Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying

The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.

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Scanning electron microscopy preparation and analysis of the cell cortex ultrastructure

10.1101/2020.01.06.896761 ◽

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.

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The Critical Point Drying Method Applied to Scanning Electron Microscopy of L-929 Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100068382 ◽

1970 ◽

Vol 28 ◽

pp. 278-279

Author(s):

Charles TurnbiLL ◽

Delbert E. Philpott

Keyword(s):

Electron Microscopy ◽

Carbon Dioxide ◽

Surface Tension ◽

Critical Point ◽

Freeze Drying ◽

Attractive Alternative ◽

Crystal Formation ◽

Critical Point Drying ◽

Time Required ◽

Scanning Electron

The advent of the scanning electron microscope (SCEM) has renewed interest in preparing specimens by avoiding the forces of surface tension. The present method of freeze drying by Boyde and Barger (1969) and Small and Marszalek (1969) does prevent surface tension but ice crystal formation and time required for pumping out the specimen to dryness has discouraged us. We believe an attractive alternative to freeze drying is the critical point method originated by Anderson (1951; for electron microscopy. He avoided surface tension effects during drying by first exchanging the specimen water with alcohol, amy L acetate and then with carbon dioxide. He then selected a specific temperature (36.5°C) and pressure (72 Atm.) at which carbon dioxide would pass from the liquid to the gaseous phase without the effect of surface tension This combination of temperature and, pressure is known as the "critical point" of the Liquid.

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Preservation of Plant Cell Surfaces For Scanning Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072800 ◽

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.

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