A targeted 3D EM and correlative microscopy method using SEM array tomography.

Area of Science:

• Cell Biology
• Developmental Biology
• Microscopy Techniques

Background:

• Localizing rare cellular events in complex tissues using electron microscopy is difficult.
• Correlative light and electron microscopy (CLEM) links fluorescent protein expression to ultrastructural detail.
• Existing methods can be complex and time-consuming for detailed 3D analysis.

Purpose of the Study:

• To present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography.
• To improve the efficiency and precision of 3D data acquisition and correlation for biological samples.
• To facilitate the identification of labeled cells or organelles in model organisms.

Main Methods:

• Developed an optimized SEM workflow for volumetric array tomography on asymmetric samples.
• Modified a diamond knife for simplified serial section array acquisition with reduced artifacts.
• Integrated light microscopy for rapid anatomical screening followed by high-resolution SEM analysis.

Main Results:

• The workflow enables efficient 3D data acquisition and correlation for model organisms like *C. elegans*, *D. melanogaster*, and *D. rerio*.
• Manual and automatic data acquisition strategies enhance simplicity and precision compared to alternatives.
• The method successfully allows for in-depth ultrastructural study of selected cells and organelles.

Conclusions:

• This optimized SEM array tomography workflow provides a powerful tool for cell and developmental biology.
• The method addresses the challenge of identifying rare cellular events or structures with high precision.
• It offers a more efficient and accurate approach for 3D ultrastructural analysis in diverse model systems.