Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy

Area of Science:

• Biological imaging
• Microscopy techniques
• Neuroscience
• Developmental biology

Background:

• Light sheet fluorescence microscopy (LSFM) has revolutionized 3D biological imaging over the last decade.
• Its application in cleared biological samples presents unique challenges for researchers.
• Despite advances, biologists often face unmet expectations with LSFM integration.

Purpose of the Study:

• To critically review challenges in LSFM for cleared biological samples.
• To propose solutions for overcoming difficulties in sample preparation, image acquisition, and data analysis.
• To provide a primer for biologists developing LSFM imaging pipelines.

Main Methods:

• Literature review of recent studies on LSFM and tissue clearing.
• Analysis of challenges identified through researcher surveys.
• Characterization of difficulties in sample clearing, labeling, image acquisition, and data analysis.

Main Results:

• Identified key challenges in LSFM for cleared samples across the entire imaging pipeline.
• Highlighted the importance of anticipating image analysis needs during sample preparation and acquisition.
• Provided practical solutions and recommendations for optimizing LSFM workflows.

Conclusions:

• Successful implementation of LSFM in cleared tissues requires careful planning and anticipation of downstream analysis.
• Addressing challenges in sample preparation, labeling, acquisition, and analysis is crucial for effective 3D imaging.
• This review serves as a guide to improve LSFM pipeline development for biologists.