CN113390695A - Method for decoloring transparent large-volume sample on line and acquiring three-dimensional data - Google Patents
Method for decoloring transparent large-volume sample on line and acquiring three-dimensional data Download PDFInfo
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- CN113390695A CN113390695A CN202110651942.6A CN202110651942A CN113390695A CN 113390695 A CN113390695 A CN 113390695A CN 202110651942 A CN202110651942 A CN 202110651942A CN 113390695 A CN113390695 A CN 113390695A
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Images
Classifications
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01—MEASURING; TESTING
- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B11/00—Measuring arrangements characterised by the use of optical techniques
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- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
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- G—PHYSICS
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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Abstract
The invention discloses a method for decoloring a transparent large-volume sample on line and acquiring three-dimensional data, which comprises an embedding step, two immersion steps before and after the embedding step, and a circulating optical imaging and slicing step, wherein an imaging solution in the immersion step at least comprises sugar or alcohol or sugar alcohol compounds; urea; and aminoalcohols. The imaging solution performs real-time light transparency operation on the sample, balances the refractive index of the tissue sample, elutes heme in the sample, removes the influence of the heme on optical imaging, improves the depth of single imaging, reduces the times of microtomy of the sample, and improves the acquisition efficiency of three-dimensional data.
Description
Technical Field
The invention relates to the technical field of biomedical optical imaging, in particular to a method for decoloring a transparent large-volume sample on line and acquiring three-dimensional data.
Background
The composition of organisms is complex and diverse, and the structure and function of the organisms are always problems explored by scientists. The acquisition of high-resolution three-dimensional structural information of biological tissues at organ or even whole body level is crucial to deeply understanding and solving a plurality of biological and medical problems, and three-dimensional data of biological sample cell resolution can be acquired by an optical imaging technology, but the opacity of the tissues causes serious scattering and absorption of optical signals, thereby not only affecting the imaging quality, but also affecting the time of three-dimensional imaging of the sample.
The tissue light transparent technology solves the problem that biological tissues are not transparent, in recent years, in the field of tissue light transparency, two strategies are provided for transparency of biological tissue samples, firstly, light scattering is reduced, light transmission and receiving are seriously influenced by the tissue scattering on light, a reagent with high refractive index is introduced into the tissue, water in the tissue is replaced by transparent solution, the refractive index matching degree inside the tissue is improved, the purpose of reducing the light scattering of the tissue is achieved, and further the penetration depth of the light in the tissue is improved. And secondly, the light absorption is reduced, many biological tissue samples contain heme, and the reduction of the light absorption is particularly important for some tissues and organs with abundant heme content, such as spleen, kidney and the like. At present, the main technology of decolorization is to remove blood through physical perfusion to realize decolorization and to remove heme by soaking with chemical reagents. However, it is very difficult for large animals such as macaques and pigs to perform the perfusion, so that a great deal of blood is always remained in biological tissues obtained from the large animals, and the efficiency of the whole immersion and decoloration is very slow, which seriously slows down the process of obtaining data.
Therefore, how to rapidly acquire three-dimensional data of a large volume sample with more hemoglobin is a problem to be solved urgently.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for decoloring a transparent large-volume sample on line and acquiring three-dimensional data.
The purpose of the invention is realized by the following technical scheme:
a method for decoloring a transparent large-volume sample on line and acquiring three-dimensional data comprises the following steps:
s1, a first immersion step, namely immersing the biological sample tissue into an imaging solution for first immersion treatment for at least 24 hours to obtain the biological sample tissue after the first immersion treatment;
s2, embedding, namely, carrying out agarose embedding on the biological sample tissue processed in the S1 to obtain an embedded biological sample tissue;
s3, a second immersion step, in which the biological sample tissue embedded in the step S2 is immersed in the imaging solution again for at least 24 hours; and slicing and imaging the biological sample tissue subjected to the second immersion.
The imaging solution comprises the following raw materials in percentage by mass:
20-40% of sugar or alcohol or sugar alcohol compounds;
10% -30% of urea;
0% -30% of penetration enhancer;
5% -10% of amino alcohol compounds;
0 to 0.5 percent of surfactant and the balance of water.
Preferably, the amino alcohol compound is one or more of N-methyldiethanolamine or N, N, N ', N' -tetra (2-hydroxypropyl) ethylenediamine.
Preferably, the surfactant is one or more of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, triton X-100 and sodium dodecyl sulfate.
Preferably, the penetration enhancer is selected from one or more of dimethyl sulfoxide and propylene glycol.
Preferably, the sugar is one or more of fructose, glucose and sucrose.
Preferably, the alcohol is one or more of glycerol and polyethylene glycol.
Preferably, the sugar alcohol compound is one or more of sorbitol and xylitol.
Preferably, the step S2 specifically includes immersing the biological sample tissue in a mold having an agarose solution, maintaining the mold at a temperature range of 45-55 ℃ so that the biological tissue sample is fully contacted with the agarose solution and cross-linked for at least 30 minutes, taking out the mold, naturally cooling, and taking out the embedded biological sample tissue.
Preferably, the "slicing the biological sample tissue after the second immersion" specifically includes,
s4, a sample installation step, namely fixing the biological sample tissue immersed for the second time in the step S3 in a sample tank, and fixing the sample tank on a three-dimensional moving platform; pouring the imaging solution into the sample tank until the biological sample tissue, the microobjective, the cutter for cutting and the shallow surface of the biological sample tissue are submerged, and the shallow surface of the biological sample tissue is transparent and decolored;
s5, an optical imaging step, namely moving the three-dimensional moving platform to move the biological sample tissue to be under an objective lens, controlling an optical microscopic imaging module to perform microscopic optical imaging on the shallow surface of the biological sample tissue, and storing the obtained image;
s6, slicing the biological sample tissue in the imaging solution, and enabling the imaging solution to transparentize the surface of the biological sample tissue which is newly exposed after slicing in real time;
cyclically alternating S5 and S6 until a three-dimensional image of the biological sample tissue is obtained;
preferably, the maximum number of cuts in step S5 is N = H/d, H is the sample height, d is the slice thickness, and the slice thickness d is 20-100 um.
Preferably, when the steps S5 and S6 are performed alternately in a circulating manner, the step S6 is preceded by a step of detecting whether the slicing number reaches the maximum cutting number, and if the maximum cutting number is reached, the whole data collection is completed.
The invention has the following beneficial effects:
1. the optical transparency and microtome tomography technology are combined, optical imaging and microtome slicing are alternately carried out in the imaging solution, meanwhile, the imaging solution can carry out real-time optical transparency operation on the sample, the refractive index of the tissue sample is balanced, meanwhile, an amino alcohol reagent in the imaging solution rapidly reacts with the biological sample tissue, heme in the sample is eluted, the influence of the heme on the optical imaging is removed, the depth of single imaging is improved, the number of times of microtome slicing of the sample is reduced, the time for obtaining three-dimensional data of the sample is obviously reduced, and the obtaining efficiency is improved;
2. the surfactant perforates cell membranes of biological tissues, so that the decoloring process can be accelerated, and the decoloring efficiency is further improved;
3. dehydrating the biological sample tissue by using sugar or alcohol or sugar alcohol compound components in the imaging solution to enable the refractive index of the biological sample tissue to be uniform;
4. the urea in the imaging solution can expand the biological sample tissue to counteract the sample shrinkage caused by the sugar or alcohol or sugar-alcohol compound, so as to reduce the original size of the biological sample tissue as much as possible;
5. the penetration enhancer in the imaging solution can further improve the transparent effect of the solution and accelerate the light transparency realization efficiency;
6. the concentration difference between the embedded biological sample tissue interstitial fluid and the imaging solution is reduced through two immersion steps, the dehydration shrinkage amplitude is reduced, the separation of the biological sample tissue from an embedding agent is avoided, and the embedded biological sample tissue can bear the cutting force of a slicing machine during slicing;
7. the use of agarose for embedding biological sample tissue improves the speed and efficiency with which biological sample tissue is optically transparent.
Drawings
The technical scheme of the invention is further explained by combining the accompanying drawings as follows:
FIG. 1: schematic diagram of step S5 of the present invention;
FIG. 2: the imaging data of 60 microns after cutting in embodiment 2 of the invention is shown schematically;
FIG. 3: the state diagram of the biological tissue sample of 100 microns soaked in the imaging solution along with the change of time in the embodiment 2 of the invention;
FIG. 4: the experimental results of comparative example 1 and example 1 of the present invention are shown in the figure.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments shown in the drawings. These embodiments are not intended to limit the present invention, and structural, methodical, or functional changes that may be made by one of ordinary skill in the art in light of these embodiments are intended to be within the scope of the present invention.
In the description of the schemes, it should be noted that the terms "center", "upper", "lower", "left", "right", "front", "rear", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. In the description of the embodiment, the operator is used as a reference, and the direction close to the operator is a proximal end, and the direction away from the operator is a distal end.
The invention discloses a method for decoloring a transparent large-volume sample on line and acquiring three-dimensional data, which comprises the following steps:
s1, a first immersion step, namely immersing the biological sample tissue into an imaging solution for first immersion treatment for at least 24 hours to obtain the biological sample tissue after the first immersion treatment;
s2, embedding, namely, carrying out agarose embedding on the biological sample tissue processed in the S1 to obtain an embedded biological sample tissue;
s3, a second immersion step, in which the biological sample tissue embedded in the step S2 is immersed in the imaging solution again for at least 24 hours;
s4, a sample installation step, namely fixing the biological sample tissue immersed for the second time in the step S3 in a sample tank, and fixing the sample tank on a three-dimensional moving platform; pouring the imaging solution into the sample tank until the biological sample tissue, the microobjective, the cutter for cutting and the shallow surface of the biological sample tissue are submerged, and the shallow surface of the biological sample tissue is transparent and decolored;
s5, an optical imaging step, namely moving the three-dimensional moving platform to move the biological sample tissue to be under an objective lens, controlling an optical microscopic imaging module to perform microscopic optical imaging on the shallow surface of the biological sample tissue, and storing the obtained image;
s6, slicing the biological sample tissue in the imaging solution, and enabling the imaging solution to transparentize the surface of the biological sample tissue which is newly exposed after slicing in real time;
cyclically alternating S5 and S6 until a three-dimensional image of the biological sample tissue is obtained.
The step S1 specifically includes fixing the biological sample tissue by 4% paraformaldehyde solution until the biological sample tissue becomes hard. Removing paraformaldehyde residues on the tissue surface of the biological sample by using 0.01M phosphate buffer solution after the completion; and then immersed in the imaging solution for at least 24 hours. The imaging solution can dehydrate the biological sample tissue to shrink to a certain degree, and can enter the biological sample tissue to replace liquid between internal tissues, so that after the biological sample tissue is embedded in the step S2, when the biological sample tissue enters the step S3, the concentration difference between the liquid inside the embedded biological sample tissue and the imaging solution can be reduced, the shrinking amplitude of the biological sample tissue is reduced, the biological sample tissue is ensured not to be separated from embedding agents on the surface of the biological sample tissue, and the embedding agents are ensured to have enough support for the biological sample tissue to bear the cutting force of the vibrating microtome on the biological sample tissue in the step S5. Since the biological sample tissue is contracted to a certain extent in the imaging solution, and the contraction amplitude is maximum in 24 hours, and is greatly reduced after exceeding 24 hours, the biological sample tissue is immersed for at least 24 hours in step S1 to ensure the stability of the biological sample tissue in the subsequent operation.
The step S2 specifically includes placing the rinsed biological sample tissue into a mold, filling the mold with the agarose solution to immerse the biological sample tissue, wherein the temperature of the agarose solution is maintained at 45-55 ℃ to ensure that the agarose solution is kept in a liquid state, and then placing the mold in a water bath at 45-55 ℃ for at least 30 minutes to fully contact and crosslink the biological sample tissue with the agarose solution; finally, the mold is removed and allowed to cool naturally for 30 minutes, and then the biological sample tissue with the agarose attached to the surface is removed so that the biological sample tissue has enough support to withstand the cutting force of the vibrating microtome in step S5.
In the step S2, an agarose solution is used for embedding, and compared with other embedding agents, agarose has larger molecules, so that agarose molecules are only cross-linked around the biological sample tissue and cannot penetrate into the biological sample tissue during the embedding process.
The step S3 is to soak the embedded biological sample tissue in the imaging solution again, and preferably for at least 24 hours, in order to dehydrate the agarose to adapt to the osmotic pressure of the imaging solution, so that the biological sample tissue is more stably linked to the agarose when proceeding to the subsequent steps S4, S5. The steps S3 and S1 are immersed for at least 24 hours, so that the stability of the biological sample tissue in the subsequent operation is further ensured.
In step S5, cutting is performed using a vibrating microtome. The maximum number of cuts in step S5 is N = H/d, and the slice thickness is preferably 20 to 100 um. The specific slice thickness is determined according to the actual volume of the biological sample tissue and the staining depth. The step S5 is shown in fig. 1, where 1 is a three-dimensional moving platform, 2 is a sample tank, 3 is a vibration cutting tool, 4 is a microscopic imaging optical path, 5 is an objective lens, 6 is a biological tissue sample, 7 is an imaging solution, 8 is a filtration water pump, 9 is a filtration barrel, and 10 is a color remover such as activated carbon.
Further, in order to ensure the complete collection of the imaging data, when the steps S5 and S6 are performed alternately in a loop, the step S6 is preceded by a step of detecting whether the number of slices reaches the maximum number of cuts, and if the maximum number of cuts is reached, the whole data collection is completed.
The imaging solution comprises the following raw materials in percentage by mass:
20-40% of sugar or alcohol or sugar alcohol compounds;
10% -30% of urea;
0% -30% of penetration enhancer;
5% -10% of amino alcohol compounds;
0 to 0.5 percent of surfactant and the balance of water.
Wherein the sugar is one or more of fructose, glucose and sucrose. The alcohol is selected from glycerol. The sugar alcohol compound is one or more of sorbitol and xylitol. The invention preferably uses fructose, which can dehydrate the biological sample tissue in contact with the fructose, homogenize the refractive index of the biological sample tissue, reduce tissue scattering, make the biological sample tissue transparent to light and improve the imaging depth.
Because the fructose makes the biological sample tissue shrink obviously, the urea is contacted with the biological sample tissue to generate hydration reaction, so that the biological sample tissue expands to counteract the sample shrinkage caused by a part of the fructose, and the original size of the biological sample tissue is restored as much as possible.
The penetration enhancer is selected from dimethyl sulfoxide or propylene glycol. The preferable penetration enhancer in the invention is dimethyl sulfoxide to further improve the penetration effect of the solution, and simultaneously, the concentration and viscosity of the imaging solution can not be greatly increased, so that the resistance received by a cutter in slicing can not be increased.
The amino alcohol compound is one or more of N-methyldiethanolamine or N, N, N ', N' -tetra (2-hydroxypropyl) ethylenediamine. The aminoalcohol compound serves to decolorize hemoglobin in a biological tissue sample, and thus is preferably soluble in water to produce a colorless aqueous solution.
The surfactant is one of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, triton X-100 and lauryl sodium sulfate. The addition of the surfactant can punch the cell membrane of the biological tissue sample, so that the amino alcohol reagent can enter the cell membrane at an accelerated speed, the effect of accelerating decoloration is achieved, and the light transparency rate is further improved.
The present invention is described in more detail below by way of examples. These examples are merely illustrative of the best mode of carrying out the invention and do not limit the scope of the invention in any way.
Example 1
The imaging solution in example 1 was composed of an aqueous solution having a concentration of 30wt% fructose, 20wt% urea and 20V/V% dimethyl sulfoxide, 5V/V% N-methyldiethanolamine and 0.5wt%3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt. The biological sample tissue is liver tissue of adult domestic pigs which do not flow through, and the acquisition of three-dimensional data is completed according to the following steps:
s1, first, liver tissue of adult domestic pig is taken as biological sample tissue, and after soaking in 4% paraformaldehyde solution, the tissue is fixed for 3 months until the sample becomes hard.
S2, the biological sample tissue is pretreated, namely, the biological sample tissue is immersed in the imaging solution for at least 24 hours, and dehydration and surface transparent and decoloration are firstly carried out on the biological sample tissue.
And S3, carrying out agarose embedding on the pretreated biological sample tissue.
And S4, soaking the embedded biological sample tissue in the transparent solution for no less than 24 hours.
And S5, adhering the embedded biological sample tissue on a sample base through glue, and fixing the sample base in a sample groove through screws.
S6, filling the sample tank with imaging solution until the biological sample tissue, the microscope objective and the cutter for cutting are submerged.
And S7, cutting the surface of the biological sample tissue flat by using a vibrating microtome, setting the maximum cutting times N = H/d according to the height H of the biological sample tissue, setting the cutting thickness to be 40 micrometers, setting the imaging step to be 10 micrometers, setting the total depth of each imaging to be 60 micrometers, and reserving the redundancy of 20 micrometers for later image calibration.
And S8, clicking to start imaging, triggering the three-dimensional translation stage to drive the sample groove to move through program control, and moving to a position 40 microns above the cutting edge of the cutting tool to perform micro thin slicing.
And S9, in the slicing process, the imaging solution quickly permeates to the newly exposed surface of the biological tissue sample, and real-time transparency and decoloration are carried out.
And S10, after slicing is completed, the three-dimensional translation stage drives the biological sample tissue to move to the focal plane position of the objective lens of the line confocal imaging system, the liver autofluorescence data is acquired by the line confocal imaging system, the translation stage drives the sample groove to move back and forth, imaging of the whole surface of the biological sample tissue is completed, then the translation stage is lifted by 10 microns, imaging of the next layer is continued until acquisition of depth information of 60 microns is completed, and the imaging result is stored in a memory of a workstation.
And S11, adding activated carbon into the filter vat in the system operation process, turning on the filter water pump, circulating the solution between the sample tank and the filter vat, and removing the pigment in the imaging solution and the cut sample slice.
And S12, detecting whether the maximum cutting layer number is reached after each imaging is completed, namely whether the cutting of the whole sample is completed, if not, repeating the step S8, the step S9 and the step S10 again until the maximum cutting layer number is reached, completing the data acquisition of the whole sample, and preprocessing the acquired image for three-dimensional reconstruction and data analysis.
Wherein step 10 is shown in the second row image of fig. 4 for imaging data 60 microns after ablation.
Example 2
The imaging solution in example 1 was prepared from an aqueous solution having a concentration of 30wt% fructose, 20wt% urea and 20V/V% dimethyl sulfoxide, 10V/V% N-methyldiethanolamine. The biological sample tissue is liver tissue of adult domestic pigs which do not flow through, and the acquisition of three-dimensional data is completed according to the following steps:
s1, first, liver tissue of adult domestic pig is taken as biological sample tissue, and after soaking in 4% paraformaldehyde solution, the tissue is fixed for 3 months until the sample becomes hard.
S2, the biological sample tissue is pretreated, namely, the biological sample tissue is immersed in the imaging solution for at least 24 hours, and dehydration and surface transparent and decoloration are firstly carried out on the biological sample tissue.
And S3, carrying out agarose embedding on the pretreated biological sample tissue.
And S4, soaking the embedded biological sample tissue in the transparent solution for no less than 24 hours.
And S5, adhering the embedded biological sample tissue on a sample base through glue, and fixing the sample base in a sample groove through screws.
S6, filling the sample tank with imaging solution until the biological sample tissue, the microscope objective and the cutter for cutting are submerged.
And S7, cutting the surface of the biological sample tissue flat by using a vibrating microtome, setting the maximum cutting times N = H/d according to the height H of the biological sample tissue, setting the cutting thickness to be 60 micrometers, setting the imaging step to be 10 micrometers, setting the total depth of each imaging to be 80 micrometers, and reserving the redundancy of 20 micrometers for later image calibration.
And S8, clicking to start imaging, triggering the three-dimensional translation stage to drive the sample groove to move through program control, and moving to a position 60 micrometers above the cutting edge of the cutting tool to perform micro thin slicing.
And S9, in the slicing process, the imaging solution quickly permeates to the newly exposed surface of the biological tissue sample, and real-time transparency and decoloration are carried out.
And S10, after slicing is completed, the three-dimensional translation stage drives the biological sample tissue to move to the focal plane position of the objective lens of the line confocal imaging system, the liver autofluorescence data is acquired by the line confocal imaging system, the translation stage drives the sample groove to move back and forth, imaging of the whole surface of the biological sample tissue is completed, then the translation stage is lifted by 10 microns, imaging of the next layer is continued until acquisition of depth information of 60 microns is completed, and the imaging result is stored in a memory of a workstation.
And S11, adding activated carbon into the filter vat in the system operation process, turning on the filter water pump, circulating the solution between the sample tank and the filter vat, and removing the pigment in the imaging solution and the cut sample slice.
And S12, detecting whether the maximum cutting layer number is reached after each imaging is completed, namely whether the cutting of the whole sample is completed, if not, repeating the step S8, the step S9 and the step S10 again until the maximum cutting layer number is reached, completing the data acquisition of the whole sample, and preprocessing the acquired image for three-dimensional reconstruction and data analysis.
Wherein step 10 is shown in figure 2 for imaging data 60 microns after ablation.
Fig. 3 is a state diagram of the biological tissue sample of 100 micrometers soaked in the imaging solution in example 2, which is placed on 2mm mesh paper and changed with time, and it can be visually seen that the mesh lines on the mesh paper are gradually clear with the passage of time, which can prove that N-methyldiethanolamine has the effect of decoloring the biological tissue sample.
Comparative example 1
The same materials and procedures as in example 1 were followed, with the only difference that the imaging solution of comparative example 1 was an aqueous solution of fructose at a concentration of 30wt%, urea at 20wt% and dimethyl sulfoxide at 20 v/v%.
Fig. 4 is a graph comparing the results of comparative example 1 and example 1, wherein the first row is the experimental result of comparative example 1 and the second row is the experimental result of example 1. It can be clearly seen that the three-dimensional data generated by the experimental result of example 1 is relatively complete and clear, the components N-methyldiethanolamine and 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt can play a good role in decoloring heme in a biological sample tissue, reduce the influence of heme in the biological sample tissue on the light transparency effect, and improve the acquisition of the three-dimensional data of the biological sample tissue, especially the biological sample tissue with much heme. Further, embodiment 1 is superior to embodiment 2 in terms of time cost and imaging efficiency, and therefore embodiment 1 in the present invention is the most preferred embodiment.
The invention combines the light transparency and microtome tomography technology, alternately performs optical imaging and microtome slicing in the imaging solution, simultaneously the imaging solution can perform real-time light transparency operation on the sample, balances the refractive index of the tissue sample, simultaneously the amino alcohol reagent in the imaging solution rapidly reacts with the biological sample tissue, elutes the heme in the sample, removes the influence of the heme on the optical imaging, improves the depth of single imaging, reduces the times of microtome slicing of the sample, thereby obviously reducing the time for obtaining three-dimensional data of the sample and improving the obtaining efficiency.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (9)
1. The method for decoloring the transparent large-volume sample on line and acquiring three-dimensional data is characterized by comprising the following steps of: the method comprises the following steps:
s1, a first immersion step, namely immersing the biological sample tissue into an imaging solution for first immersion treatment for at least 24 hours to obtain the biological sample tissue after the first immersion treatment;
s2, embedding, namely, carrying out agarose embedding on the biological sample tissue processed in the S1 to obtain an embedded biological sample tissue;
s3, a second immersion step, in which the biological sample tissue embedded in the step S2 is immersed in the imaging solution again for at least 24 hours; slicing and imaging the biological sample tissue after the second immersion,
the imaging solution comprises the following raw materials in percentage by mass:
20-40% of sugar or alcohol or sugar alcohol compounds;
10% -30% of urea;
0% -30% of penetration enhancer;
5% -10% of amino alcohol compounds;
0 to 0.5 percent of surfactant and the balance of water.
2. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 1, wherein: the amino alcohol compound is one or more of N-methyldiethanolamine or N, N, N ', N' -tetra (2-hydroxypropyl) ethylenediamine.
3. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 1, wherein: the surfactant is one or more of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, triton X-100 and lauryl sodium sulfate.
4. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 1, wherein: the penetration enhancer is selected from one or more of dimethyl sulfoxide and propylene glycol.
5. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 1, wherein: the sugar is one or more of fructose, glucose and sucrose; the alcohol is one or more of glycerol and polyethylene glycol; the sugar alcohol compound is one or more of sorbitol and xylitol.
6. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 1, wherein: the step S2 specifically includes immersing the biological tissue sample in a mold having an agarose solution, maintaining the mold at a temperature range of 45-55 ℃ to allow the biological tissue sample to be in sufficient contact with the agarose solution and cross-linked for at least 30 minutes, removing the mold, naturally cooling, and removing the embedded biological tissue sample.
7. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 1, wherein: the "slicing imaging of the biological sample tissue after the second immersion" specifically includes,
s4, a sample installation step, namely fixing the biological sample tissue immersed for the second time in the step S3 in a sample tank, and fixing the sample tank on a three-dimensional moving platform; pouring the imaging solution into the sample tank until the biological sample tissue, the microobjective, the cutter for cutting and the shallow surface of the biological sample tissue are submerged, and the shallow surface of the biological sample tissue is transparent and decolored;
s5, an optical imaging step, namely moving the three-dimensional moving platform to move the biological sample tissue to be under an objective lens, controlling an optical microscopic imaging module to perform microscopic optical imaging on the shallow surface of the biological sample tissue, and storing the obtained image;
s6, slicing the biological sample tissue in the imaging solution, and enabling the imaging solution to transparentize the surface of the biological sample tissue which is newly exposed after slicing in real time;
cyclically alternating S5 and S6 until a three-dimensional image of the biological sample tissue is obtained.
8. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 7, wherein: the maximum number of cuts in step S5 is N = H/d, H is the sample height, d is the slice thickness, and the slice thickness d is 20-100 um.
9. The method for the on-line destaining of transparent bulk samples and acquiring three-dimensional data according to claim 1, wherein: when the steps S5 and S6 are performed alternately in a circulating manner, the step S6 is preceded by a detection step of detecting whether the slicing number reaches the maximum cutting number, and if the maximum cutting number is reached, the whole data acquisition is completed.
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