Spatial analysis of tissue immunity and vascularity by light sheet fluorescence microscopy

Area of Science:

• Biomedical Imaging
• Pathology
• Microscopy

Background:

• The tissue microenvironment plays a crucial role in the pathogenesis of cancer and cardiovascular diseases.
• Understanding the spatial distribution of immune cells, vasculature, and hypoxia is vital for disease progression and therapy resistance.
• Current imaging techniques often face limitations in depth and resolution for large tissues.

Purpose of the Study:

• To describe a detailed protocol for light sheet fluorescence microscopy (LSFM) for whole-organ imaging.
• To enable multiplex visualization of immune cells, vasculature, and hypoxia within the tissue microenvironment.
• To facilitate the spatial illustration of tissue structure and function in disease contexts.

Main Methods:

• Tissue clarification and immunostaining followed by LSFM.
• Utilizing a transgenic vascular labeling system for visualization.
• Detailed procedures for tissue collection, semi-clearing, immunostaining, and data analysis.

Main Results:

• Demonstrated 3D high-resolution imaging at a whole-organ level.
• Successful visualization of immune cell composition, vascularization, perfusion, and hypoxia in mouse brains, hearts, and brain tumors.
• Established standard methods for analyzing tissue immunity and vascularity.

Conclusions:

• LSFM offers a powerful approach for detailed spatial analysis of the tissue microenvironment.
• This method can significantly advance the understanding of complex diseases like cancer and cardiovascular conditions.
• The described procedure is feasible within 1-2 weeks for researchers with general molecular biology expertise.