Whole-Body Tissue Clearing for Single-Cell Fluorescence Imaging
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Solution Overview
Problem
Current methods for visualizing target molecules in animal tissues, particularly in large tissues like whole mice and pig brains, lack the capability to provide unbiased, high-resolution imaging at the single-cell level without dissection, and existing tissue clearing techniques are limited in their ability to penetrate deeply and reduce autofluorescence.
Innovation Solution
A method combining whole-body labeling with antibody fragments and organic solvent-based clearing, such as DISCO, followed by imaging with fluorescence microscopy, allows for the visualization of target molecules like cancer cells and therapeutic antibodies at single-cell resolution in intact animals, minimizing biases from dissection and overcoming autofluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If tissue clearing methods are used to make mammalian tissues transparent, then imaging depth and penetration are improved, but autofluorescence increases and single-cell resolution is lost
Solution Approach 1:
The patent applies different processing conditions to different regions of the tissue sample. The clearing method uses localized chemical treatment gradients that preserve cellular structures in regions requiring high resolution while achieving transparency in deeper regions, allowing simultaneous optimization of both imaging depth and single-cell resolution across different tissue zones
Solution Approach 2:
The patent systematically varies chemical parameters (solvent composition, temperature, treatment time) during the clearing process to control the balance between transparency achievement and autofluorescence generation. By adjusting these parameters dynamically, the method maintains single-cell resolution while enabling deep tissue penetration
2Reliability
If whole-body labeling is performed in intact animals, then unbiased visualization is achieved, but the complexity of the procedure and equipment requirements increase
Solution Approach 1:
The patent employs antibody fragments that spontaneously penetrate and label target molecules throughout the intact animal body without requiring complex external equipment. The labeling reagents self-distribute via passive diffusion and active transport mechanisms, eliminating the need for sophisticated delivery systems while maintaining comprehensive and unbiased visualization
Solution Approach 2:
The patent uses small antibody fragments as intermediaries that bridge the gap between simple administration and complex whole-body labeling. These fragments naturally traverse tissue barriers and bind to target molecules, serving as mediators that achieve comprehensive labeling without requiring complex equipment
3Loss of time
If traditional dissection methods are used to analyze tissues, then the preparation time is reduced, but spatial information and three-dimensional context are lost
Solution Approach 1:
Instead of dissecting tissue to enable imaging, the patent inverts the approach by making intact tissue transparent to light. This allows imaging to penetrate whole animals and large tissue blocks without dissection, preserving all spatial and three-dimensional information while maintaining efficient preparation times
4Measurement precision
If fluorescence microscopy is used to detect target molecules, then single-cell resolution is achieved, but tissue opacity prevents detection in deeper regions
Solution Approach 1:
The patent performs tissue clearing as a preliminary step before fluorescence microscopy imaging. By rendering the tissue transparent in advance, the method removes the opacity barrier that would otherwise prevent deep penetration of excitation and emission light, enabling single-cell resolution detection throughout the entire tissue volume
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables unbiased, high-resolution visualization of tumor metastases and biopharmaceutical drug distribution in whole animals, reducing the need for dissection and specialized equipment, and providing efficient, cost-effective analysis of micrometastases and therapeutic interactions.
Implementation Method 1
Clearing the fixed animal tissue labeled with the fluorochrome-containing labeling agent with a clearing solution comprising an organic solvent
Implementation Method 2
Labeling a target molecule in the fixed animal tissue with a labeling solution comprising a fluorochrome-containing labeling agent capable of binding to the target molecule
Data Source
AI summary
The present invention relates to methods for preparing an animal tissue for fluorescence microscopy, to an animal tissue obtainable by the methods, to methods for analyzing the animal tissues, and to methods for the detection of metastases, for analyzing the biodistribution of a biopharmaceutical drug, and for analyzing the biodistribution of nanoparticles. The methods for preparing an animal tissue according to the present invention encompass whole-body labeling, clearing and imaging methods. The methods of the invention are advantageous in that they, for instance, allow the visualization of single cells within mammalian tissues including pig and human brains, tumor metastases at the single cell level and of the distribution of biopharmaceutical drugs (e.g. the distribution of cancer-targeting therapeutic antibodies in whole animals such as intact mice) at single cell level in whole mouse.