Biopharma Insights Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.
Total time
~3–5 days per sample (including fixation, processing, embedding, and imaging)
Steps
1
Fix and process tissue for electron microscopy
Apply chemical fixation and post-fixation protocols to preserve ultrastructural morphology. Prepare samples according to standard electron microscopy preparation standards.
▶ 00:51
2
Embed samples in resin for sectioning
Infiltrate fixed tissue with embedding resin and polymerize to create blocks suitable for ultramicrotomy and electron microscopy imaging.
▶ 03:51
3
Image samples using serial block face SEM
Acquire serial cross-sectional images using SBF-SEM to obtain 3D volumetric data and identify regions of interest in large tissue fields.
▶ 05:03
4
Perform targeted imaging with focused ion beam SEM
Use FIB-SEM to acquire high-resolution 3D ultrastructural data at targeted areas identified during SBF-SEM survey imaging.
▶ 06:22
5
Analyze and interpret volumetric microscopy data
Process and visualize combined SBF-SEM and FIB-SEM datasets to characterize rare cellular structures and events in three dimensions.
▶ 07:49