 1 fluorescence excitation light source, 2 imaging device, 3 motorized objective lens converter, 4 fluorescence objective lens assembly, 5 detection platform, 6 slice clamp tray; Figure 2 Schematic diagram of a method for collecting all rectangular images in a tissue area in an embodiment of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below with reference to the accompanying drawings.

[0019] It should be noted that the directions “up, down, front, back, left and right” in the embodiments of the present invention are relative to the operator and are not absolute limitations.

[0020] Example: Figure 1As shown, a device for obtaining a fluorescent area image in a biological slice comprises a fluorescent excitation light source 1 for exciting the biological slice to generate fluorescence, a detection platform 5 for placing a slice clamp tray 6, an imaging device 2 for collecting tissue area images, and a fluorescent objective lens assembly 4, wherein the fluorescent objective lens assembly 4 is composed of an electric objective lens converter 3 and a low-power fluorescent objective lens and a high-power fluorescent objective lens arranged on the electric objective lens converter 3. In this embodiment, the low-power fluorescent objective lens is a 2-power fluorescent objective lens, and the high-power fluorescent objective lens is a 2-power fluorescent objective lens. The electric objective lens converter 3 is used to switch the low-power fluorescent objective lens or the high-power fluorescent objective lens into the optical path, and the detection platform 5 can move up and down in the Z-axis direction, move forward and backward in the Y-axis direction, and move left and right in the X-axis direction. The slice clamp tray 6 is provided with five biological sample placement cavities, and the slice clamp tray 6 is placed on the detection platform 5 by magnetic attraction, and can be pulled out when used.

[0021] The method of acquiring a fluorescent region image in a biological slice in this embodiment includes the following steps: Step 1: Place the biological slice to be scanned on a detection platform 5 that can move in the horizontal plane along the mutually perpendicular X and Y directions, and turn on the fluorescence excitation light source 1 to excite the biological slice to generate fluorescence; Step 2: insert a 2x fluorescent objective lens into the optical path, move the detection platform 5 to place the biological slice to be scanned within the imaging field of view of the 2x fluorescent objective lens, and focus to obtain a clear biological slice image; Step 3: ① Move the detection platform 5 to ensure that the upper left corner of the tissue area is placed within the observable imaging field of view; ② Move the detection platform backward along the Y axis according to the predetermined Y axis step distance to bring the operator closer, and collect a rectangular image for each step. When the lower edge of the tissue area appears in the imaging field of view, stop moving the detection platform 5; ③ Move the detection platform 5 to the left along the X-axis. When the lower right corner of the tissue area appears in the imaging field of view, stop moving the detection platform 5. It can be determined that the length of the long side of the rectangular image is greater than or equal to the distance the detection platform moves along the X-axis. ④ Move the detection platform 5 forward along the Y-axis away from the operator according to the predetermined Y-axis step, and collect a rectangular image with each step. When the upper edge of the tissue area appears in the imaging field of view, stop moving the detection platform 5 to complete the rectangular image collection of all tissue areas.

[0022] Step 4: In the tissue area of ​​the biological slice, all the acquired rectangular images are stitched together. The specific method is as follows: ① Read the rectangular images from left to right and from front to back. Read the first rectangular image first. If the rectangular image read subsequently is the first row or the first column, splice it according to the default method, that is, the rectangular image of the first row is spliced ​​with the offset in the left direction, and the rectangular image of the first column is spliced ​​with the offset in the back direction. At the same time, for each rectangular image, according to its splicing path relative to the first rectangular image, record the cumulative registration degree and cumulative offset coordinates on the path. ② The rectangular images that are not in the first row and the first column are judged from the registration degree of the back direction and the left direction, and the direction with the larger registration degree is used to stitch the rectangular images; when the registration degree of the left direction and the back direction is not greater than 0, if the standard registration degree of the left direction is 0, the back direction is used for stitching; in other cases, the left direction is selected for stitching; then, according to the stitching path of the current rectangular image relative to the first rectangular image, the cumulative registration degree and the cumulative offset coordinates on the path are recorded; ③ Determine the position of the current rectangular image relative to the first rectangular image by accumulating the offset coordinates, and gradually process each rectangular image to complete the stitching of all rectangular images; When stitching two adjacent rectangular images, first select the overlapping area in the adjacent rectangular images, and then perform the following operations: ①According to the prior information, the overlapping areas of adjacent visual field images are intercepted and converted into grayscale images; ②Use SURF algorithm to find feature points and calculate descriptors; ③ Obtain matching pairs through Brute-Force matching method; ④ Set the threshold by finding the minimum distance, and get a good match pair through threshold filtering; ⑤ Use the random sampling consistency algorithm to screen for better matching pairs and obtain the final offset and registration. Step 5: Based on the complete biological slice image, calculate the X and Y coordinate values ​​of the image boundary of the tissue area where fluorescence occurs in the biological slice; Step 6: Replace the 2x fluorescent objective lens with a 20x fluorescent objective lens and focus; Step 7: According to the X, Y coordinate values ​​of the image boundary of the fluorescent tissue area obtained in step 5, the detection platform 5 is moved according to the method of step 3 to scan the entire fluorescent tissue area and collect all rectangular images in the fluorescent tissue area through the imaging device. The size of the rectangular image is the same as that in step 4. Step 8: In the tissue area where fluorescence occurs, all the collected rectangular images are spliced ​​according to the method of step 4 to obtain an image of the fluorescent area in the complete biological slice.

[0023] The above description shows and describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept of this article through the above teachings or the technology or knowledge of the relevant field. Changes and variations made by non-technical personnel do not depart from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for obtaining a fluorescent area image in a biological slice, characterized in that The following steps are involved: Step 1: Place the biological slice to be scanned on a detection platform that can move in the horizontal plane along the mutually perpendicular X and Y directions, and turn on the fluorescent excitation light source to excite the biological slice to produce fluorescence; Step 2: Insert a low-magnification fluorescent objective lens into the optical path, move the detection platform to place the biological slice to be scanned within the imaging field of view of the low-magnification fluorescent objective lens, and focus to obtain a clear biological slice image; Step 3: The mobile detection platform scans the tissue area of ​​the complete biological slice and collects all rectangular images in the tissue area of ​​the biological slice through an imaging device; Step 4: In the tissue region of the biological slice, all the acquired rectangular images are stitched together to obtain a complete tissue region image of the biological slice; Step 5: Based on the complete biological slice image, calculate the X and Y coordinate values ​​of the image boundary of the tissue area where fluorescence occurs in the biological slice; Step 6: Replace the low-power fluorescence objective lens with a high-power fluorescence objective lens and focus; Step 7: According to the X, Y coordinate values ​​of the image boundary of the fluorescent tissue area obtained in step 5, the mobile detection platform scans the entire fluorescent tissue area and collects all rectangular images in the fluorescent tissue area through an imaging device, and the size of the rectangular image is the same as that in step 4; Step 8: In the tissue area where fluorescence occurs, all the collected rectangular images are stitched together to obtain an image of the fluorescent area in the complete biological slice.

2. A method for obtaining a fluorescent area image in a biological slice according to claim 1, characterized in that The low-power fluorescence objective lens is a 1x or 2x fluorescence objective lens, and the high-power fluorescence objective lens is a 20x or 40x fluorescence objective lens.

3. A method for obtaining a fluorescent area image in a biological slice according to claim 1, characterized in that In the step 3, the specific method of collecting all rectangular images in the tissue area of ​​the biological slice by the imaging device is: ① Move the detection platform to ensure that the upper left corner of the tissue area is within the observable imaging field of view; ② Move the detection platform backward along the Y axis according to the predetermined Y axis step distance to get closer to the operator, and collect a rectangular image with each step. When the lower edge of the tissue area appears in the imaging field of view, stop moving the detection platform; ③ Move the detection platform to the left along the X-axis. When the lower right corner of the tissue area appears in the imaging field of view, stop moving the detection platform. ④ Move the detection platform forward along the Y-axis away from the operator according to the preset Y-axis step distance, and collect a rectangular image with each step. When the upper edge of the tissue area appears in the imaging field of view, stop moving the detection platform to complete the rectangular image collection of all tissue areas.

4. A method for obtaining a fluorescent area image in a biological slice according to claim 3, characterized in that The length of the long side of the rectangular image is greater than or equal to the distance the detection platform moves along the X-axis.

5. A method for obtaining a fluorescent area image in a biological slice as claimed in claim 3, characterized in that The specific method for stitching all the collected rectangular images is: ① Read the rectangular images from left to right and from front to back. Read the first rectangular image first. If the rectangular image read subsequently is the first row or the first column, splice it according to the default method, that is, the rectangular image of the first row is spliced ​​with the offset in the left direction, and the rectangular image of the first column is spliced ​​with the offset in the back direction. At the same time, for each rectangular image, according to its splicing path relative to the first rectangular image, record the cumulative registration degree and cumulative offset coordinates on the path. ② The rectangular images that are not in the first row and the first column are judged from the registration degree of the back direction and the left direction, and the direction with the larger registration degree is used to stitch the rectangular images; when the registration degree of the left direction and the back direction is not greater than 0, if the standard registration degree of the left direction is 0, the back direction is used for stitching; in other cases, the left direction is selected for stitching; then, according to the stitching path of the current rectangular image relative to the first rectangular image, the cumulative registration degree and the cumulative offset coordinates on the path are recorded; ③ Determine the position of the current rectangular image relative to the first rectangular image by accumulating the offset coordinates, and gradually process each rectangular image to complete the stitching of all rectangular images.

6. A method for obtaining a fluorescent area image in a biological slice according to claim 1, characterized in that The specific method of stitching two adjacent rectangular images is: select the overlapping area in the adjacent rectangular images, and then perform the following operations: ①According to the prior information, the overlapping areas of adjacent visual field images are intercepted and converted into grayscale images; ②Use SURF algorithm to find feature points and calculate descriptors; ③ Obtain matching pairs through Brute-Force matching method; ④ Set the threshold by finding the minimum distance, and get a good match pair through threshold filtering; ⑤ Use the random sampling consistency algorithm to screen for better matching pairs and obtain the final offset and alignment.

7. A device for acquiring an image of a fluorescent region in a biological slice, characterized in that The invention comprises a fluorescence excitation light source for exciting biological slices to generate fluorescence, a detection platform for placing a slice clamp tray, an imaging device for adopting tissue area images and a fluorescence objective lens assembly, wherein the fluorescence objective lens assembly is composed of an electric objective lens converter and at least one low-power fluorescence objective lens and at least one high-power fluorescence objective lens arranged on the electric objective lens converter, and the electric objective lens converter is used to switch the low-power fluorescence objective lens or the high-power fluorescence objective lens into the light path, and the detection platform 5 can move up and down in the Z-axis direction, move forward and backward in the Y-axis direction, and move left and right in the X-axis direction.

8. The device for acquiring an image of a fluorescent region in a biological slice according to claim 7, characterized in that The slice clamp tray is placed on the detection platform by magnetic attraction.

9. The device for acquiring an image of a fluorescent region in a biological slice according to claim 7, characterized in that The slice clamp tray is provided with five biological sample placement cavities.

10. The device for acquiring an image of a fluorescent region in a biological slice according to claim 7, characterized in that The low-power fluorescence objective lens is a 1x or 2x fluorescence objective lens, and the high-power fluorescence objective lens is a 20x or 40x fluorescence objective lens.

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