Online optical transparent solution for three-dimensional structure imaging of large-volume biological sample

Abstract

The invention discloses an online optical clear solution for three-dimensional structure imaging of a large-volume biological sample, which is characterized in that the solution comprises iohexol, urea, 1, 2-propylene glycol, dimethyl sulfoxide, 3-[3-(cholamidopropyl) dimethylamino] propanesulfonic acid inner salt and water. The invention further discloses a three-dimensional imaging method for the large-volume biological sample. The method comprises the following steps: carrying out transparentizing treatment on the biological sample by using the online optical transparentizing solution, and carrying out three-dimensional imaging on the transparentized biological sample by using a fluorescence microscope. The fluorescence microscope comprises a confocal microscope and / or a light sheet microscope. When the online optical transparent solution is used for imaging, the transparent depth of a surface sample is large, the single imaging depth can be increased, and the cutting thickness can be improved, so that the cutting frequency is reduced, and finally the data acquisition efficiency of a large-volume sample is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical optical imaging, and in particular relates to an online optically transparent solution for three-dimensional structural imaging of large-volume biological samples. Background Art

[0002] In the fields of medical diagnosis and pathology, obtaining three-dimensional histopathological information of organs is of great significance for disease diagnosis, treatment planning, and medical research. Prior art has disclosed a variety of different methods, such as fixing the tissue in a fixative, then embedding it in paraffin or a polymer, then cutting the sample into slices several microns thick and obtaining projection images using optical or electron microscopy. This method of visualizing thick tissue essentially involves continuously cutting it into thin slices, and then reconstructing a three-dimensional image from these slices through computational methods. However, this method is not only laborious but also has limitations when the true three-dimensional properties of the tissue cannot be determined from the thin slices.

[0003] To preserve the true three-dimensional structure of tissues, tissue clearing technology has been developed in this field. Tissue clearing involves treating biological tissues or organs with one or more special chemical reagents (clearing agents) to reduce the tissue's light attenuation, thereby rendering the tissue optically transparent and facilitating optical imaging and observation of the tissue. The principle behind this technology is to reduce light scattering and absorption in biological tissues. By using various chemical or physical methods to minimize light absorption and scattering, the light beam propagates deeper into the material, significantly increasing the imaging depth of microscopes. This technology is gaining increasing popularity in fields such as neuroscience, tumor systems, and three-dimensional pathology testing.

[0004] However, existing online optically transparent solutions have two major defects: first, the solution viscosity is too high, which will inhibit the amplitude and frequency of the vibrating slicing equipment, thereby affecting the cutting quality; second, the transparency depth of the surface sample is insufficient. Sufficient transparency depth can not only increase the single imaging depth, but also increase the cutting thickness, thereby reducing the number of cuts and ultimately improving the data acquisition efficiency of large-volume samples.

[0005] Therefore, it is necessary to further explore and study new online optically transparent solutions to improve their transparency effect on biological tissues and imaging stability. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, we provide a new online optically transparent solution and a method for performing three-dimensional imaging of biological samples after processing using the transparent solution.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an online optically transparent solution for three-dimensional structural imaging of large-volume biological samples, wherein the solution comprises iohexol, urea, 1,2-propylene glycol, dimethyl sulfoxide, 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt and water.

[0009] In one or more embodiments, in the solution, the concentration of iohexol is 35-45 wt / vol%, the concentration of urea is 8-12 wt / vol%, the concentration of 1,2-propylene glycol is 8-12 wt / vol%, the concentration of dimethyl sulfoxide is 8-12 wt / vol%, and the concentration of 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt is 4-6 wt / vol%.

[0010] In one or more embodiments, the solution has a refractive index of 1.4-1.5 and a viscosity of 4.5-5.5 mPa·s.

[0011] Preferably, the refractive index of the solution is 1.44 and the viscosity is 5 mPa·s.

[0012] In a second aspect, the present invention provides a method for preparing the online optically transparent solution, comprising the following steps: weighing iohexol, urea, 1,2-propylene glycol, dimethyl sulfoxide, and 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt according to the concentrations, with the remainder being water, mixing all the ingredients together and stirring until the solution becomes clear.

[0013] In a third aspect, the present invention provides the use of the online optical clearing solution in clearing large-volume biological samples and three-dimensional structure imaging.

[0014] In a fourth aspect, the present invention provides a method for clearing a large volume of biological samples, the method comprising the steps of immersing the biological sample in the online optical clearing solution of the present invention and incubating the sample.

[0015] Preferably, the biological sample is immersed in the optically transparent solution of the present invention for 24 hours (the duration is adjusted according to the sample size) to achieve refractive index matching and intra-tissue material exchange.

[0016] In one or more embodiments, the biological sample is pretreated before being cleared, and the pretreatment is to fix the biological sample by perfusion and then wash it with PBS solution to remove residual fixative.

[0017] Preferably, the pretreatment is: fixing the biological sample by perfusion with 4% PFA, and then washing with PBS solution for 12 hours to remove residual PFA.

[0018] In a fifth aspect, the present invention provides a three-dimensional imaging method for large-volume biological samples, the method comprising the steps of transparentizing the biological sample according to the method of the present invention, and performing three-dimensional imaging of the transparentized biological sample using a fluorescence microscope; the fluorescence microscope comprises a confocal microscope and / or a light sheet microscope.

[0019] In one or more embodiments, before performing three-dimensional imaging, the method further includes embedding the cleared biological sample in agarose.

[0020] In one or more embodiments, the method further comprises the step of performing GFP fluorescence / immuno-staining on the biological sample or incorporating fluorescent beads into the sample as a resolution reference.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The present invention optimizes the penetration enhancers (DMSO and 1,2-propylene glycol) and introduces a surfactant (CHAPS) so that the online optically transparent solution can achieve deep and uniform transparency of large-volume biological samples, allowing a single imaging depth of 81 μm. This depth advantage is reflected in: reducing the number of cutting times: a larger single imaging thickness reduces the cutting frequency and shortens the total data acquisition time; reducing the impact of cutting errors: sufficient transparency reduces imaging errors caused by uneven cutting; maintaining sample structural stability: the solution controls the deformation of the sample within 18 μm, ensuring data continuity and reducing the workload of subsequent image stitching and processing. In summary, the increase in imaging depth and the reduction in deformation significantly optimize cutting efficiency, equipment switching time and data processing flow, greatly improving the overall efficiency of three-dimensional imaging of large-volume samples (such as the whole brain).

[0023] (2) By using low-viscosity, high-refractive-index iohexol as the main component, the present invention can achieve effective refractive index matching at a relatively low concentration, thereby reducing the overall viscosity of the solution (5 mPa·s). The low viscosity characteristic brings the following key advantages: stable cutting process: reducing interference with the vibrating microtome equipment, maintaining stable amplitude and frequency, and ensuring cutting quality; avoiding bubble interference: low-viscosity solutions are less likely to generate bubbles, preventing obstruction of the objective lens and ensuring the continuity of signal acquisition; convenient operation: low viscosity makes it easier for the solution to flow and change, improving experimental operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 1 is the imaging result of the mouse brain sample after being treated with different optically transparent solutions in Example 5.

[0025] Figure 2 This is a comparison of imaging depths of mouse brain samples treated with different optically transparent solutions in Example 5.

[0026] Figure 3 This is a diagram of the imaging results of a single data acquisition of a Thy1-EGFP mouse brain sample in Example 6.

[0027] Figure 4 This is a normalized RMS line graph of the Thy1-EGFP mouse brain sample in Example 6 as the imaging depth changes.

[0028] Figure 5 This is the imaging result of a single data acquisition of a pig cerebral blood vessel sample in Example 7.

[0029] Figure 6 This is a normalized RMS line graph of the pig brain blood vessel sample in Example 7 as the imaging depth changes. DETAILED DESCRIPTION

[0030] In this field, online optical clearing solutions must meet the following key requirements: (1) maintain the stability of fluorescent protein signals and avoid quenching; (2) achieve rapid transparency of the sample surface and transparency at a certain depth; (3) minimize sample deformation and maintain tissue structural integrity; (4) have low viscosity and biosafety. In the present invention, after system optimization, the final online optical clearing solution determined includes: iohexol, urea, 1,2-propylene glycol, dimethyl sulfoxide (DMSO), and 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid salt (CHAPS). Compared with previous online optical clearing solutions, the online optical clearing solution of the present invention significantly improves the data acquisition efficiency of large-volume samples through the synergistic effect between the various reagents. For example, this formula uses the high-refractive-index, low-viscosity refractive-index matching reagent iohexol, which can achieve effective refractive-index matching at a relatively low concentration, thereby reducing the overall viscosity of the solution; urea can reduce the refractive index of lipids and other substances through hydration; DMSO can synergistically enhance permeability with 1,2-propylene glycol and the like; at the same time, a surfactant (CHAPS) is introduced to enhance cell membrane permeability through its amphiphilic structure, promote rapid penetration of transparent reagents, and increase the depth of transparency while maintaining the stability of cell morphology.

[0031] It should be understood that the online optically clear solution described herein does not limit the content of each component, and those skilled in the art may adjust the content of each component according to actual circumstances. As a preferred example, the online optically clear solution described herein may comprise the following components: iohexol at a concentration of 40 wt / vol%, urea at a concentration of 10 wt / vol%, 1,2-propylene glycol at a concentration of 10 wt / vol%, dimethyl sulfoxide at a concentration of 10 wt / vol%, and 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt at a concentration of 5 wt / vol%. In this preferred optically clear solution, the refractive index of the solution is 1.44, and the viscosity is 5 mPa·s.

[0032] The preparation of the online optically transparent solution provided by the present invention is simple and clear, and only requires mixing all components in proportion, and then adding water and stirring until the solution becomes clear.

[0033] In the present invention, the three-dimensional imaging method for a large volume biological sample may include the following steps:

[0034] (1) Preprocessing of biological samples;

[0035] (2) immersing the pretreated biological sample in the online optically transparent solution of the present invention for incubation;

[0036] (3) embedding the cleared biological sample in agarose;

[0037] (4) Using a fluorescence microscope to perform three-dimensional imaging of the biological sample after the transparentization treatment.

[0038] It should be understood that the tissues, biological tissues, biological samples, biological tissue samples, etc. mentioned in the present invention all have the same meaning. The biological tissues can be any tissue or organ of any organism, such as mice, pigs, and human organs, including but not limited to complete brain tissue, embryonic tissue, kidney tissue, spinal tissue, liver tissue, intestinal tissue, and ovarian tissue.

[0039] It should be understood that in the present invention, the step of pre-treating the biological sample in step (1) is not essential, and the absence of this step does not affect the effect of the optically transparent solution and the three-dimensional imaging effect of the present invention. In the present invention, the pre-treatment method of the biological sample is not limited. Those skilled in the art can perform any pre-treatment according to actual needs and existing technologies, such as perfusion fixation of the biological sample with 4% PFA and then washing with PBS solution for 12 hours to remove residual PFA.

[0040] It should be understood that, in the present invention, the step of embedding the transparentized biological sample in agarose in step (3) is not essential, and the absence of this step does not affect the final three-dimensional imaging effect of the biological sample. In addition, those skilled in the art may also use other embedding techniques to embed the transparentized biological sample according to actual needs and existing technology, such as using a commercially available embedding kit.

[0041] It should be understood that when step (3) is used, the sample can be soaked for a further 3-5 days to ensure that the refractive index of the agarose matches that of the solution.

[0042] It should be understood that in the present invention, the fluorescence microscope in step (4) is not limited. Those skilled in the art can use fluorescence microscopes of different types or specifications for three-dimensional imaging according to actual needs and existing technologies, such as confocal microscopes and / or light sheet microscopes.

[0043] Furthermore, to enhance visualization of 3D imaging, biological samples can be stained with GFP fluorescence or immunostained, or fluorescent beads can be incorporated into the sample to enhance resolution. These steps can be performed before or after clearing the biological sample. For example, when embedding the cleared biological sample in agarose, 0.2 μm green fluorescent beads can be incorporated as a resolution reference.

[0044] When the online optically transparent solution of the present invention is used for imaging, the transparent depth of the surface sample is large, which can not only increase the single imaging depth, but also improve the cutting thickness, thereby reducing the number of cutting times and ultimately improving the data acquisition efficiency of large-volume samples.

[0045] Example

[0046] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0047] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0048] When numerical ranges are given in the examples, it should be understood that, unless otherwise specified herein, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0049] The Thy1-EGFP mouse brain samples used in the following examples were purchased from the biological platform of Suzhou Brain Space Information Institute, Huazhong University of Science and Technology; the Tek mouse brain slice samples were from the biological platform of Suzhou Brain Space Information Institute, Huazhong University of Science and Technology; the virus-labeled C57 mouse brain samples were purchased from the biological platform of Suzhou Brain Space Information Institute, Huazhong University of Science and Technology; and the porcine brain blood vessel samples were purchased from the biological platform of the MOST team of the School of Biomedical Engineering, Hainan University.

[0050] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages mentioned are by mass unless otherwise specified.

[0052] Example 1:

[0053] An online optically transparent solution is provided, comprising: 40 wt / vol% iohexol, 10 wt / vol% urea, 10 wt / vol% 1,2-propylene glycol, 10 wt / vol% dimethyl sulfoxide, 5 wt / vol% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, and the remainder being water. The components are mixed and stirred until the solution becomes clear. Testing revealed that the solution had a refractive index of 1.44 and a viscosity of 5 mPa·s.

[0054] Example 2:

[0055] An online optically transparent solution is provided, comprising: 35 wt / vol% iohexol, 12 wt / vol% urea, 8 wt / vol% 1,2-propylene glycol, 8 wt / vol% dimethyl sulfoxide, 6 wt / vol% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, and the remainder being water. The components are mixed and stirred until the solution becomes clear. Testing revealed that the solution has a refractive index of 1.40 and a viscosity of 4.5 mPa·s.

[0056] Example 3:

[0057] An online optically transparent solution is provided, comprising: 45 wt / vol% iohexol, 8 wt / vol% urea, 12 wt / vol% 1,2-propylene glycol, 12 wt / vol% dimethyl sulfoxide, 4 wt / vol% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, and the remainder being water. The components are mixed and stirred until the solution becomes clear. Testing revealed that the solution has a refractive index of 1.50 and a viscosity of 5.5 mPa·s.

[0058] Example 4:

[0059] A three-dimensional imaging method for large-volume biological samples may include the following steps:

[0060] (1) Sample pretreatment: After perfusion fixation of biological samples with 4% PFA, the samples were washed with PBS solution for 12 hours to remove residual PFA;

[0061] (2) Transparent treatment: Soak the biological sample in an optically transparent solution for 24 hours to achieve refractive index matching and intra-tissue material exchange;

[0062] (3) Agarose embedding: The biological tissue samples after transparent treatment were embedded in agarose embedding technology, and 0.2 μm green fluorescent beads were simultaneously incorporated as resolution reference;

[0063] First, prepare a 5% agarose solution, protect it from light, place it on a magnetic stirrer and stir it to fully oxidize the agarose. Then use a filtration device to filter the agarose solution. Place the filtered agarose solution in a microwave oven to melt it into a gel. Place the melted agarose gel in a 55°C water bath to cool it down. After cooling, add the fluorescent ball solution and stir it thoroughly with a glass rod. Then pour the agarose gel with added fluorescent balls into a mold, place the sample in the mold, and wait for it to cool down before taking it out.

[0064] (4) Refractive index balance: The embedded sample is immersed in the optically transparent solution for 3-5 days to ensure that the refractive index of agarose matches that of the solution;

[0065] (5) Three-dimensional imaging: Structural imaging is performed using a confocal microscope.

[0066] Example 5:

[0067] To further verify the synergistic effect between the various reagents in the online light-clearing solution, four different light-clearing solutions were prepared in this study: the first group consisted of 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol, 10% (wt / vol) DMSO, and 5% (wt / vol) CHAPS; the second group consisted of 40% (wt / vol) iohexol, 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol, and 5% (wt / vol) CHAPS. ) CHAPS, the third group consisted of 40% (wt / vol) iohexol, 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol, and 10% (wt / vol) DMSO, and the fourth group consisted of 40% (wt / vol) iohexol, 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol, 10% (wt / vol) DMSO, and 5% (wt / vol) CHAPS (i.e., the optically clear solution in Example 1). This example used 100 μm thick Tek mouse brain slices as model samples, in which vascular structures were labeled to facilitate more intuitive evaluation of the permeation-enhancing effect.

[0068] Figure 1(Pseudocolor) shows vascular signals in different optically transparent solutions acquired using confocal microscopy Z-stack mode. Among them, A is the imaging result under 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol, 10% (wt / vol) DMSO and 5% (wt / vol) CHAPS solution, B is the imaging result under 40% (wt / vol) iohexol, 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol and 5% (wt / vol) CHAPS solution, C is the imaging result under 40% (wt / vol) iohexol, 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol and 10% (wt / vol) DMSO solution, and D is the imaging result under 40% (wt / vol) iohexol, 10% (wt / vol) urea, 10% (wt / vol) 1,2-propylene glycol, 10% (wt / vol) DMSO and 5% (wt / vol) CHAPS solution.

[0069] This example also uses the normalized RMS value as a measure of image clarity, and uses the point where the RMS drops to 70% of the maximum value as the definition standard for imaging depth, and calculates the imaging depth of each solution. The results are as follows: Figure 2 As shown in the bar chart.

[0070] pass Figure 1 and Figure 2 It can be seen that the components of the online optically transparent solution of the present invention have a synergistic effect, which can improve the data acquisition efficiency of large-volume samples.

[0071] Example 6:

[0072] The biological sample used in this example is a mouse brain array: containing two Thy1-EGFP transgenic mice and four virus-labeled C57 mouse brains, forming a 60×50×35mm 3 Imaging model. ( Figure 3 and Figure 4 Imaging of Thy1-EGFP transgenic mice)

[0073] This embodiment uses the online optically transparent solution in Example 2 and the three-dimensional imaging method in Example 4, and the imaging parameters are set as follows: single imaging depth 81 μm, cutting thickness 63 μm, and retaining an 18 μm redundant layer for image registration.

[0074] The mouse brain array imaging took 110 hours (including imaging time, cutting time, and switching time from imaging position to cutting position). Figure 3 (without pseudo color) shows the signal area layered imaging quality of the Thy1-EGFP transgenic mouse in this example, Figure 4The normalized RMS curve for a Thy1-EGFP transgenic mouse in this example shows that, while signal quality gradually decreases (RMS value decreases) with increasing imaging depth, it still maintains a high quality standard of over 0.7 at a depth of 81 μm, confirming the effectiveness of this method. Furthermore, in this example, sample deformation was less than 18 μm, ensuring 3D data continuity and stitching accuracy.

[0075] Example 7:

[0076] The biological sample used in this example is a pig hemibrain sample: FITC perfusion labeled blood vessels, constructed 60×50×40mm 3 Agarose embedded blocks

[0077] This embodiment uses the online optically transparent solution in Example 3 and the three-dimensional imaging method in Example 4, and the imaging parameters are set as follows: single imaging depth 81 μm, cutting thickness 63 μm, and retaining an 18 μm redundant layer for image registration.

[0078] The mouse brain array imaging took 150 hours (including imaging time, cutting time, and switching time from imaging position to cutting position). Figure 5 (without pseudo-color) shows the vascular region layered imaging quality of this embodiment, Figure 6 The normalized RMS curve for this example shows that while signal quality gradually decreases (RMS value decreases) with increasing imaging depth, it still maintains a high quality standard of over 0.7 at a depth of 81 μm, confirming the effectiveness of this method. Furthermore, in this example, sample deformation was less than 18 μm, ensuring 3D data continuity and stitching accuracy.

[0079] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An online optically transparent solution for three-dimensional structural imaging of large-volume biological samples, characterized in that: The solution contains iohexol, urea, 1,2-propylene glycol, dimethyl sulfoxide, 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt and water.

2. The online optically transparent solution according to claim 1, wherein In the solution, the concentration of iohexol is 35-45 wt / vol%, the concentration of urea is 8-12 wt / vol%, the concentration of 1,2-propylene glycol is 8-12 wt / vol%, the concentration of dimethyl sulfoxide is 8-12 wt / vol%, and the concentration of 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt is 4-6 wt / vol%.

3. The online optically transparent solution according to claim 1 or 2, wherein: The refractive index of the solution is 1.4-1.5, and the viscosity is 4.5-5.5 mPa·s.

4. The method for preparing the online optically transparent solution according to claim 2, characterized in that: Iohexol, urea, 1,2-propylene glycol, dimethyl sulfoxide, and 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt were weighed according to the concentrations, and the remainder was water. All the ingredients were mixed together and stirred until the solution became clear.

5. Use of the online optical clearing solution according to claim 1 or 2 in clearing large-volume biological samples and three-dimensional structure imaging.

6. A method for transparentizing a large volume biological sample, characterized in that: The method comprises the steps of immersing the biological sample in the online optically transparent solution of claim 1 or 2 for incubation.

7. The transparentizing method according to claim 6, wherein: The biological sample is pretreated before being transparentized. The pretreatment is to fix the biological sample by perfusion and then wash it with PBS solution to remove residual fixative.

8. A three-dimensional imaging method for large-volume biological samples, characterized in that: The method comprises the steps of transparentizing the biological sample according to the method of claim 6 or 7, and performing three-dimensional imaging of the transparentized biological sample using a fluorescence microscope; the fluorescence microscope comprises a confocal microscope and / or a light sheet microscope.

9. The three-dimensional imaging method according to claim 8, wherein: Before performing three-dimensional imaging, the method also includes a step of embedding the transparent biological sample in agarose.

10. The three-dimensional imaging method according to claim 9, wherein: The method further comprises the steps of performing GFP fluorescence / immuno-staining on the biological sample or incorporating fluorescent beads into the sample as a resolution reference.

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