Light-Sheet Fluorescence Microscopy: Chemical Clearing and Labeling Protocols for Ultramicroscopy

Area of Science:

• Neuroscience
• Developmental Biology
• Cancer Research

Background:

• Light-sheet microscopy requires translucent specimens for 3D imaging.
• Tissue clearing is essential for visualizing cellular networks and organs.

Purpose of the Study:

• To provide 3DISCO-based protocols for preparing cleared, immuno-stained samples for light-sheet microscopy.
• To enable long-term archiving of cleared biological specimens.

Main Methods:

• Utilized 3DISCO clearing with tetrahydrofuran (THF) and dibenzylether (DBE).
• Implemented transcardial perfusion and peroxide-free chemicals for tissue transparency and GFP preservation.
• Developed a resin embedding protocol for long-term sample archiving.

Main Results:

• Successfully prepared cleared samples of immuno-stained neural networks, vascular networks, and beta-amyloid plaques.
• Achieved transparent tissues with preserved endogenous GFP signal.
• Demonstrated long-term archiving of cleared specimens with minimal signal loss.

Conclusions:

• 3DISCO protocols offer an effective method for preparing diverse biological samples for light-sheet microscopy.
• The developed protocols facilitate detailed analysis of neural, vascular, and pathological structures in 3D.
• Resin embedding ensures sample integrity for future research.