3D imaging with enhanced transparency, signal-to-background ratios, and antigen detection using HyPer-3D

Area of Science:

• Biomedical Imaging
• Tissue Clearing
• Fluorescence Microscopy

Background:

• 3D imaging of cleared tissues faces challenges like high autofluorescence and low signal-to-background ratios (SBRs).
• Existing methods struggle with dense and autofluorescent tissues, limiting detection of specific cellular components.

Purpose of the Study:

• To develop and validate a chemical treatment, HyPer-3D (hydrogen peroxide-NaN3-DMSO), for enhanced 3D imaging of cleared tissues.
• To improve signal detection, reduce autofluorescence, and increase optical clearing capacity in various biological specimens.

Main Methods:

• Developed HyPer-3D treatment comprising hydrogen peroxide, sodium azide, and DMSO.
• Applied HyPer-3D to stem cell, zebrafish, murine, and human tissue samples.
• Evaluated improvements in SBR, optical clearing, antigen detection, and fluorescent reporter detection.

Main Results:

• HyPer-3D increased SBR by 30× in animal models and 5× in human tissues.
• Potentiated optical clearing capacity by 6× and increased antigen detection by 4.5×.
• Enabled enhanced detection of fluorescent reporters and multiplex imaging with cellular resolution.

Conclusions:

• HyPer-3D effectively quenches tissue autofluorescence and enhances imaging quality in diverse biological samples.
• The treatment facilitates high-contrast 3D imaging of challenging, autofluorescent, and dense tissues.
• HyPer-3D offers a robust solution for advanced 3D imaging in both basic research and clinical settings.