Single-cell phenotyping within transparent intact tissue through whole-body clearing

Area of Science:

• Neuroscience
• Biotechnology
• Anatomical Imaging

Background:

• 3D anatomical and phenotypical maps are crucial for understanding structure-function relationships across scales.
• A significant knowledge gap exists due to the lack of whole-organ imaging methods.
• Current limitations hinder comprehensive mapping of cellular structures and connectivity within organs.

Purpose of the Study:

• To present novel techniques for whole-organ and whole-body tissue clearing and imaging.
• To enable high-resolution, high-content mapping and phenotyping of biological structures.
• To address the absence of methods for creating detailed 3D anatomical maps of intact organs.

Main Methods:

• Passive Clarity Technique (PACT) for passive tissue clearing and immunostaining of intact organs.
• Refractive Index Matching Solution (RIMS) as a mounting medium for imaging thick tissues.
• Perfusion-Assisted Agent Release In Situ (PARS) for whole-body clearing and immunolabeling.

Main Results:

• PACT, RIMS, and PARS render whole organs and bodies macromolecule-permeable and optically transparent.
• Methods are compatible with endogenous fluorescence, immunohistochemistry, and RNA single-molecule FISH in rodents.
• Demonstrated cellular and subcellular resolution imaging with long-term storage capabilities.

Conclusions:

• The developed techniques overcome limitations in whole-organ imaging, enabling detailed 3D anatomical mapping.
• These methods facilitate high-resolution, high-content phenotyping of normal and pathological elements within intact biological systems.
• The PACT, RIMS, and PARS protocols offer a versatile toolkit for advancing biological and medical research through enhanced imaging capabilities.