Tissue Dehydration and Staining for Deep Imaging
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Solution Overview
Problem
Current methods for tissue processing in pathology are limited by the need for traditional wax-embedding and cutting, which result in incomplete sample evaluation, artifacts, and a trade-off between tissue preservation and imaging quality, especially for deep tissue imaging and rapid diagnostics.
Innovation Solution
A method involving fixation with a fixative solution containing fluorescent dyes, followed by clearing with a benzyl alcohol and benzyl benzoate solution, allowing for deep imaging without destructive sectioning, and converting fluorescence intensity values to optical densities for image reconstruction similar to traditional histology stains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional wax-embedding and microtome-cutting methods are used, then tissue samples can be prepared for routine pathologic evaluation, but the process is time-consuming, results in incomplete sample evaluation, and creates artifacts
Solution Approach 1:
The invention extracts and eliminates the wax-embedding and microtome-cutting steps from the traditional tissue processing workflow. By using optical clearing agents to make intact tissue blocks transparent, the method removes the need for physical sectioning, thereby preventing artifacts and enabling complete 3D sample evaluation while significantly reducing processing time
Solution Approach 2:
The invention replaces the mechanical microtome-cutting system with an optical clearing system. Instead of physically slicing tissue with a microtome, the method uses chemical/optical agents (clearing solutions like BABB or Scale) to render the entire tissue block transparent, allowing non-destructive deep imaging without mechanical sectioning
2Speed
If traditional histology methods are used, then rapid processing is achieved, but deep tissue imaging beyond 50 μm is not possible
Solution Approach 1:
The invention performs preliminary optical clearing of the tissue block before imaging. By pre-treating the tissue with clearing agents that penetrate and render the entire specimen transparent, the method enables deep imaging capabilities from the outset without requiring time-consuming serial sectioning or ablation during the imaging process
Solution Approach 2:
The invention changes the optical parameters of the tissue by applying clearing agents that alter the refractive index matching between tissue components and the surrounding medium. This parameter change (from opaque to transparent) enables light penetration to depths of hundreds of micrometers while maintaining tissue integrity and allowing rapid imaging
3Speed
If serial sectioning or serial tissue ablation is used to image deeper tissue, then imaging depth increases, but the tissue specimen is destroyed during imaging
Solution Approach 1:
The invention extracts and eliminates the destructive elements (serial sectioning or ablation) from the imaging process. By using optical clearing to enable direct deep imaging of intact tissue, the method removes the need to physically remove or destroy tissue layers to access deeper structures
Solution Approach 2:
The invention replaces the destructive mechanical approaches (sectioning knives or ablation lasers) with a non-destructive optical clearing approach. The clearing agents chemically modify the tissue optics without physical damage, allowing repeated imaging and subsequent use of the same intact specimen for other diagnostic purposes
4Measurement precision
If fluorescent dyes are used for staining, then tissue structures can be visualized, but the staining process is time-consuming and complex
Solution Approach 1:
The invention merges the optical clearing process with the staining process by incorporating fluorescent dyes directly into the clearing solution or applying them during the clearing protocol. This combination allows simultaneous tissue transparency and molecular labeling, eliminating separate staining steps and reducing overall processing time while maintaining excellent visualization quality
Solution Approach 2:
The clearing solution serves multiple functions simultaneously: it renders the tissue transparent for deep imaging, acts as a mounting medium for microscopy, and delivers fluorescent dyes for specific tissue structure labeling. This multi-functionality consolidates multiple traditional separate steps into a single integrated process
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 high-resolution, deep tissue imaging up to 500 μm with preserved tissue integrity, facilitating rapid and accurate diagnostic evaluation and maintaining compatibility with subsequent traditional processing and staining methods.
Implementation Method 1
contacting the tissue sample with a fixative solution comprising at least one fixative and at least one fluorescent dye
Implementation Method 2
contacting the tissue sample with a clearing solution... imaging the tissue sample to produce a visual image... high-resolution, deep tissue imaging up to 500 μm with preserved tissue integrity
Implementation Method 3
converting fluorescence intensity values to optical densities for image reconstruction
Data Source
AI summary
A biopsy-sized tissue sample is stained for quick imaging. A significant amount of permeation enhancer is included in a mixed solution of permeant enhancer, fixative or dehydrant, and one or two fluorescent dyes to simultaneously dehydrate and dye the tissue sample. The permeation enhancer, e.g., 10% to 50% in the mixed solution, achieves an image of dyed tissue in the contacted tissue sample at a depth of at least 200 um within no more than 1.5 hours. One of the fluorescent dyes is a fluorescent nuclear dye such as DAPI, SYTOX green, acridine orange, propidium iodide, or a Hoechst dye. The other fluorescent dye is a fluorescent protein dye such as eosin or rhodamine B. The tissue sample is cleared with a clearing agent having a refractive index of at least 1.4[R2], e.g., using BABB. The mixed solution may further include Chloroform or other morphology preservative.