CN117054182B - Shapable tissue transparentizing agent and preparation method and application method thereof - Google Patents
Shapable tissue transparentizing agent and preparation method and application method thereof Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N1/36—Embedding or analogous mounting of samples
Abstract
The invention discloses a shapable tissue transparentizing reagent, a preparation method and a use method thereof, belonging to the technical field of medical materials. The tissue transparentizing agent comprises imidazole, sorbitol and water. The tissue transparentizing agent provided by the invention has excellent optical clearing capacity and good tissue clearing effect, can self-assemble hydrogel, realizes double effects of tissue transparentization and embedding, can play a good shaping role on biological tissues, and is beneficial to the fixation and imaging of the biological tissues; the tissue transparentizing agent can well preserve biological tissues and can realize recycling through thermal reversible phase transformation. The tissue transparentizing agent provided by the invention has simple components, takes water as a solvent and an adhesive, and does not need to add an additional cross-linking agent; the preparation process is controllable, the preparation and use methods are simple, and the preparation method is green, safe and pollution-free.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a shapable tissue transparentizing reagent, a preparation method and a use method thereof.
Background
The tissue transparentization technology which is emerging in recent years can realize complete tissue or organ transparentization, and has wide application prospect. The tissue transparentization technology can reduce light scattering and light absorption to make a sample transparent under vision, and can realize three-dimensional reconstruction of a tissue structure when being combined with an optical microscope and a fluorescent marking technology, so that the defects of discontinuity, difficult image reconstruction and the like of the traditional tissue section technology are overcome.
Currently, the ultra-fast optical clear-out (FOCM) technology, SCALE, CUBIC, SEEDB, MACS and other transparentization technologies are widely applied in the field of life science. The hydrogel embedding and transparentizing technology is widely focused on shaping soft tissues, and is divided into an active type and a passive type, but the hydrogel forming and transparentizing operations are independently carried out, and the operation steps are relatively complicated. Therefore, the development of the transparentizing agent which is used for rapidly shaping biological tissues and is simple to operate has great significance.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a shapable tissue transparentizing agent, a preparation method and a use method thereof. The tissue transparentizing agent provided by the invention not only can transparentize biological tissues, but also can rapidly shape the biological tissues.
The invention provides a shapable tissue transparentizing agent.
Specifically, a tissue transparentizing agent comprises imidazole, sorbitol and water.
Preferably, the tissue transparentizing agent comprises, in mass percent: 2% -15% of imidazole, 80% -95% of sorbitol and 1% -10% of water.
Further preferably, the tissue transparentizing agent comprises, in mass percent: 4% -8% of imidazole, 85% -95% of sorbitol and 2% -10% of water.
More preferably, the tissue transparentizing agent comprises, in mass percent: 4% -6% of imidazole, 86% -93% of sorbitol and 2% -8% of water.
The tissue transparentizing agent provided by the invention uses imidazole, sorbitol and water as raw materials, and the amino groups of the imidazole can be combined with the lipid of the tissue, so that the lipid seeps out from the tissue, the scattering of biological tissue is rapidly reduced, and the refractive indexes of the components of the tissue are similar. When sorbitol acts on transparent tissues, the fibril structure of the collagen can be decomposed into microfibers, and the solubility of the collagen is increased, so that the transparent effect is achieved. The water molecules interact with the hydrophilic groups to enter the interior of the molecules, and the refractive index of the sample is homogenized by removing, replacing and modifying some components in the tissue, so that the biological tissue can be made transparent, and the shaping effect is achieved.
Preferably, the amino group of the imidazole and the sorbitol C 2 The hydroxyl groups at the positions are subjected to hydrogen bond interaction to form hydrogel; the hydrogen bond interaction is shown as a formula (1):
preferably, the refractive index of the hydrogel is 1.500-1.600; further preferably, the refractive index of the hydrogel is 1.505-1.550; more preferably, the refractive index of the hydrogel is 1.505-1.520.
The invention also provides a preparation method of the tissue transparentizing agent.
Specifically, a preparation method of the tissue transparentizing agent comprises the following steps:
mixing imidazole, sorbitol and water according to a certain proportion, heating and dissolving to obtain the tissue transparentizing agent.
After mixing both imidazole and sorbitol with water, intermolecular hydrogen bonding causes it to form a stable three-dimensional network. When heating, the intermolecular thermal motion is aggravated, so that the hydrogen bond action is broken, the intermolecular structure is changed, the structure is gradually loosened along with the rise of the temperature, and after the temperature reaches a certain value, the intermolecular structure is completely broken, and the molecules start to move freely.
Preferably, the heating temperature is 70 ℃ to 80 ℃.
The invention also provides a use method of the tissue transparentizing agent.
Specifically, the application method of the tissue transparent reagent comprises the following steps:
and (3) placing the biological tissue into a tissue transparentizing reagent, and incubating to obtain the hydrogel-embedded transparentizing tissue. The hydrogel-embedded transparentized tissue is shaped and transparentized.
When biological tissues are placed in the tissue transparentizing agent for incubation, due to the combined action of imidazole, sorbitol and water, the amino group of the imidazole can form hydrogen bond interaction with the hydroxyl group at the C2 position of the sorbitol to form hydrogel; and at the incubation temperature, the movement between molecules is slowed down, the hydrogen bond and the hydrophobic effect are enhanced, and a net structure is formed again, so that the tissue is embedded by the hydrogel while being transparent, and the high-viscosity thermoreversible gel with strong mechanical property is formed. Especially for thin-wall soft tissues such as small intestine, esophagus and the like, the molding is stable, and the preparation of tissue slices and the like is convenient.
Preferably, the temperature of the incubation is 20-37 ℃ and the time of the incubation is 6-48 h.
Compared with the prior art, the invention has the following beneficial effects:
(1) The tissue transparentizing agent provided by the invention has excellent optical clearing capacity and good tissue clearing effect, can self-assemble hydrogel, realizes double effects of tissue transparentization and embedding, can play a good shaping role on biological tissues, and is beneficial to the fixation and imaging of the biological tissues; the tissue transparentizing agent can well preserve tissues and can realize recycling through thermal reversible phase transformation.
(2) The tissue transparentizing agent provided by the invention has high refractive index and excellent optical clearing capacity, is immersed into a tissue structure in a small molecular form to be self-assembled into gel, is more fully immersed, is more tightly combined, is uniformly distributed in the tissue, and has the effect of immediately supporting and shaping.
(3) The tissue transparentizing agent provided by the invention has simple components, takes water as a solvent and an adhesive, and does not need to add an additional cross-linking agent; the preparation process is controllable, the preparation and use methods are simple, and the preparation method is green, safe and pollution-free.
Drawings
FIG. 1 is a visual appearance of a gel formed by adding different mass percentages of water in experiment 2;
FIG. 2 is a graph showing the comparison of the gel of the small intestine tissue before the small intestine is transparent in example 1;
FIG. 3 is a graph showing the comparison of the tissue before and after the tissue transparentization treatment of example 2;
FIG. 4 is a fluorescent CLSM image of example 3 after embedding the tissue-clearing agent in the small intestine of a mouse.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Experiment 1
The gel formation of the different reagent compositions was studied by first setting an experimental group (No. i) and 7 control groups (No. ii, iii, iv, v, vi, vii, viii), the reagent compositions and concentrations of each group, and the gel formation results of the solutions are shown in table 1. In 7 control groups, glucose, fructose, mannose, methanol, ethanol, propanol and butanol were used in place of sorbitol, respectively. The mass ratio of imidazole to sugar or alcohol is controlled to be 6%/88%, and the water content is controlled to be 6%.
TABLE 1 composition and concentration of different reagents, state of solution
| Reagent set | Mass ratio | Water content (%) | Whether or not to gel |
| Ⅰ | Imidazole/sorbitol 6%/88% | 6％ | Can be used for |
| Ⅱ | Imidazole/glucose 6%/88% | 6％ | Cannot be used |
| Ⅲ | Imidazole/fructose 6%/88% | 6％ | Cannot be used |
| Ⅳ | Imidazole/mannose 6%/88% | 6％ | Cannot be used |
| Ⅴ | Imidazole/methanol 6%/88% | 6％ | Cannot be used |
| Ⅵ | Imidazole/ethanol 6%/88% | 6％ | Cannot be used |
| Ⅶ | Imidazole/propanol 6%/88% | 6％ | Cannot be used |
| Ⅷ | Imidazole/butanol 6%/88% | 6％ | Cannot be used |
As can be seen from table 1, imidazole cannot form gel with glucose, fructose, mannose, methanol, ethanol, propanol and butanol, and thus even though imidazole can have a certain tissue transparentizing effect in combination with a part of components, it cannot form gel, cannot have a good shaping effect on biological tissues, and is unfavorable for fixation and imaging of biological tissues.
In the reagent group I, imidazole is combined with sorbitol, the amino group of the imidazole and the hydroxyl group of the sorbitol have hydrogen bonding effect, and further water molecules interact with hydrophilic groups of the imidazole, so that the hydrogel can be formed under the condition that no additional cross-linking agent is added. Therefore, the tissue transparent reagent prepared from imidazole, sorbitol and water can have a good shaping effect on biological tissues, not only can cut and not deform, but also can shape the parenchyma, and is beneficial to the fixation and imaging of the biological tissues.
Although mannose and sorbitol are isomers, the hydroxyl space structure is different only at the C2 position, the chirality of the C2 atom is different, and the sorbitol is S, R, R and R; mannitol is R, R, R and R are affected by a blocking effect, and the amino group of imidazole can form hydrogen bond interaction with the hydroxyl group at the C2 position of sorbitol, but cannot form hydrogen bond interaction with mannitol. The remaining sugar, alcohol and imidazole also cannot undergo the above-mentioned hydrogen bonding, and thus cannot form a gel. The results demonstrate that only sorbitol-based tissue transparentization and transparent solution, while not too high in viscosity, are beneficial to tissue shaping, and that fructose, mannose and glucose do not produce similar effects.
Experiment 2
The effect of water content, and mass ratio of imidazole to sorbitol on the properties of tissue transparency agents, mass ratio of imidazole to sorbitol, water content, and transparency effect of the solution, refractive index, and gel forming results were studied, see table 2.
TABLE 2 refractive index of imidazole/sorbitol solutions of different Mass ratios and gel formation results
As is clear from Table 2, when the water content is 0, the transparency of the gel formed is extremely low, which is not conducive to tissue observation, and when the water content is too high, the solution cannot form a gel state, and the tissue cannot be shaped. Fig. 1 shows the gel formation state with different mass percentages of water, and fig. 1 (a) and (b) show gel state diagrams after the tissue transparent agent is put forward and inverted, respectively. By adjusting the amount of water added, tissue transparentizing agents with different refractive indices are designed. When the water content is too low, the reagent is more viscous, the refractive index is higher, the degree of transparentization of the tissue is low, and the subsequent observation effect is affected. When the water content is too high, the viscosity of the reagent is low, and the formed gel is loose, so that the shaping of the thin-wall soft tissue is not facilitated.
Example 1
A tissue transparentizing agent, which consists of the following components in percentage by mass: imidazole 6.0%, sorbitol 90.0% and water 4.0%.
The preparation method of the tissue transparentizing agent comprises the following steps:
the imidazole, sorbitol and water are mixed evenly and heated and dissolved at 75 ℃ to obtain the tissue transparentizing agent. The prepared tissue transparentizing agent has high transparency, and has a refractive index of 1.5138, a hardness of 310.68N and a viscosity of 3.2mJ.
Experiments were performed on the small intestine tissue of the mice using the tissue clearing reagent prepared in example 1, and the small intestine tissue of the mice was soaked in the tissue clearing reagent at 25 deg.c for 10 hours.
As shown in fig. 2, in fig. 2 a, (a) is a state diagram before the small intestine is transparent, (b) is a schematic view of the transparency of the hydrogel, and (c) is a tissue hydrogel for the small intestine; in fig. 2, B (a) is a state diagram before the small intestine is transparent, (B) is a diagram of the effect of the tissue hydrogel after the small intestine is transparent, (c) is a diagram of the use of the small intestine tissue hydrogel sheet after the small intestine is transparent for confocal splint fixation, and (d) is a schematic diagram of the thickness of the small intestine tissue transparent hydrogel sheet. As can be seen from fig. 2, imidazole/sorbitol has a strong tissue clearance ability to tissues; the formed hydrogel has high transparency and no color (B (B) in fig. 2), meanwhile, the specimen has high compatibility with self-polymerized hydrogel (B (c) in fig. 2), the transparent specimen cannot be influenced, more importantly, the hydrogel block formed by the tissue transparentizing agent has high plasticity and stable forming, can stably maintain a specific shape, can also be moved at will (A in fig. 2), and provides convenience for subsequent experiments and preservation. In addition, the thickness of the hydrogel formed by the tissue transparentizing agent is controllable, the thickness of the hydrogel can be made to be as thin as 2mm, and the hydrogel can be suitable for the thickness of a confocal microscope slice loading tool, and is convenient for placement and observation.
Example 2
A tissue transparentizing agent, which consists of the following components in percentage by mass: imidazole 5.6%, sorbitol 89.4% and water 5.0%.
The preparation method of the tissue transparentizing agent comprises the following steps:
mixing imidazole, sorbitol and water uniformly, and heating and dissolving at 80 ℃ to obtain the tissue transparentizing agent. The prepared tissue transparentizing agent has high transparency, and has a refractive index of 1.5123, a hardness of 280.93N and a viscosity of 3.6mJ. The transparentizing agent is used for transparentizing treatment of kidneys, small intestines, esophagus, half brains, lungs, livers and spleens of mice, and when the soaking temperature is controlled to be 25 ℃, the soaking time is respectively as follows: the kidney of the mouse was 18h, the small intestine of the mouse was 6h, the esophagus of the mouse was 8h, the half brain of the mouse was 12h, the lung of the mouse was 16h, and the liver of the mouse was 24h.
Fig. 3 is a comparison image taken before and after the tissue is subjected to the transparentization treatment. From left to right, kidney, small intestine, esophagus, hemi-brain, lung, liver and spleen tissues of a mouse are respectively, an upper-row image is an image before tissue transparentization treatment, and a line lattice below the tissue is hidden from view by the tissue due to the turbidity characteristic of the tissue; the lower row image is an image of the same tissue after the transparentization treatment, at the moment, the tissue becomes highly transparent, and the line lattice below the tissue is clearly visible (the background line lattice is 2mm x 2 mm). The tissue transparentizing agent provided by the invention can realize a better transparentizing effect by simple immersion on the basis of degreasing, has an excellent effect on clearing tubular tissue with thinner wall such as small intestine and esophagus, and has the shortest time and unchanged tissue form; the method has certain transparency to hollow viscera with thicker myocardial wall and complex result, and the method has good clearance to the substantial viscera with thicker tissues such as liver, spleen and lung, and can realize transparency to brain tissues with rich lipid.
Example 3
In this example, the group II tissue transparentizing agent of table 2 in experiment 2 was used to treat the small intestine tissue of the mouse, and after transparentizing treatment, the small intestine tissue was sectioned, and then placed under a confocal laser scanning microscope for observation, and the fluorescence signal appeared green under the microscope. The CLSM image (laser scanning confocal microscope image) after the mice small intestine is cleared and formed by the organization transparent method and the hydrogel is embedded in the hydrogel is shown in fig. 4, a and b in fig. 4 are DiO-dyed mice small intestine tissue CLSM images, a is tiled small intestine tissue, and obvious blood vessel distribution and trend can be observed; panel b shows the distribution of signals in the circular small intestine. In fig. 4, c and d are CLSM images of EGFP (enhanced green fluorescent protein) mouse small intestine tissue, c is a single-layer fluorescence imaging image, and d is a Z-axis superimposed stereoscopic image. The images are collected after being embedded by the gel formed by the tissue transparentizing agent, the gel formed by the tissue transparentizing agent can fix the position of the tissue, so that the tissue is prevented from floating and shaking in the imaging liquid, and imaging is facilitated; the gel has high transparency and good imaging capability.
Claims (4)
1. The application of the tissue transparentizing hydrogel reagent in tissue transparentizing is characterized in that,
the tissue transparentizing hydrogel reagent comprises the following substances in percentage by mass: 4% -6% of imidazole, 86% -93% of sorbitol and 2% -8% of water; amino groups of the imidazole and the sorbitol C 2 The hydroxyl groups at the positions are subjected to hydrogen bond interaction to form hydrogel; the hydrogen bond interaction is shown as a formula (1):
formula (1);
the amino groups of the imidazole are combined with the lipid of the tissue, so that the lipid seeps out of the tissue, the scattering of biological tissue is reduced rapidly, and the refractive indexes of the components of the tissue are similar; sorbitol breaks down the fibrillar structure of collagen into microfibrils and increases the solubility of collagen; the water molecules interact with the hydrophilic groups of sorbitol to enter the interior of the molecules, and the refractive index of the sample is homogenized by removing, replacing and modifying some components in the tissue, so that the biological tissue can be made transparent and the shaping effect can be achieved.
2. The use according to claim 1, wherein the tissue-transparentizing hydrogel agent has a refractive index of 1.505-1.520.
3. A method of using the tissue-clearing hydrogel reagent in any one of claims 1-2, comprising the steps of:
placing the immobilized biological tissue into a tissue transparentizing hydrogel reagent, and incubating to obtain a transparentizing tissue embedded by the hydrogel;
the amino groups of the imidazole are combined with the lipid of the tissue, so that the lipid seeps out of the tissue, the scattering of biological tissue is reduced rapidly, and the refractive indexes of the components of the tissue are similar; sorbitol breaks down the fibrillar structure of collagen into microfibrils and increases the solubility of collagen; the water molecules interact with the hydrophilic groups of sorbitol to enter the interior of the molecules, and the refractive index of the sample is homogenized by removing, replacing and modifying some components in the tissue, so that the biological tissue can be made transparent and the shaping effect can be achieved.
4. A method of use according to claim 3, wherein the incubation is at a temperature of 20 ℃ to 37 ℃ and the incubation time is 6h to 48h.
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| CN101979133A (en) * | 2010-11-17 | 2011-02-23 | 天津大学 | Imidazole ionic liquid gel and preparation method thereof |
| CN105392363A (en) * | 2013-03-01 | 2016-03-09 | A·S·戈尔兹伯勒 | Sample fixation and stabilisation |
| GB201402347D0 (en) * | 2014-02-11 | 2014-03-26 | Apacor Ltd | Fixative solution for materials or samples of biological origin |
| CN106556582A (en) * | 2016-10-31 | 2017-04-05 | 华中科技大学 | A kind of method of smooth transparence biological tissue |
| JP2020083860A (en) * | 2018-11-30 | 2020-06-04 | ライオン株式会社 | Transparent gel composition for rubbing tongue and cleaning method using the same |
| WO2021095718A1 (en) * | 2019-11-11 | 2021-05-20 | 公立大学法人大阪 | Method for making biological tissue transparent and reagent for same |
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