Bone Tissue Clearing via Convective Flow and Amino Alcohol

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Solution Overview

Problem

Current methods for visualizing bone tissue at the cellular level are limited by the hard, fibrous nature of osseous tissue, which makes it difficult to access three-dimensional information and risks extensive damage to the tissue sample, and existing bone clearing techniques do not effectively maintain endogenous fluorescence or achieve deep imaging within intact bone.

Innovation Solution

A specialized bone tissue clearing method incorporating continuous convective flow, amino alcohol to minimize autofluorescence, and an imaging procedure that minimizes refractive index variations in light-sheet microscopy, using a protocol that includes decalcification with EDTA, hydrogel stabilization, lipid removal, and refractive index matching to achieve whole-bone clearing with an imaging depth of up to 1.5 mm while preserving fluorescence and signal-to-noise ratio.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If bone tissue is sectioned for visualization, then cellular level information can be obtained, but extensive damage to the tissue sample occurs and three-dimensional information is lost

Engineering Contradiction:

Improvecellular level visualizationVSAvoidtissue sample integrity

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent changes the physical state of bone tissue by decalcifying it (removing calcium minerals) and embedding it in hydrogel, transforming hard, opaque tissue into soft, transparent tissue that can be imaged in three dimensions without sectioning, thereby preserving tissue integrity while enabling cellular level visualization

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent replaces mechanical sectioning with optical clearing and imaging techniques. By making the tissue transparent through chemical treatment and hydrogel embedding, the method eliminates the need for physical slicing while achieving comparable or superior visualization of cellular structures

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Shape

If traditional clearing methods are used on bone tissue, then transparency is achieved, but endogenous fluorescence is lost and imaging depth is limited

Engineering Contradiction:

Improvetissue transparencyVSAvoidfluorescence signal quality

Core Design Contradiction:

ShapeVSMeasurement precision

Solution Approach 1:

The patent applies different treatments to different components of the tissue: lipids are removed for transparency while proteins and nucleic acids are preserved in the hydrogel matrix to maintain fluorescence. This selective treatment achieves both transparency and fluorescence preservation

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent creates a composite structure by embedding bone tissue in hydrogel matrix. This composite material combines the transparency benefits of clearing with the fluorescence preservation benefits of hydrogel, enabling both properties to coexist

Inventive Principle:

Principle #40Composite materials

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 comprehensive visualization and quantification of cellular morphology and biomolecular properties in intact bone tissue, allowing for the detection and 3D placement of single cells with improved precision and reduced tissue damage, while maintaining native fluorescence and deep imaging capabilities.

Implementation Method 1

decalcifying the fixed bone tissue sample by applying a decalcifying solution including a calcium chelating agent

Methodology Applied

Scientific EffectChelation:

Implementation Method 2

initiating polymerization of the monomer solution to generate a decalcified bone tissue sample hydrogel matrix

Methodology Applied

Scientific EffectPolymerization: Photopolymerisation

Implementation Method 3

removing lipids from the washed decalcified bone tissue sample hydrogel matrix by applying a detergent solution

Methodology Applied

Scientific EffectSolubilization: Solvation

Implementation Method 4

removing heme from the substantially cleared bone tissue sample by applying a removal solution including an amino alcohol to generate a cleared bone tissue sample

Methodology Applied

Scientific EffectChemical reduction: Reduction

Implementation Method 5

serially applying refractive index matching solutions (RIMS) with progressively higher refractive indexes (RIs) to the cleared bone tissue sample

Methodology Applied

Scientific EffectRefraction: Refraction

Data Source

PatentUS10955322B2Methods and devices for soft and osseous tissue clearing and fluorescent imaging

Publication Date: 2021.03.23 CALIFORNIA INST OF TECH

AI summary

Described herein is a bone tissue clearing method with enhanced optical access. Compositions and techniques for bone tissue clearing include continuous convective flow during the clearing process, amino alcohol to minimize tissue autofluorescence, and an imaging procedure that minimizes refractive index variations in light-sheet microscopy. These improvements allowed the Inventors to achieve whole-bone clearing with an imaging depth of up to about 1.5 mm while maintaining fluorescence and a signal-to-noise ratio (SNR) that permits detection and 3D placement of single cells. In various embodiments, the present application teaches methods and kits for clearing and optionally subsequently visualizing tissue containing bone.