A method and apparatus for acquiring fluorescent region images in a biological section

Abstract

The application discloses a method and device for acquiring a fluorescent region image in a biological section, and low-power fluorescent objective lenses and high-power fluorescent objective lenses are integrally arranged on an electric objective converter. The two kinds of fluorescent objective lenses can be flexibly switched in the same light path through the electric objective converter. The low-power fluorescent objective lenses can be used to scan (image acquisition) and splice a complete tissue region image of the biological section under illumination of a fluorescent excitation light source, and X and Y coordinate values of the image boundary of the tissue region generating fluorescence in the biological section are calculated. After the region needing to be scanned is determined, the high-power fluorescent objective lenses are used to accurately scan the tissue region generating fluorescence in the biological section and splice the image, so that a complete fluorescent region image in the biological section is obtained, the scanning region is reduced, and the work efficiency is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a method and device for obtaining a fluorescence region image in a biological section. BACKGROUND

[0002] The prior art method for obtaining a fluorescence region image in a biological section is to use a common fluorescence section scanner to scan. Since the fluorescence objective lens in the fluorescence section scanner is a 20x or 40x high-power fluorescence objective lens, a marker pen must be used to mark the tissue region on the biological section, and then the fluorescence section scanner is used to collect the biological section to obtain an image including the marked tissue region. After edge detection of the image, the edge profile of the marked tissue region is obtained, and then connected region detection is performed on the edge profile to obtain a set composed of a plurality of connected regions. Convex hull detection is performed on each connected region to finally identify the tissue region marked by the marker pen. After the tissue region is obtained, the fluorescence objective lens of the fluorescence section scanner is used to perform subsequent scanning and detection on the tissue region of the biological section to obtain the fluorescence region image in the biological section.

[0003] The prior art method requires the user to mark the biological section to be scanned in advance using a marker pen. When multiple biological sections need to be scanned, this step increases the workload. The tissue on the biological section needs to be irradiated by a specific waveband light source to emit fluorescence so that the section scanner can collect the image. When the user marks the region using a marker pen (in a natural light state), only the approximate position of the tissue region can be marked, which increases the scanning range of the subsequent section scanning work. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method and device for obtaining a fluorescence region image in a biological section, which can accurately obtain the pixel position of the tissue in the biological section without using a fluorescence marker pen and accurately position the scanning range of the subsequent tissue.

[0005] The technical solution adopted by the present application to solve the above technical problem is a method for obtaining a fluorescence region image in a biological section, comprising the following steps:

[0006] Step 1: Place the biological section to be scanned on a detection platform that can move in the X direction and the Y direction perpendicular to each other on a horizontal plane, and turn on a fluorescence excitation light source to excite the biological section to generate fluorescence;

[0007] Step 2: Insert a low-power fluorescence objective lens into the optical path, move the detection platform to place the biological section to be scanned in the imaging field of view of the low-power fluorescence objective lens, and focus to obtain a clear image of the biological section;

[0008] Step three: the moving detection platform scans the whole tissue area of the biological section and collects all the rectangular images in the tissue area of the biological section by the imaging device;

[0009] Step four: all the collected rectangular images are spliced to obtain the image of the whole tissue area of the biological section;

[0010] Step five: according to the image of the whole biological section, the X and Y coordinate values of the image boundary of the tissue area with fluorescence in the biological section are calculated;

[0011] Step six: the low-power fluorescence objective lens is replaced by a high-power fluorescence objective lens and focused;

[0012] Step seven: according to the X and Y coordinate values of the image boundary of the tissue area with fluorescence obtained in step five, the moving detection platform scans the whole tissue area with fluorescence and collects all the rectangular images in the tissue area with fluorescence by the imaging device, and the size of the rectangular image is the same as that in step four;

[0013] Step eight: all the collected rectangular images are spliced to obtain the image of the whole fluorescence area in the biological section.

[0014] Compared with the prior art, the advantages of the present application are that the whole biological section is scanned (image collection) under the illumination of a fluorescence excitation light source by using a low-power fluorescence objective lens to splice the image of the whole tissue area of the biological section and calculate the X and Y coordinate values of the image boundary of the tissue area with fluorescence in the biological section, so as to determine the area to be scanned, and then a high-power fluorescence objective lens is used to accurately scan the tissue area with fluorescence in the biological section and splice the image, so as to obtain the image of the whole fluorescence area in the biological section, thereby reducing the scanning area and improving the work efficiency.

[0015] Preferably, the low-power fluorescence objective lens is a 1-power or 2-power fluorescence objective lens, and the high-power fluorescence objective lens is a 20-power or 40-power fluorescence objective lens.

[0016] Preferably, the specific method for collecting all the rectangular images in the tissue area is as follows:

[0017] ①Move the detection platform to ensure that the left upper corner of the tissue area is in the observable imaging field of view;

[0018] ②Move the detection platform backward along the Y axis at a predetermined Y axis step distance to approach the operator, and collect a rectangular image every time the detection platform moves one step, and stop moving the detection platform when the lower edge of the tissue area appears in the imaging field of view;

[0019] ③ Move the detection platform to the left along the X axis, and stop when the lower right corner of the tissue region appears in the imaging field of view;

[0020] ④ Move the detection platform forward along the Y axis at a predetermined Y axis step distance away from the operator, and acquire a rectangular image every time the detection platform moves one step, and stop when the upper edge of the tissue region appears in the imaging field of view, thereby completing the acquisition of the rectangular images of all the tissue regions.

[0021] Preferably, the length of the long side of the rectangular image is greater than or equal to the distance of the movement of the detection platform along the X axis.

[0022] Preferably, the specific method for splicing all the acquired rectangular images is as follows:

[0023] ① Read the rectangular images in the order from left to right and from front to back, read the first rectangular image first, and if the subsequently read rectangular image is in the first row or the first column, splice it according to the default method, that is, splice the rectangular image in the first row with the offset in the left direction, and splice the rectangular image in the first column with the offset in the rear direction; at the same time, record the cumulative registration degree and the cumulative offset coordinates on the path according to the splicing path of each rectangular image relative to the first rectangular image;

[0024] ② Determine the registration degree of the rectangular image in the non-first row and the non-first column from the rear direction and the left direction, and splice the rectangular image in the direction with the greater registration degree; when the registration degrees in the left direction and the rear direction are both not greater than 0, if the standard registration degree in the left direction is 0, splice in the rear direction; otherwise, splice in the left direction; then record the cumulative registration degree and the cumulative offset coordinates on the path according to the splicing path of the current rectangular image relative to the first rectangular image;

[0025] ③ Determine the position of the current rectangular image relative to the first rectangular image through the cumulative offset coordinates, and process each rectangular image step by step to complete the splicing of all the rectangular images.

[0026] Preferably, the specific method for splicing the adjacent two rectangular images is as follows:

[0027] Select the overlapping region of the adjacent rectangular images, and the specific splicing method is as follows:

[0028] ① According to the prior information, intercept the overlapping region of the adjacent field of view images, and convert it into a gray-scale image;

[0029] ② Find the feature points by using the SURF algorithm, and calculate the descriptors;

[0030] ③ Obtain the matching pairs by using the Brute-Force brute force matching method;

[0031] IV. Setting a threshold by searching for the minimum distance, and filtering the good matching pairs by the threshold;

[0032] V. Using the random sample consensus algorithm to screen the better matching pairs, and obtaining the final offset and registration degree.

[0033] The device for acquiring the fluorescent region image in the biological section of the method disclosed in the application comprises a fluorescent excitation light source for exciting the biological section to generate fluorescence, a detection platform for placing a section holder tray, an imaging device for acquiring the tissue region image, and a fluorescent objective assembly, the fluorescent objective assembly is composed of an electric animal lens converter and at least one low-power fluorescent objective and at least one high-power fluorescent objective arranged on the electric animal lens converter, the electric animal lens converter is used for switching the low-power fluorescent objective or the high-power fluorescent objective 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.

[0034] The device integrates the low-power fluorescent objective and the high-power fluorescent objective on the electric animal lens converter, the two fluorescent objectives can be flexibly switched in the same optical path through the electric animal lens converter, the region needing to be scanned can be determined through the low-power fluorescent objective, and then the high-power fluorescent objective is used to accurately scan the tissue region generating fluorescence in the biological section and splice the images.

[0035] Preferably, the section holder tray is placed on the detection platform in a magnetic suction mode. The section holder tray can be pulled out for use.

[0036] Preferably, the section holder tray is provided with five biological section placing cavities.

[0037] Preferably, the low-power fluorescent objective is a 1-power or 2-power fluorescent objective, and the high-power fluorescent objective is a 20-power or 40-power fluorescent objective. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 FIG. 1 is a structural schematic diagram of the device for acquiring the fluorescent region image in the biological section in the embodiment of the application;

[0039] In the figure, 1 is a fluorescent excitation light source, 2 is an imaging device, 3 is an electric animal lens converter, 4 is a fluorescent objective assembly, 5 is a detection platform, and 6 is a section holder tray.

[0040] Figure 2 FIG. 2 is a method schematic diagram of acquiring all rectangular images in the tissue region in the embodiment of the application. DETAILED DESCRIPTION

[0041] The application will be further described in detail in combination with the embodiments of the drawings.

[0042] It should be noted that the directions "up, down, forward, backward, left, and right" in the embodiments of the present invention are all relative to the operator and are not an absolute limitation.

[0043] Example: Figure 1 As shown, an apparatus for acquiring images of fluorescent regions in biological slides includes a fluorescence excitation light source 1 for exciting fluorescence in the biological slides, a detection platform 5 for placing a slide holder tray 6, an imaging device 2 for acquiring images of the tissue region, and a fluorescence objective assembly 4. The fluorescence objective assembly 4 consists of an electro-optical endoscope converter 3 and a low-power fluorescence objective and a high-power fluorescence objective mounted on the electro-optical endoscope converter 3. In this embodiment, the low-power fluorescence objective is a 2x fluorescence objective, and the high-power fluorescence objective is also a 2x fluorescence objective. The electro-optical endoscope converter 3 is used to switch the low-power or high-power fluorescence objective into the optical path. The detection platform 5 can move up and down in the Z-axis direction, forward and backward in the Y-axis direction, and left and right in the X-axis direction. The slide holder tray 6 is provided with five biological sample placement cavities and is placed on the detection platform 5 by magnetic attraction, allowing it to be pulled out for use.

[0044] The method for obtaining fluorescent region images in biological slices in this embodiment includes the following steps:

[0045] Step 1: Place the biological slice to be scanned on the detection platform 5, which can move horizontally along mutually perpendicular X and Y directions, and turn on the fluorescence excitation light source 1 to excite the biological slice to produce fluorescence.

[0046] Step 2: Insert the 2x fluorescence objective into the optical path, move the detection platform 5 to place the biological slice to be scanned within the imaging field of the 2x fluorescence objective, and focus to obtain a clear image of the biological slice.

[0047] Step 3:

[0048] ① The mobile detection platform 5 ensures that the upper left corner of the tissue area is within the observable imaging field of view;

[0049] ② Move the detection platform backward along the Y-axis at a predetermined Y-axis step distance to bring it closer to the operator. Acquire a rectangular image with each step. Stop moving the detection platform when the lower edge of the tissue area appears in the imaging field of view.

[0050] ③ Move the detection platform 5 to the left along the X-axis. When the lower right corner of the tissue region 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.

[0051] ④ Move the detection platform 5 forward along the Y-axis away from the operator according to the predetermined Y-axis step distance. Acquire 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 acquisition of rectangular images of all tissue areas.

[0052] Step 4: Within the tissue area of ​​the biological slice, stitch together all the acquired rectangular images. The specific method is as follows:

[0053] ① Read the rectangular images in order from left to right and from front to back. Read the first rectangular image first. If the rectangular image read later is in the first row or the first column, it is stitched according to the default method. That is, the rectangular images in the first row are stitched with the left offset and the rectangular images in the first column are stitched with the back offset. At the same time, for each rectangular image, record the cumulative registration degree and cumulative offset coordinates on the stitching path relative to the first rectangular image.

[0054] ② For rectangular images that are neither the first row nor the first column, the registration is determined by the back and left directions. The direction with the larger registration is selected for stitching the rectangular image. When the registration of both the left and back directions is not greater than 0, if the standard registration of the left direction is 0, the back direction is selected for stitching. Otherwise, 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 and cumulative offset coordinates on the path are recorded.

[0055] ③ The position of the current rectangular image relative to the first rectangular image is determined by accumulating offset coordinates, and each rectangular image is processed step by step to complete the stitching of all rectangular images;

[0056] When stitching two adjacent rectangular images, first select the overlapping area in the adjacent rectangular images, and then perform the following operations:

[0057] ① Based on prior information, the overlapping area of ​​adjacent visual field images is extracted and converted into a grayscale image;

[0058] ② Use the SURF algorithm to find feature points and calculate descriptors;

[0059] ③ Matching pairs are obtained using the brute-force matching method;

[0060] ④ Set a threshold by finding the minimum distance, and obtain good matching pairs by filtering through the threshold;

[0061] ⑤ Use the random sampling consistency algorithm to filter out better matching pairs and obtain the final offset and registration degree. Step 5: Based on the complete biological slice image, calculate the X and Y coordinate values ​​of the image boundary of the fluorescent tissue region in the biological slice;

[0062] Step six: replace the 2X fluorescence objective with a 20X fluorescence objective and focus;

[0063] Step seven: according to the X, Y coordinate values of the image boundary of the fluorescent tissue area obtained in step five, move the detection platform 5 according to the method of step three to scan the entire fluorescent tissue area and collect all rectangular images in the fluorescent tissue area by the imaging device, the size of the rectangular image is the same as that of step four;

[0064] Step eight: in the fluorescent tissue area, splice all the collected rectangular images according to the method of step four to obtain the image of the fluorescent area in the complete biological section.

[0065] The above description shows and describes the preferred embodiments of the present application, it should be understood that the present application is not limited to the forms disclosed herein, should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the inventive concept disclosed herein, through the above teaching or related technical or knowledge. The modifications and changes made by those skilled in the art without departing from the spirit and scope of the present application shall be within the scope of the appended claims of the present application.

Claims

1. A method for obtaining a fluorescent region image in a biological section, comprising the following steps: Step 1: placing the biological section to be scanned on a detection platform that can move in the X and Y directions perpendicular to each other in the horizontal plane, and turning on a fluorescent excitation light source to excite the biological section to generate fluorescence; Step 2: inserting a low-power fluorescent objective lens into the optical path, moving the detection platform to place the biological section to be scanned in the imaging field of view of the low-power fluorescent objective lens, and focusing to obtain a clear image of the biological section; Step 3: moving the detection platform to scan the entire tissue region of the biological section and collecting all rectangular images in the tissue region of the biological section by an imaging device; Step 4: in the tissue region of the biological section, stitching all the collected rectangular images to obtain an image of the entire tissue region of the biological section; Step 5: calculating the X and Y coordinate values of the image boundary of the tissue region that generates fluorescence in the biological section according to the complete image of the biological section; Step 6: replacing the low-power fluorescent objective lens with a high-power fluorescent objective lens and focusing; Step 7: moving the detection platform to scan the entire tissue region that generates fluorescence according to the X and Y coordinate values of the image boundary of the tissue region that generates fluorescence obtained in Step 5, and collecting all rectangular images in the tissue region that generates fluorescence by an imaging device, the size of the rectangular image being the same as that in Step 4; Step 8: in the tissue region that generates fluorescence, stitching all the collected rectangular images to obtain an image of the fluorescent region in the complete biological section; characterized in that in Step 3, the specific method for collecting all rectangular images in the tissue region of the biological section by an imaging device is as follows: ① moving the detection platform to ensure that the left upper corner of the tissue region is placed in the observable imaging field of view; ② moving the detection platform backward along the Y axis at a predetermined Y axis step distance to move closer to the operator, collecting a rectangular image every time the detection platform moves one step, and stopping moving the detection platform when the lower edge of the tissue region appears in the imaging field of view; ③ moving the detection platform leftward along the X axis, and stopping moving the detection platform when the right lower corner of the tissue region appears in the imaging field of view; ④ moving the detection platform forward along the Y axis at a predetermined Y axis step distance to move away from the operator, collecting a rectangular image every time the detection platform moves one step, and stopping moving the detection platform when the upper edge of the tissue region appears in the imaging field of view, thereby completing the collection of all rectangular images of the tissue region.

2. The method of claim 1, wherein The low-power fluorescent objective lens is a 1x or 2x fluorescent objective lens, and the high-power fluorescent objective lens is a 20x or 40x fluorescent objective lens.

3. The method of claim 1, wherein the method further comprises: The length of the long side of the rectangular image is greater than or equal to the distance of the detection platform moving along the X axis. ​ 4. The method of claim 1, wherein the method further comprises: The specific method for stitching all the collected rectangular images is as follows: ​ ① Read the rectangular images in the order 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 according to the default method, that is, splice the rectangular image of the first row with the offset in the left direction, splice the rectangular image of the first column with the offset in the back direction; at the same time, record the cumulative registration degree and the cumulative offset coordinates on the path according to the splicing path of each rectangular image relative to the first rectangular image; ② Judge the rectangular image which is not the first row and not the first column from the registration degrees in the back direction and the left direction, splice the rectangular image in the direction with the greater registration degree; when the registration degrees in the left direction and the back direction are both not greater than 0, if the standard registration degree in the left direction is 0, splice in the back direction; otherwise, splice in the left direction; then record the cumulative registration degree and the cumulative offset coordinates on the path according to the splicing path of the current rectangular image relative to the first rectangular image; ③ Determine the position of the current rectangular image relative to the first rectangular image through the cumulative offset coordinates, process each rectangular image step by step to complete the splicing of all rectangular images.

5. The method of claim 1, wherein The specific method of splicing the adjacent two rectangular images is as follows: select the overlapping area of the adjacent rectangular images, and then perform the following operations: ① According to the prior information, intercept the overlapping area of the adjacent field of view images and convert it into a gray-scale image; ② Find the feature points by using the SURF algorithm and calculate the descriptors; ③ Get the matching pairs by using the Brute-Force brute force matching method; ④ Set the threshold value by finding the minimum distance, and filter the good matching pairs by the threshold value; ⑤ Screen the better matching pairs by using the random sample consensus algorithm to get the final offset and registration degree.

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