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.

3D X-Ray Nanotomography of Cells Grown on Electrospun Scaffolds

“…For comparison, the value for cytoplasm was calculated from the measured electron density of frozen hydrated cells to be similar at 8.2 × 10 −6 , while that of organelles was estimated to be higher at 1 × 10 −5 . The sample preparation technique used in this study can lead to cell shrinkage, and hence an increase in the real part of the refractive index in comparison to frozen hydrated cells. This would give rise to similar overall contrast for scaffold and cells at the current imaging resolution, as observed, while increasing the contrast of clearly resolved organelles.…”

Imaging analysis of the interface between osteoblasts and microrough surfaces of laser‐sintered titanium alloy constructs

“…However, we still observed artefacts in the cell membrane. Although HMDS has been shown to induce less cell shrinkage than critical point drying, research has also shown that increasing HMDS exposure time correlates with increased cell shrinkage (Katsen-Globa et al, 2016). In addition, handling of samples for confocal imaging, including mounting and creating an orientation marker using epoxy resin and securing a coverslip with PBS for better optical resolution, may affect cells.…”

Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification

“…The postfixation implemented in consecutive steps composed of incubation in 2% osmium tetroxide (Carl Roth), 1% tannic acid (Sigma‐Aldrich), and 1% uranyl acetate (Ted Pella, Redding, CA) solutions. Subsequently, dehydration in increased ethanol series and drying in hexamethyldisilazane (Sigma‐Aldrich) ensued according to Katsen‐Globa et al . The prepared samples were investigated at a 5 kV accelerating voltage and work distance of 10 mm in Philips FESEM XL30 (FEI, Eindhoven, Netherlands).…”