Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy

• Published in: PloS one Vol. 12; no. 5; p. e0176839
• Main Authors: Zhang, Ying, Huang, Tao, Jorgens, Danielle M, Nickerson, Andrew, Lin, Li-Jung, Pelz, Joshua, Gray, Joe W, López, Claudia S, Nan, Xiaolin
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31.05.2017; Public Library of Science (PLoS)
• Subjects: Adhesion; Animals; Biological properties; Biological samples; Biology and Life Sciences; Biomedical engineering; Cancer; Cell adhesion molecules; Cell differentiation; Cell Line; Cell morphology; Cloning, Molecular; Coating; Correlation; Critical point; Cytology; Distortion; Drying; Electron microscopy; Engineering and Technology; Fibronectin; Fibronectins; Fixation; Health aspects; Image acquisition; Medicine and Health Sciences; Methods; Microscopy, Electron, Scanning - methods; Morphology; Optimization; Osmium; Osmium tetroxide; Physical Sciences; Plating; Proteins; Research and Analysis Methods; Sample preparation; Scanning electron microscopy; Science; Specimen Handling; Temporal variations; Three dimensional imaging; Oregon
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0176839

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.