Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest.

Abstract

Background: For decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm³ large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample's morphology, thus making navigation to the ROI difficult.

Results: Using the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems-charging and ROI localization-that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition.

Conclusions: In MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique.