High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging

Area of Science:

• Biotechnology
• Microscopy
• Histology

Background:

• Understanding complex organ structure-function relationships necessitates in situ reconstruction of molecular properties within 3D tissue architecture.
• Existing methods often struggle with preserving tissue integrity, reporter molecule fluorescence, and compatibility with multiplexed labeling for large-volume imaging.

Purpose of the Study:

• To provide detailed protocols for the Clearing-Enhanced 3D (Ce3D) tissue clearing method.
• To enable high-resolution, quantitative 3D visualization of cellular and molecular spatial relationships within intact tissues.
• To introduce RNA detection compatibility and optimize volumetric histocytometry analysis for Ce3D-processed samples.

Main Methods:

• Development and optimization of the Ce3D tissue clearing technique for rapid (within 24h) and effective tissue transparency.
• Integration of direct immunolabeling for multiplexed fluorescence microscopy of large tissue volumes.
• Application of volumetric histocytometry for quantitative spatial analysis of cells based on phenotypic and functional markers.

Main Results:

• Ce3D achieves excellent tissue transparency while preserving cell morphology, architecture, and fluorescence signals.
• The method is compatible with direct immunolabeling and enables high-quality multiplex fluorescence microscopy of large tissue samples.
• Optimized workflows and new RNA detection protocols enhance the utility of Ce3D for comprehensive cellular and molecular profiling.

Conclusions:

• Ce3D provides a robust and efficient solution for high-resolution volumetric imaging and quantitative analysis of cellular spatial organization.
• This technique facilitates the exploration of structure-function relationships in diverse tissue environments.
• The detailed procedures and addressed limitations empower researchers to overcome challenges in large-volume microscopy and data analysis.