A practical guide to starting SEM array tomography-An accessible volume EM technique.

Area of Science:

• * Microscopy
• * Ultrastructural Biology
• * Materials Science

Background:

• * Volume electron microscopy (vEM) encompasses various techniques with distinct pros and cons.
• * Scanning Electron Microscopy array tomography (SEM-AT) is a flexible, cost-effective vEM approach.
• * SEM-AT requires microtomy skills and post-imaging data alignment, presenting user challenges.

Purpose of the Study:

• * To describe the general principles, advantages, and disadvantages of SEM-AT.
• * To provide a step-by-step guide to the SEM-AT workflow.
• * To highlight the potential for SEM-AT to become a routine vEM technique.

Main Methods:

• * Detailed workflow from block trimming, sectioning, and collection on coverslips.
• * Alignment of high-resolution 3D datasets.
• * Utilization of Scanning Electron Microscopy (SEM) with backscatter electron detectors and specialized imaging software.

Main Results:

• * SEM-AT enables 3D ultrastructural data acquisition using standard electron microscopy equipment.
• * Appropriate software can automate the imaging of hundreds to thousands of sections.
• * The technique is inherently non-destructive, offering significant advantages.

Conclusions:

• * SEM-AT is a versatile and accessible vEM technique for regular EM facilities.
• * Advances in hardware and software are simplifying the process.
• * SEM-AT has the potential to become a globally routine vEM method.