CN118311016B - Method and system for detecting position and morphology of dendritic spines of high-resolution complete neurons - Google Patents
Method and system for detecting position and morphology of dendritic spines of high-resolution complete neurons Download PDFInfo
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Abstract
The invention discloses a method and a system for detecting the position and the form of a dendritic spine of a high-resolution complete neuron. The method comprises obtaining an in vitro brain tissue with neurons carrying fluorescent signals; fixing the isolated brain tissue, slicing, and performing light removal treatment on the brain slice; performing overturn imaging on the brain slice subjected to light removal treatment by using a fluorescence microscope to obtain a brain slice image block; according to the fluorescence signal of the overlapping area of the brain slice image blocks, performing three-dimensional image stitching on the brain slice image blocks to obtain complete target brain slice image blocks; reconstructing the neuron dendritic spines by using the target brain slice image block to obtain the position and form information of the neuron dendritic spines. The method can provide the position and form information of the neuron dendritic spine structure, and the method for detecting the position and form of the high-resolution complete neuron dendritic spine can realize the analysis of a single complete neuron dendritic spine within a few hours, and is simple to operate and easy to implement.
Description
Technical Field
The invention belongs to the technical field of fluorescence microscopic imaging and reconstruction, and particularly relates to a method and a system for detecting the position and the shape of a dendritic spine of a high-resolution complete neuron.
Background
Neurons are connected by synaptic structures, constituting millions of overlapping and intersecting neural loops, which are thought to be the basis for the brain to perform a specific function. Most excitatory synapses in the mammalian central nervous system are located on dendritic spines, which makes the dendritic spines morphological markers of excitatory synapses. The morphology of the dendritic spines is closely related to the characteristics of synaptic strength, plasticity, signal transmission and the like, analysis of the dendritic spines on the whole neuron is an important basis for understanding a nerve loop, however, the dendritic spines belong to a fine structure of the neuron, and analysis of the micron-sized dendritic spines on the whole neuron scale brings high requirements for marking, imaging and image processing methods.
At present, an electron microscope or an optical microscope is commonly used for carrying out morphological analysis on the dendritic spines, however, although the resolution of the electron microscope can reach the nanometer level, the field of view is small, and meanwhile, the sample preparation process is complex. For an optical microscope, the numerical aperture and the field of view of the objective lens are also a pair of contradictions, and the fine structure of the dendritic spines needs to be analyzed by the objective lens with high resolution, so that most of researches on the dendritic spines are concentrated on a small scale, the field of view size and the analysis range are very limited, and often, the dendritic spines on a certain section of dendrites on the neuron are researched, and reconstruction and quantitative analysis cannot be performed on all the dendritic spines on the complete neuron.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide a method and a system for detecting the position and the shape of a dendritic spine of a high-resolution complete neuron, which solve the problems of small field of view, limited imaging range and low fineness in the process of detecting the dendritic spine structure.
The technical scheme adopted by the invention is as follows, comprising the following steps:
step S1: aiming at an in-vitro brain tissue, the in-vitro brain tissue adopts virus-labeled neurons and dendritic spine structures of the neurons, so that the neurons carry fluorescent signals;
Step S2: fixing the isolated brain tissue, slicing, and performing light removal treatment on the brain slice;
Step S3: performing overturn imaging on the brain slice subjected to light removal treatment by using a fluorescence microscope to obtain a brain slice image block;
step S4: according to the fluorescence signal of the overlapping area of the brain slice image blocks, performing three-dimensional image stitching on the brain slice image blocks to obtain complete target brain slice image blocks;
Step S5: reconstructing the neuron dendritic spines by using the target brain slice image block to obtain the position and form information of the neuron dendritic spines.
The step S2 specifically comprises the following steps:
S2.1, firstly, fixing isolated brain tissue in a sample groove of an oscillation slicer, and slicing the brain tissue by using the oscillation slicer to cut the brain tissue into brain slices with the thickness of 400-600 mu m;
and S2.2, placing the brain slice in one light removal reagent of 1-2 ml for light removal treatment, taking out the brain slice after 2-3 hours, transferring the brain slice into the other light removal reagent of 1-3 ml, and taking out the brain slice after 4-6 minutes of light removal treatment to obtain the brain slice after light removal treatment.
The photo-scavenging reagent is Triton X-100 or a mixed solution prepared by mixing antipyrine and nicotinamide.
In the step S3, the specific method for performing flip imaging on the brain slice by using the objective lens is as follows:
Step S3.1, placing a prepared mould on a first cover glass, placing a brain slice in a square hole in the middle of the mould, then filling a light removal reagent into the square hole, and then covering a second cover glass on the mould to seal the square hole of the mould so as to prepare a brain slice sample; the upper and lower surfaces of the brain slice are respectively contacted with two cover slips;
S3.2, selecting a silicone oil objective lens with the power of 40 times of 1.25-NA, and placing the upper surface of the brain slice sample at the focus of the silicone oil objective lens;
Step S3.3, selecting an imaging view field of a silicone oil objective lens by taking a neuron cell body as a center, wherein the specific selection mode of the imaging view field of the objective lens is as follows:
firstly, selecting a plurality of imaging fields by taking cell bodies of neurons as central points, namely taking the centers of all imaging fields as neuron cell bodies, wherein pictures corresponding to all imaging fields comprise all fluorescence signals of neurons in brain slices, the size of an overlapping area between every two adjacent imaging fields is 8% -12% of the size of the imaging fields, and the sizes of all imaging fields are consistent;
s3.4, imaging the brain slice by using a silicone oil objective lens of the fluorescence microscope according to each selected imaging view field to obtain a brain slice image block under each imaging view field;
In the imaging process, the silicone oil objective lens starts to image the brain slice from the upper surface of the brain slice sample, the imaging depth is 260-280 mu m, the imaging step distance is 0.33 mu m, and the brain slice image blocks under each imaging visual field before overturning are obtained through imaging.
And S3.5, overturning the brain slice sample, placing the upper surface of the overturned brain slice sample at the focus of the objective lens, and repeating the steps S3.3-S3.4 to obtain brain slice image blocks under each imaging view after overturning.
In the step S4, the method for performing three-dimensional image stitching on the brain slice image blocks specifically includes:
S4.1, all the brain slice image blocks are moved into a brain slice three-dimensional coordinate system, wherein in the brain slice three-dimensional coordinate system, the depth direction of the brain slice image blocks is defined as a Z axis, and the length direction and the width direction of the brain slice image blocks are respectively defined as an X axis and a Y axis;
Step S4.2, splicing all brain slice image blocks before overturning to obtain a target image block before overturning; splicing all the overturned brain slice image blocks to obtain an overturned target image block;
Step S4.3, splicing the target image block before overturning and the target image block after overturning:
Firstly, turning the turned target image block up and down by 180 degrees, and then acquiring the relative displacement of the turned target image block relative to the turned target image block in the X axis, the relative displacement of the turned target image block in the Y axis and the relative displacement of the turned target image block in the Z axis according to fluorescent signals of the turned target image block and the turned target image block in the overlapping area of the visual field, wherein the relative displacement is respectively used as three-way displacement one delta X ', three-way displacement two delta Y ' and three-way displacement three delta Z '; shifting the turned target image block by one delta X ' along the negative direction translation distance three-way of the X axis, shifting by two delta Y ' along the negative direction translation distance three-way of the Y axis and shifting by three delta Z ' along the negative direction translation distance three-way of the Z axis, so that the turned target image block and the turned target image block overlap in a visual field overlapping area, and then performing image stitching on the overlapped turned target image block and the turned target image block, and obtaining a complete brain slice image block after stitching is completed; the complete brain slice image block contains complete neurons of a brain slice;
And S4.4, inputting the complete brain slice image block into a convolutional neural network CNN to denoise the complete brain slice image block, and taking the denoised brain slice image block output by the convolutional neural network CNN as the complete target brain slice image block.
In the step S5, the specific method for reconstructing the neuron is as follows:
Step S5.1, firstly, inputting a complete target brain slice image block into image analysis software, and reconstructing the whole neuron in the brain slice by using the image analysis software: firstly, selecting and drawing a dendritic structure in a neuron in a complete target brain slice image block by utilizing image analysis software, then automatically identifying and reconstructing a dendritic spine structure on the neuron by utilizing the image analysis software, further completing reconstruction of the whole neuron, and obtaining a three-dimensional model containing complete neuron and all dendritic spine information after the reconstruction of the neuron is completed;
S5.2, acquiring position information and morphological information of the dendritic spines in the neurons according to the three-dimensional model; the position information of the dendritic spines comprises the space coordinate position of the dendritic spines and the relative position of each dendritic spine relative to a dendritic structure in the whole neuron, and the morphological information comprises the volume, the area and the straightness information of the dendritic spines.
The specific way of splicing all the brain slice image blocks before overturning in the step S4.2 is as follows:
Step S4.2.1, firstly, selecting any brain slice image block from all brain slice image blocks before overturning to serve as a seed image block;
Step S4.2.2, selecting one brain slice image block overlapped with the imaging area of the seed image block from the rest brain slice image blocks before overturning as a moving image block, and acquiring the relative displacement of the moving image block relative to the seed image block in the X axis and the relative displacement of the moving image block in the Y axis according to fluorescent signals of the seed image block and the moving image block in the field of view overlapped area, wherein the relative displacement is respectively used as a first displacement Deltax before overturning and a second displacement Deltay before overturning; translating the moving image block along the negative direction of the X axis by a distance delta X before first overturning and translating the moving image block along the negative direction of the Y axis by a distance delta Y before second overturning, so that the seed image block and the moving image block are overlapped in a visual field overlapping area, then performing image stitching on the overlapped seed image block and the moving image block, and taking the stitched image block as a new seed image block;
step S4.2.3, repeating step S4.2.2 for a plurality of times until all the pre-overturn brain slice image blocks are subjected to image stitching, and obtaining a pre-overturn target image block.
The specific way of splicing all the flipped brain slice image blocks in the step S4.2 is as follows:
Step S4.2.1, firstly, selecting any brain slice image block from all the turned brain slice image blocks to serve as a seed image block;
Step S4.2.2, selecting one brain slice image block overlapped with the imaging area of the seed image block from the rest brain slice image blocks after overturning as a moving image block, and acquiring the relative displacement of the moving image block relative to the seed image block in the X axis and the relative displacement of the moving image block in the Y axis according to fluorescent signals of the seed image block and the moving image block in the field of view overlapped area, wherein the relative displacement is respectively used as a first overturning displacement Deltax 0 and a second overturning displacement Deltay 0; then, translating the moving image block along the negative direction of the X axis by a distance delta X 0 after first overturning and translating the moving image block along the negative direction of the Y axis by a distance delta Y 0 before second overturning, so that the seed image block and the moving image block are overlapped in a visual field overlapping area, then performing image stitching on the overlapped seed image block and the moving image block, and taking the stitched image block as a new seed image block;
Step S4.2.3, repeating step S4.2.2 for a plurality of times until all the flipped brain slice image blocks are subjected to image stitching, and obtaining flipped target image blocks.
The invention relates to a high-resolution complete neuron dendritic spine position and morphology detection system:
Comprises a high-resolution imaging module which is used for carrying out overturn imaging on the brain slice after the light removal treatment, acquiring brain slice image blocks;
Comprises an image processing module for performing three-dimensional image stitching on brain slice image blocks, obtaining a complete target brain slice image block;
The system comprises a dendritic spine reconstruction module, a neural spine reconstruction module and a neural spine reconstruction module, wherein the dendritic spine reconstruction module is used for carrying out three-dimensional reconstruction according to a target brain slice image block to obtain the position and the form information of a neuron dendritic spine structure.
The invention provides a marking method of a single complete neuron and a dendritic spine structure thereof, and a light clearing technology is combined to realize the transparency of a thick tissue sample, so that a turnover neuron high-resolution imaging and multi-view three-dimensional image stitching method is provided, and the reconstruction and quantitative analysis of all dendritic spines on the complete neuron are realized.
The detection method of the position and the form of the dendritic spines of the complete neuron comprises single neuron and dendritic spines structure marks, brain slice sample preparation, overturning neuron high-resolution imaging, multi-view three-dimensional image stitching and neuron dendritic spines reconstruction. Complete morphological labeling of individual neurons and their dendritic structures was achieved within 24 hours using sindbis virus; the preparation of brain slice samples comprises the steps of perfusion, fixation, slicing and the like; the adopted turnover imaging method ensures the collection of fluorescent signals with different depths; three-dimensional image stitching is performed by using overlapping signals among a plurality of images, so that the integrity of neurons in a large-view-field image is ensured; the neuronal dendritic spine reconstruction can provide positional and morphological information of the neuronal dendritic spine structure. The high-resolution complete neuron dendritic spine analysis method can realize analysis of single complete neuron dendritic spine within a few hours, and has extremely high application and research values.
The beneficial effects of the invention are as follows:
1. the invention realizes the combination of large visual field and high resolution through the high resolution imaging of the turnover neurons, and has simple steps and simple and easy method.
2. The three-dimensional image stitching and reconstructing method can obtain the structural position and morphological information of the dendritic spines of the neurons, and high-resolution complete analysis of the dendritic spines of the neurons is realized.
3. The invention can realize the rapid light removal of the thick brain slice by a simple sample processing method.
4. The marking of the neuron and the dendritic spine thereof can be realized within 24 hours, and the operation is simple and the speed is high.
Drawings
FIG. 1 is a flow chart of the high resolution full neuron dendritic spine resolution of the present invention;
FIG. 2 is a graph of the effect of 300 nl on Sindbis virus markers at a titer of 1.44×10 6 FFU/ml;
FIG. 3 is the light scavenging effect of a thick brain slice;
FIG. 4 is a schematic view of an XY-direction three-dimensional image stitching;
FIG. 5 is a schematic view of Z-direction three-dimensional image stitching;
fig. 6 is a graph of the results of reconstruction of intact neurons and dendritic spines of the hippocampus.
Detailed Description
The invention will now be described in detail with reference to specific examples which will assist those skilled in the art in further understanding the invention, but which are not intended to be limiting in any way.
The steps of the embodiment of the invention are as follows, as shown in fig. 1:
Step S1, injecting virus, and marking hippocampal single neurons and dendritic spines of the hippocampal single neurons:
Adult male or female mice were selected, the body weight of the mice was recorded, and a sodium pentobarbital solution (anesthetic) having a mass to volume ratio of 1% was injected into the abdominal cavity of the mice. The volume of anesthetic is regulated according to the weight of the mice, and is generally 0.1 ml/kg; the anesthetized mice are fixed on a stereotactic instrument by using an ear rod, the skin is disinfected by using alcohol after the hair on the skin is shaved, the scalp is cut along a middle seam, a cotton swab is used for dipping in normal saline, and then the skull is gently rubbed, so that fascia is removed. The virus injection needle is fixed on a stereotactic instrument, the head of the mouse is leveled, and finally, the bregma is set as the bregma front-rear AP: 0mm, ML at right and left middle joint: 0mm, skull dura mater plane down DV: 0 mm; the injection site of the hippocampus was AP: -1.9 mm, ml:1.25 mm, DV: -1.5 mm. The virus injection needle is used for sucking virus liquid 500 nl with titer of 1.44 multiplied by 10 6 FFU/ml, the needle is slowly and smoothly inserted into brain tissue, the needle stays for about 5 minutes, the virus 300 nl is injected at the speed of 80 nl/min, the needle stays for 10 minutes after the virus injection is finished, and the needle is slowly and smoothly pulled out. The marking effect is shown in fig. 2.
After 24 hours, the mice were again injected with 1% sodium pentobarbital solution intraperitoneally, after confirmation of complete anesthesia, the mice were fixed on foam plates with their ventral side facing upwards, and were perfused with heart, the blood was washed by first perfusing about 30 ml PBS, then perfused with 4% paraformaldehyde continuously for 30 ml, after the catalepsy, the perfusion was stopped, brain tissue was obtained, and the isolated brain tissue was immersed in 10ml of 4% paraformaldehyde solution for fixation for 12 hours.
Step S2: fixing the isolated brain tissue, slicing, and performing light removal treatment on the brain slice;
step S2.1, firstly, fixing the isolated brain tissue in a sample groove of an oscillation slicer, and slicing the brain tissue at a speed of 1.0 mm/S by using the oscillation slicer, so that the brain tissue is cut into brain slices with a thickness of 500 mu m, and the thickness of the slices of 500 mu m ensures the integrity of neurons and avoids excessive invalid information in the imaging and image processing processes.
S2.2, transferring brain slices into 24-well plates containing polybutylene succinate PBS, and slicing one brain slice in each well; 1 ml Triton X-100 (first light scavenging reagent) is used for replacing PBS, a 24-pore plate is wrapped by tinfoil and protected from light, and the mixture is placed on a shaking table for 2 hours; the first photo-stripping reagent was replaced with 1 ml antipyrine-nicotinamide solution (second photo-stripping reagent) and the stripping continued for 5 minutes. Finally, the light-removed brain slice is sealed on a cover glass containing a metal mold, and the light removal effect is shown in fig. 3.
The first photo-cleaning reagent adopts polyethylene glycol octyl phenyl ether Triton X-100, the mass volume ratio of the polyethylene glycol octyl phenyl ether is 15%, and the solvent is water; the second photo-scavenging reagent is mainly prepared by mixing antipyrine and nicotinamide, wherein the mass volume ratio of the antipyrine is 50%, the mass volume ratio of the nicotinamide is 30%, and the solvent is water.
Step S3: and (3) performing overturn imaging on the brain slice subjected to light removal treatment by using an objective lens to obtain a brain slice image block:
Step S3.1, preparing a die with a square through hole in the middle and a thickness of 500 mu m, placing the prepared die on a first cover glass, placing a brain slice in a square hole in the middle of the die, filling a second light removal reagent into the square hole, and then covering the second cover glass on the die to seal the square hole of the die to obtain a brain slice sample; the upper surface and the lower surface of the brain slice are respectively contacted with two cover slips, and the two surfaces of the brain slice contacted with the cover slips are respectively used as a first surface (A surface) and a second surface (B surface) of the brain slice;
and S3.2, selecting brain slices near the injection site, searching the positions of the complete neurons under a 10-fold mirror, ensuring that the cell bodies of the neurons are positioned in the middle of the sample, and recording the positions of signals to be acquired. And placing the surface A of the sample at the focus of an objective lens of an imaging system, switching and selecting a 40-time silicone oil lens with 1.25-NA, moving the neuron to the center of the field of view, selecting 8 imaging fields with cell bodies as the center, wherein the selected fields need to cover all fluorescent signals of the neuron, and the overlapping area between adjacent fields is about 10% of the size of the imaging fields. Imaging is started from the position where the surface A of the sample contacts the cover glass, the imaging depth is about 270 mu m, the imaging time is about 1.5 hours, the imaging step distance is 0.33 mu m, and a three-dimensional brain slice image block under each imaging view before overturning is obtained, the size of the horizontal section of the brain slice image block is equal to the size of the imaging view, and the depth of the brain slice image block is equal to the imaging depth.
And S3.3, turning the sample, placing the surface B of the sample at the focus of an objective lens of an imaging system, finding out the selected neuron under a low power mirror, switching to a 40-power silicone oil mirror with 1.25-NA, selecting 8 fields of view, and repeating the steps to obtain brain slice image blocks under each turned imaging field of view.
Specifically, the overlapping area of the image blocks before and after inversion in the Z-axis direction is about 40 μm.
Step S4: according to the fluorescence signals of the overlapping area of the brain slice image blocks, performing three-dimensional image stitching on the brain slice image blocks to obtain complete target brain slice image blocks, thereby realizing high-resolution complete neuron dendritic spine analysis:
s4.1, moving all the brain slice image blocks into a brain slice three-dimensional coordinate system, wherein in the brain slice three-dimensional coordinate system, the depth direction of the brain slice image blocks is defined as a Z axis, the length direction and the width direction of the brain slice image blocks are respectively defined as an X axis and a Y axis, and all the brain slice image blocks are arranged in parallel at intervals;
step S4.2, as shown in FIG. 4, all brain slice image blocks before overturning are spliced according to an overlapping area of 10% between adjacent visual fields, so as to obtain a target image block before overturning;
The step S4.2 specifically comprises the following steps:
Step S4.2.1, firstly, selecting any brain slice image block from all brain slice image blocks before overturning to serve as a seed image block;
Step S4.2.2, selecting one brain slice image block overlapped with the imaging area of the seed image block from the rest brain slice image blocks before overturning as a moving image block, and acquiring the relative displacement of the moving image block relative to the seed image block in the X axis and the relative displacement of the moving image block in the Y axis according to fluorescent signals of the seed image block and the moving image block in the field of view overlapped area, wherein the relative displacement is respectively used as a first displacement Deltax before overturning and a second displacement Deltay before overturning; translating the moving image block along the negative direction of the X axis by a distance delta X before first overturning and translating the moving image block along the negative direction of the Y axis by a distance delta Y before second overturning, so that the seed image block and the moving image block are overlapped in a visual field overlapping area, then performing image stitching on the overlapped seed image block and the moving image block, and taking the stitched image block as a new seed image block;
step S4.2.3, repeating step S4.2.2 for a plurality of times until all the pre-overturn brain slice image blocks are subjected to image stitching, and obtaining a pre-overturn target image block.
Then, splicing all the overturned brain slice image blocks according to the 10% overlapping area between the adjacent visual fields to obtain an overturned target image block;
Step S4.2.4, firstly, selecting any brain slice image block from all the turned brain slice image blocks to serve as a seed image block;
Step S4.2.5, selecting one brain slice image block overlapped with the imaging area of the seed image block from the rest brain slice image blocks after overturning as a moving image block, and acquiring the relative displacement of the moving image block relative to the seed image block in the X axis and the relative displacement of the moving image block in the Y axis according to fluorescent signals of the seed image block and the moving image block in the field of view overlapped area, wherein the relative displacement is respectively used as a first overturning displacement Deltax 0 and a second overturning displacement Deltay 0; then, translating the moving image block along the negative direction of the X axis by a distance delta X 0 after first overturning and translating the moving image block along the negative direction of the Y axis by a distance delta Y 0 before second overturning, so that the seed image block and the moving image block are overlapped in a visual field overlapping area, then performing image stitching on the overlapped seed image block and the moving image block, and taking the stitched image block as a new seed image block;
Step S4.2.6, repeating step S4.2.5 for a plurality of times until all the flipped brain slice image blocks are subjected to image stitching, and obtaining flipped target image blocks.
Step S4.3, as shown in FIG. 5, splicing the target image block before overturning and the target image block after overturning by using fluorescence signals of the overlapping area of the three-dimensional images of the A face and the B face in the Z axis direction of 40 mu m:
Firstly, turning the turned target image block up and down by 180 degrees, and then acquiring the relative displacement of the turned target image block relative to the turned target image block in the X axis, the relative displacement of the turned target image block in the Y axis and the relative displacement of the turned target image block in the Z axis according to fluorescent signals of the turned target image block before turning and the turned target image block after turning up and down in a Z axis visual field overlapping area, wherein the relative displacement is respectively used as three-way displacement one delta X ', three-way displacement two delta Y ' and three-way displacement three delta Z '; shifting the turned target image block by one delta X ' along the negative direction translation distance three-way of the X axis, shifting by two delta Y ' along the negative direction translation distance three-way of the Y axis and shifting by three delta Z ' along the negative direction translation distance three-way of the Z axis, so that the turned target image block and the turned target image block overlap in a visual field overlapping area, and then performing image stitching on the overlapped turned target image block and the turned target image block, and obtaining a complete brain slice image block after stitching is completed; the complete brain slice image block contains the complete neurons of the brain slice;
And S4.4, inputting the complete brain slice image block into a denoising convolutional neural network Unet so as to denoise the complete brain slice image block, weakening noise in the imaging process, enhancing signal quality, and outputting the denoised brain slice image block as a complete target brain slice image block by the convolutional neural network.
Step S5: reconstructing the neuron dendritic spines by using the target brain slice image blocks to obtain the structural position and morphological information of the neuron dendritic spines:
Step S5.1, firstly, inputting the complete target brain slice image block into the image analysis software imaris, and reconstructing the whole neuron in the brain slice by using the image analysis software:
Selecting and drawing a dendritic structure in the neuron from a complete target brain slice image block by using an image analysis software imaris with a cell body of the hippocampal neuron as a starting point, ensuring that each dendritic is selected, automatically identifying and reconstructing the dendritic spine structure on the dendritic of the neuron by using the image analysis software, further completing reconstruction of the whole neuron, and obtaining a complete neuron and a three-dimensional model with all dendritic spine information after the reconstruction of the neuron is completed; the image and its reconstructed structure are shown in fig. 6.
S5.2, acquiring position information and morphological information of the dendritic spines in the neurons according to the three-dimensional model; the position information of the dendritic spines comprises absolute space coordinate positions of the dendritic spines and relative positions of the dendritic spines relative to the dendritic structures in the whole neuron, and the morphological information comprises the volume, the area, the straightness and the like of the dendritic spines.
The foregoing embodiments have described the technical solutions and advantages of the present invention in detail, and it should be understood that the foregoing embodiments are merely illustrative of the present invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like that fall within the principles of the present invention should be included in the scope of the invention.
Claims (8)
1. The method for detecting the position and the morphology of the dendritic spines of the high-resolution complete neurons is characterized by comprising the following steps of:
Step S1: aiming at an in-vitro brain tissue with neurons carrying fluorescent signals, the in-vitro brain tissue adopts virus-labeled neurons and dendritic spine structures of the neurons;
Step S2: fixing the isolated brain tissue, slicing, and performing light removal treatment on the brain slice;
Step S3: performing overturn imaging on the brain slice subjected to light removal treatment by using a fluorescence microscope to obtain a brain slice image block;
In the step S3, the specific method for performing flip imaging on the brain slice by using the fluorescence microscope is as follows:
Step S3.1, placing a prepared mould on a first cover glass, placing a brain slice in a square hole in the middle of the mould, then filling a light removal reagent into the square hole, and then covering a second cover glass on the mould to seal the square hole of the mould so as to prepare a brain slice sample; the upper and lower surfaces of the brain slice are respectively contacted with two cover slips;
S3.2, selecting a silicone oil objective lens of a fluorescence microscope, and placing the upper surface of a brain slice sample at a focus of the silicone oil objective lens;
Step S3.3, selecting an imaging view field of a silicone oil objective lens by taking a neuron cell body as a center, wherein the specific selection mode of the imaging view field of the objective lens is as follows:
firstly, selecting a plurality of imaging fields by taking the cell bodies of neurons as central points, wherein the selected imaging fields comprise all fluorescence signals of the neurons in brain slices;
s3.4, imaging the brain slice by using a silicone oil objective lens according to each selected imaging view field to obtain a brain slice image block under each imaging view field;
Step S3.5, overturning the brain slice sample, placing the upper surface of the overturned brain slice sample at the focus of the objective lens, and repeating the steps S3.3-S3.4 to obtain brain slice image blocks under each imaging view after overturning;
step S4: according to the fluorescence signal of the overlapping area of the brain slice image blocks, performing three-dimensional image stitching on the brain slice image blocks to obtain complete target brain slice image blocks;
In the step S4, the method for performing three-dimensional image stitching on the brain slice image blocks specifically includes:
S4.1, all the brain slice image blocks are moved into a brain slice three-dimensional coordinate system, wherein in the brain slice three-dimensional coordinate system, the depth direction of the brain slice image blocks is defined as a Z axis, and the length direction and the width direction of the brain slice image blocks are respectively defined as an X axis and a Y axis;
Step S4.2, splicing all brain slice image blocks before overturning to obtain a target image block before overturning; splicing all the overturned brain slice image blocks to obtain an overturned target image block;
Step S4.3, splicing the target image block before overturning and the target image block after overturning:
Firstly, turning the turned target image block up and down by 180 degrees, and then acquiring the relative displacement of the turned target image block relative to the turned target image block in the X axis, the relative displacement of the turned target image block in the Y axis and the relative displacement of the turned target image block in the Z axis according to fluorescent signals of the turned target image block and the turned target image block in the overlapping area of the visual field, wherein the relative displacement is respectively used as three-way displacement one delta X ', three-way displacement two delta Y ' and three-way displacement three delta Z '; shifting the turned target image block by one delta X ' along the negative direction translation distance three-way of the X axis, shifting by two delta Y ' along the negative direction translation distance three-way of the Y axis and shifting by three delta Z ' along the negative direction translation distance three-way of the Z axis, so that the turned target image block and the turned target image block overlap in a visual field overlapping area, and then performing image stitching on the overlapped turned target image block and the turned target image block, and obtaining a complete brain slice image block after stitching is completed; the complete brain slice image block contains complete neurons in a brain slice;
S4.4, inputting the complete brain slice image block into a convolutional neural network to denoise the complete brain slice image block, and taking the denoised brain slice image block output by the convolutional neural network as a complete target brain slice image block;
Step S5: reconstructing the neuron dendritic spines by using the target brain slice image block to obtain the position and form information of the neuron dendritic spines.
2. The method for detecting the position and the morphology of the dendritic spines of the high-resolution complete neurons according to claim 1, wherein the method comprises the following steps of: the step S2 specifically comprises the following steps:
Step S2.1, firstly, fixing the isolated brain tissue in a sample groove of an oscillation slicer, and slicing the brain tissue by using the oscillation slicer so that the brain tissue is cut into brain slices with a certain thickness;
and S2.2, placing the brain slice in a light removal reagent, taking out the brain slice after 2-3 hours, transferring the brain slice into another light removal reagent, and taking out the isolated brain tissue after 4-6 minutes to obtain the brain slice after light removal treatment.
3. The method for detecting the position and the morphology of the dendritic spines of the high-resolution complete neurons according to claim 1, wherein the method comprises the following steps of: in the step S5, the specific method for reconstructing the neuron is as follows:
Step S5.1, firstly, inputting a complete target brain slice image block into image analysis software, and reconstructing the whole neuron in the brain slice by using the image analysis software: firstly, selecting and drawing a dendritic structure in a neuron in a complete target brain slice image block by utilizing image analysis software, then automatically identifying and reconstructing a dendritic spine structure on the neuron by utilizing the image analysis software, further completing reconstruction of the whole neuron, and obtaining a three-dimensional model containing complete neuron and all dendritic spine information after the reconstruction of the neuron is completed;
s5.2, acquiring position information and morphological information of the dendritic spines in the neurons according to the three-dimensional model; the position information of the dendritic spines comprises the relative positions of the dendritic spines relative to the dendritic structures in the whole neuron, and the morphological information comprises the volume, the area and the straightness information of the dendritic spines.
4. The method for detecting the position and the morphology of the dendritic spines of the high-resolution complete neurons according to claim 1, wherein the method comprises the following steps of: the specific way of splicing all the brain slice image blocks before overturning in the step S4.2 is as follows:
Step S4.2.1, firstly, selecting any brain slice image block from all brain slice image blocks before overturning to serve as a seed image block;
Step S4.2.2, selecting one brain slice image block overlapped with the imaging area of the seed image block from the rest brain slice image blocks before overturning as a moving image block, and acquiring the relative displacement of the moving image block relative to the seed image block in the X axis and the relative displacement of the moving image block in the Y axis according to fluorescent signals of the seed image block and the moving image block in the field of view overlapped area, wherein the relative displacement is respectively used as a first displacement Deltax before overturning and a second displacement Deltay before overturning; translating the moving image block along the negative direction of the X axis by a distance delta X before first overturning and translating the moving image block along the negative direction of the Y axis by a distance delta Y before second overturning, so that the seed image block and the moving image block are overlapped in a visual field overlapping area, then performing image stitching on the overlapped seed image block and the moving image block, and taking the stitched image block as a new seed image block;
step S4.2.3, repeating step S4.2.2 for a plurality of times until all the pre-overturn brain slice image blocks are subjected to image stitching, and obtaining a pre-overturn target image block.
5. The method for detecting the position and the morphology of the dendritic spines of the high-resolution complete neurons according to claim 1, wherein the method comprises the following steps of: the specific way of splicing all the flipped brain slice image blocks in the step S4.2 is as follows:
Step S4.2.1, firstly, selecting any brain slice image block from all the turned brain slice image blocks to serve as a seed image block;
Step S4.2.2, selecting one brain slice image block overlapped with the imaging area of the seed image block from the rest brain slice image blocks after overturning as a moving image block, and acquiring the relative displacement of the moving image block relative to the seed image block in the X axis and the relative displacement of the moving image block in the Y axis according to fluorescent signals of the seed image block and the moving image block in the field of view overlapped area, wherein the relative displacement is respectively used as a first overturning displacement Deltax 0 and a second overturning displacement Deltay 0; then, translating the moving image block along the negative direction of the X axis by a distance delta X 0 after first overturning and translating the moving image block along the negative direction of the Y axis by a distance delta Y 0 before second overturning, so that the seed image block and the moving image block are overlapped in a visual field overlapping area, then performing image stitching on the overlapped seed image block and the moving image block, and taking the stitched image block as a new seed image block;
Step S4.2.3, repeating step S4.2.2 for a plurality of times until all the flipped brain slice image blocks are subjected to image stitching, and obtaining flipped target image blocks.
6. A system for carrying out the method of any one of claims 1-5, characterized in that:
Comprises a high-resolution imaging module which is used for carrying out overturn imaging on the brain slice after the light removal treatment, acquiring brain slice image blocks;
Comprises an image processing module for performing three-dimensional image stitching on brain slice image blocks, obtaining a complete target brain slice image block;
The system comprises a dendritic spine reconstruction module, a neural spine reconstruction module and a neural spine reconstruction module, wherein the dendritic spine reconstruction module is used for carrying out three-dimensional reconstruction according to a target brain slice image block to obtain the position and the form information of a neuron dendritic spine structure.
7. A computer device comprising a memory and a processor, the memory storing a computer program, characterized in that the processor implements the steps of the method of any of claims 1-5 when the computer program is executed.
8. A computer readable storage medium, on which a computer program is stored, characterized in that the computer program, when being executed by a processor, implements the steps of the method of any of claims 1-5.
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