Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology

Area of Science:

• Cell Biology
• Microscopy Techniques
• Virology

Background:

• Correlative imaging of small cellular targets within large fields presents a significant challenge in cell biology.
• Existing methods often lack the speed, resolution, or automation required for comprehensive analysis.

Purpose of the Study:

• To develop and demonstrate technical advances in focused ion beam scanning electron microscopy (FIB-SEM) for efficient correlative imaging.
• To introduce a novel "keyframe imaging" approach for simultaneous large-field and high-resolution sub-volume imaging.
• To apply these advances to the 3D imaging of HIV core structures within infected cells.

Main Methods:

• Implemented technical improvements in FIB-SEM for increased speed, robustness, and automation.
• Achieved consistent z-slice thickness of approximately 3 nm.
• Developed "keyframe imaging" to combine large field-of-view imaging with high-resolution 3D sub-volume acquisition.
• Utilized fluorescence microscopy to create target maps of HIV-infected cells with labeled components.
• Generated correlated 3D electron microscopy volumes of entire cells and individual HIV cores.

Main Results:

• Demonstrated significant increases in the speed, robustness, and automation of FIB-SEM imaging.
• Achieved consistent and high-resolution (∼3 nm) z-slice thickness.
• Successfully applied "keyframe imaging" to obtain correlated 3D EM volumes across a 10^9 volume scale.
• Visualized post-fusion cytoplasmic intermediates of the HIV core with high fidelity.

Conclusions:

• The developed FIB-SEM technical advances and "keyframe imaging" provide an efficient solution for correlative imaging challenges.
• This approach enables high-resolution 3D structural analysis of specific targets, such as HIV cores, within their cellular context.
• The methodology facilitates a multi-scale imaging approach within a single automated experiment.