Resin comparison for Serial Block Face Scanning Electron Microscopy

Session

Stream 3: Volume Microscopy in Physical and Life Sciences

Authors

Dr Anna Kremer (1, 2, 3), Peter Borghgraef (1, 2, 3), Michiel De Bruyne (1, 2, 3), Dr Chris Guerin (1), Dr Saskia Lippens (1, 2, 3)

Affiliations

1. VIB Bioimaging Core
2. VIB Center for Inflammation Research
3. Department of Biomedical Molecular Biology, Ghent University

Keywords

SBF-SEM, VolumeEM, Resin

Abstract text

Volume electron microscopy allows for the automated acquisition of serial section imaging data that can be reconstructed in 3D to provide a detailed, geometrically accurate view of cellular ultrastructure. In the case of serial block face scanning electron microscopy (SBF-SEM), an ultra-microtome is used inside the SEM chamber to subsequently image and remove sections from the block face (Denk and Horstmann, 2004), resulting in high resolution datasets at nanometer scale. Sample preparation for SBF-SEM includes several incubations in heavy metals (Osmium, Uranyl Acetate, Lead) to provide contrast, followed by dehydration and embedding in non-conductive resins to stabilize samples in the vacuum and to enable ultra-thin sectioning. One downside of the use of these resins in SBF-SEM is that the viscosity of the resin before polymerization determines the time and ease of infiltration of the resin into the sample, which for some samples can take up to several days. Also, resin-embedded samples can be prone to charging effects, particularly when the samples are low in contrast or contain large regions of bare resin e.g. cell culture monolayers, highly vascularized tissues or plant tissue (Kremer et al., 2015; Peddie & Collinson, 2014). Charging effects compromise image quality, cause a low signal to noise ratio and result in distortion. Despite efforts to develop a conductive resin, there is no ideal resin for SBF-SEM and although most charging issues can now be eliminated by the use of a focal charge compensator (FCC, (Deerinck et al., 2018), an optimal resin choice can benefit SBF-SEM imaging significantly.

In this study, we aim to compare different resins on their performance in SBF-SEM. We are looking for a resin with low viscosity for infiltration into any tissue, animal or plant based, that is easy to handle (good sectioning) and that has minimal charging effects in the electron microscope. In literature, SBF-SEM has been combined with a range of different resins, but never comparatively (only for Focused Ion Beam -SEM, Kizilyaprak et al., 2015). In our comparison we therefore compared Durcupan, Embed 812, Spurr’s, TAAB Embedding hard plus, LX112 and LR White. We have analysed the viscosity of these resins after mixing their components, handling and sectioning after polymerization and their behavior in the microscope and compared their performance for SBF-SEM.

References

Denk W and Horstmann H  (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11):e329

Kremer et al, Developing 3D SEM in a broad biological context, J Microsc 2015, 259(2):80-96 

Peddie & Collinson, Exploring the third dimension: volume electron microscopy comes of age, Micron 2014,61:9-19

Deerinck TJ et al., High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation, J Microsc 2018, 270(2):142-149 

Kizilyaprak C et al., Investigation of resins suitable for the preparation of biological samples for 3-d electron microscopy, J Struct Biol 2015, 189(2):135-46