Comparison of hexamethyldisilazane (HMDS), Peldri II, and critical-point drying methods for scanning electron microscopy of biological specimens

1993 ◽  
Vol 26 (6) ◽  
pp. 489-495 ◽  
Author(s):  
D. F. Bray ◽  
J. Bagu ◽  
P. Koegler
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Drying Methods ◽  
Critical Point Drying ◽  
Scanning Electron

scholarly journals Scanning electron microscopy preparation and analysis of the cell cortex ultrastructure

2020 ◽  
Author(s):  
D. Flormann ◽  
M. Schu ◽  
E. Terriac ◽  
M. Koch ◽  
S. Paschke ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Structural Defects ◽  
Cell Cortex ◽  
Drying Methods ◽  
Actin Network ◽  
Actin Cortex ◽  
Critical Point Drying ◽  
Scanning Electron

AbstractThe cellular cortex is a 200-nm-thick actin network that lies beneath the cell membrane. It is responsible for the mechanical properties of the cell and is involved in many cellular processes, such as cell migration and interactions with the environment. To develop a clear view of the structure of this meshwork, high resolution imaging is essential, such as electron microscopy. This technique requires complex sample preparation that can lead to artifacts like shrinkage or hole formation. We present a preparation method that reduces artifacts significantly. Here, the final drying step that is typically performed by critical point drying is replaced by hexamethyldisilazane drying. We quantitatively investigated sample integrity after both preparation methods, and show that there are significant advantages of hexamethyldisilazane drying compared to critical point drying. Furthermore, automated analysis of a network is classically performed by thresholding-based software programs, which are sensitive to noise and uneven brightness of images. The here presented analysis that we have developed is based on a vectorial node algorithm. It reproduces all kinds of networks sufficiently to allow derivation of quantitative network-specific parameters, such as mesh hole size. We use this analysis to compare the network structure of cells prepared by these two drying methods, and show that hexamethyldisilazane drying leads to fewer artificial mesh holes compared to critical point drying. We thus present here a significantly improved method to quantitatively investigate the actin cortex of cells, and show that hexamethyldisilazane drying leads to more accurate imaging compared to critical point drying.Insight BoxThe highest resolution for imaging the cellular actin cortex is provided by electron microscopy. Scanning electron microscopy samples require a drying process, usually achieved by critical point drying, which is critical for the sample integrity. We compare the structural defects in the actin cortex of hTert RPE1 cells after critical point drying and a chemical based method, namely hexamethyldisilazane drying. In order to characterize the actin network, we also developed a new vectorial based tracing software. We bring here new tool, both experimental and analytical, which will help to streamline studies of the actin cortex.

Preservation of Plant Cell Surfaces For Scanning Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 472-473
Author(s):  
Linda M. Sicko ◽  
Thomas E. Jensen
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Filter Paper ◽  
Freeze Drying ◽  
Cell Surfaces ◽  
Air Drying ◽  
Popular Method ◽  
Critical Point Drying ◽  
Scanning Electron

The use of critical point drying is rapidly becoming a popular method of preparing biological samples for scanning electron microscopy. The procedure is rapid, and produces consistent results with a variety of samples. The preservation of surface details is much greater than that of air drying, and the procedure is less complicated than that of freeze drying. This paper will present results comparing conventional air-drying of plant specimens to critical point drying, both of fixed and unfixed material. The preservation of delicate structures which are easily damaged in processing and the use of filter paper as a vehicle for drying will be discussed.

Chemical and bacteriological composition of granular methanogenic sludge

1985 ◽  
Vol 31 (8) ◽  
pp. 744-750 ◽  
Author(s):  
J. Dolfing ◽  
A. Griffioen ◽  
A. R. W. van Neerven ◽  
L. P. T. M. Zevenhuizen
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Buoyant Density ◽  
Extraction Methods ◽  
Drying Methods ◽  
Extracellular Material ◽  
Extracellular Polymers ◽  
Intact Cells ◽  
Critical Point Drying ◽  
Scanning Electron

Methanogenic sludge granules grown on waste water from a sugar refinery consisted of several bacterial morphotypes embedded in a matrix of extracellular material. Comparison of critical point drying and freeze-drying methods for preparing samples for scanning electron microscopy to determine the presence of extracellular material indicated that the former method permitted observations of extracellular material and intact cells. The effects of different extraction methods used for isolation of these extracellular polymers was also investigated by scanning electron microscopy. Of the various extraction procedures (EDTA, NaOH, autoclaving, water–phenol), water–phenol left most of the cells intact and was found to be a very efficient method of extraction. Extracellular polymers equivalent to 10–20 mg hexose/g of granules were extracted. The high resistance of the granules against disintegration by various chemical methods suggested that different extracellular polymers and probably different groups of organisms contributed to the matrix in which the bacteria were embedded. The chemical composition of the granules did not differ from the composition of bacteria in general. The buoyant density of 1.00–1.05 g∙g−1of the granules indicated that a simple agglomeration was the mechanism by which these methanogenic consortia improved their settling characteristics.

Alternative Method of Rapid Drying Vascular Specimens for Scanning Electron Microscopy

2003 ◽  
Vol 10 (2) ◽  
pp. 285-287 ◽  
Author(s):  
DáŠa Slížová ◽  
Otakar Krs ◽  
Blanka PospíŠilová
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Micrographs ◽  
Low Cost ◽  
High Rate ◽  
Drying Methods ◽  
Scanning Electron Micrographs ◽  
Air Drying ◽  
Critical Point Drying ◽  
Scanning Electron

Purpose: To report the use of hexamethyldisilazane (HMDS) as an alternative to critical point drying for preparing stented canine peripheral vessels for scanning electron microscopy (SEM). Technique: Vascular specimens were fixed in 4% formaldehyde overnight, dehydrated in a graded ethanol series, followed by immersion in 100% hexamethyldisilazane. After air drying, the specimens were mounted on stainless steel stubs, coated with gold, and examined in the SEM. The electron micrographs were of high quality, showing the layers of the vascular wall and the incorporated stent covered by a neointimal layer. The micrographs were comparable to corresponding histological sections, but detailed endothelial patterns were more visible. Conclusions: HMDS treatment and subsequent air drying provides good quality scanning electron micrographs that reveal both endothelial patterns and the layered architecture of stented vessels. The disadvantage of HMDS drying may be a shrinkage and distortion similar to other drying agents. Ease of handling, low cost, and a high rate of success are advantages that favor HMDS desiccation over other drying methods.

Delicate Botanical Specimens Preserved for Scanning Electron Microscopy by Critical Point Drying

1971 ◽  
Vol 29 ◽  
pp. 450-451
Author(s):  
Arthur L. Cohen ◽  
Gerald E. Garner
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Freeze Drying ◽  
Animal Cell ◽  
Pollen Grains ◽  
Scanning Electron Micrographs ◽  
Animal Cells ◽  
Critical Point Drying ◽  
Scanning Electron

The surface forms and structures of animal cells have been strikingly preserved for scanning electron microscopy by freeze-drying and by critical point drying both by the method with CO2 used as the transitional fluid and the later procedure which uses a fluorocarbon (Freon 13) as a medium for the transition from the liquid to the gaseous environment. Freeze-drying is often prolonged (5-12 hours as compared with an hour or less by the critical point method) and in our experience with mold cultures on agar, the substrate shrivels and cracks and hyphal filaments are distorted.Despite, and possibly because of a flexible but inelastic cell wall, plant cells often show greater distortion than do animal cells after evaporative drying or replacement dehydration for mixrotechnical work. The animal cell membrane can contract more or less uniformly on drying - as shown by the numerous micrographs of well-preserved erythrocytes, while plant cell walls often crumple. The many scanning electron micrographs of partially collapsed pollen grains bear witness to this fact.

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