title: Study of SEM preparation artefacts with correlative microscopy: Cell shrinkage of adherent cells by HMDS-drying.
abstract: One of the often reported artefacts during cell preparation to scanning electron microscopy (SEM) is the shrinkage of cellular objects, that mostly occurs at a certain time-dependent stage of cell drying. Various methods of drying for SEM, such as critical point drying, freeze-drying, as well as hexamethyldisilazane (HMDS)-drying, were usually used. The latter becomes popular since it is a low cost and fast method. However, the correlation of drying duration and real shrinkage of objects was not investigated yet. In this paper, cell shrinkage at each stage of preparation for SEM was studied. We introduce a shrinkage coefficient using correlative light microscopy (LM) and SEM of the same human mesenchymal stem cells (hMSCs). The influence of HMDS-drying duration on the cell shrinkage is shown: the longer drying duration, the more shrinkage is observed. Furthermore, it was demonstrated that cell shrinkage is inversely proportional to cultivation time: the longer cultivation time, the more cell spreading area and the less cell shrinkage. Our results can be applicable for an exact SEM quantification of cell size and determination of cell spreading area in engineering of artificial cellular environments using biomaterials. SCANNING 38:625-633, 2016. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department for Cryo- and Stem Cell Technology, Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany.
authors.forename: Alisa
authors.initials: A
authors.lastname: Katsen-Globa
authors.affiliation: Chair of Anatomy, Saarland University, Homburg, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Puetz
authors.affiliation: Department for Cryo- and Stem Cell Technology, Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Gepp
authors.affiliation: Department for Cryo- and Stem Cell Technology, Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany.
authors.forename: Julia C
authors.initials: JC
authors.lastname: Neubauer
authors.affiliation: Department for Cryo- and Stem Cell Technology, Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany.|Chair of Molecular and Cellular Biotechnology, Saarland University, Saarbrücken, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Zimmermann
mesh_terms: D016477:Artifacts; D002448:Cell Adhesion; D048429:Cell Size; D002478:Cells, Cultured; D006801:Humans; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D017646:Organosilicon Compounds
chemical_list: D017646:Organosilicon Compounds; C024548:hexamethylsilazane
keywords: cell shrinkage; cell spreading area; hexamethyldisilazane (HMDS); light microscopy (LM); scanning electron microscopy (SEM)