Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures

Area of Science:

• Cell Biology
• Microscopy
• Biotechnology

Background:

• Correlative light and electron microscopy (CLEM) integrates data from different resolutions for holistic structural analysis.
• Advancements in 3D imaging enable nanometer-scale morphological studies across diverse sample volumes.
• Combining light and electron microscopy data provides deeper insights into cellular and tissue organization.

Purpose of the Study:

• To present two alternative approaches for correlating 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data.
• To describe an adapted sample preparation method using high-pressure freezing and freeze-substitution for multimodal imaging.
• To demonstrate the applications and advantages of these CLEM methods on various biological samples.

Main Methods:

• High-pressure freezing for structure preservation, followed by freeze-substitution for en-bloc or serial-section imaging.
• Confocal laser scanning microscopy (CLSM) coupled with focused ion beam-SEM (FIB-SEM) for targeted 3D correlation.
• Serial-section LM and SEM imaging for large-area or volume screening, compatible with immunocytochemistry.

Main Results:

• Successful correlation of 3D LM and SEM data across diverse biological samples including cells, organisms, and tissues.
• Demonstration of distinct advantages for each CLEM approach: CLSM/FIB-SEM for small volumes, serial-section for large volumes.
• Validation of the sample preparation method for preserving ultrastructure during multimodal imaging.

Conclusions:

• Two robust CLEM approaches enable detailed 3D structural analysis of biological samples.
• These methods facilitate the extraction of statistically relevant structural data for systems biology.
• The presented techniques expand the capabilities of correlative microscopy for comprehensive biological investigation.