Tissue Sample Preparation System Morphology Preservation
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Solution Overview
Problem
Existing tissue clearing methods using organic solvents cause significant shrinkage and morphological changes, leading to loss of biologically important 3D information, while water-based methods do not provide sufficient optical clarity.
Innovation Solution
A method involving fixation, delipidation with a chaotropic agent, dehydration, and organic clearing, which includes a chaotropic agent to induce swelling to counteract shrinkage, ensuring minimal deviation in volume and morphology, combined with selective photo bleaching to enhance clarity and reduce autofluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If organic solvent based clearing methods are used, then optical clarity is improved, but tissue shrinkage and morphological changes occur
Solution Approach 1:
The clearing process is divided into multiple sequential steps: dehydration with organic solvent, clearing with high-refractive-index organic solvent, and post-clearing treatment with water-based solution. This segmentation allows each step to optimize for its specific function while minimizing overall tissue damage
Solution Approach 2:
Dehydration with organic solvent is performed as a preliminary step before clearing to remove water and prepare the tissue for subsequent clearing steps, preventing water-solvent mixing issues and improving clearing efficiency
2Manufacturing precision
If water based clearing methods are used, then tissue morphology is preserved, but optical clarity is insufficient
Solution Approach 1:
The refractive index of the clearing medium is changed from water-based (low RI) to organic solvent-based (high RI matching tissue), dramatically improving optical clarity while post-clearing treatments restore morphological fidelity
3Illumination intensity
If organic solvents are used for clearing, then light scattering is reduced, but fluorescent protein emission is quenched
Solution Approach 1:
The harmful quenching effect of organic solvents on fluorescent proteins is converted into a beneficial timing strategy: clearing is performed first to optimize light transmission, then fluorescent staining is applied afterward when the tissue is already cleared but fluorescent proteins have not yet been exposed to quenching conditions
Data Source
Figure 1
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Figure 3A~3B
AI summary
The invention relates to a method (100) of preparing a biological tissue sample (301) for optical analysis such that a deviation of the volume and/or morphology of the prepared sample from the volume or morphology of the original tissue is minimized. The method comprises: − a) fixing (102) the tissue sample with a first fixation solution (202); − b) immersing (104) the tissue sample in a delipidation solution (204) comprising a chaotropic agent adapted to induce a swelling of the sample; − d) staining (108) the tissue sample; − f) dehydrating (112) the tissue sample in an organic dehydration solution (210), the dehydration solution adapted to induce a shrinking of the sample; and − g) immersing (114) the fixed tissue sample in an organic clearing solution (212).