Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells

Our plain-English summary of Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells, by B. Weyn, W. Kalle, S. Kumar-Singh, et al (Journal of Microscopy, 1998).

The study

• Authors: B. Weyn, W. Kalle, S. Kumar-Singh, E. Van Marck, H. Tanke, W. Jacob
• Journal: Journal of Microscopy
• Published: 1998;189(Pt 2):172-80
• Cited by: 25 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The influence of fixation, air-drying and liquid-imaging on the morphology as well as on the viscoelasticity of malignant mesothelioma cells was studied by atomic force microscopy. In this study, dehydrated cells were more easily scanned and offered faster data recording than hydrated cells. However, the influence of fixation strength was more noticeable. Strong fixation induced flattening of the cytoplasm and loss of nuclear structure, resulting in a clearly visible cytoskeleton which could be easily seen as fibres orientated in the direction of the cell growth. By contrast, the morphology of hydrated cells was influenced to a lesser degree on fixation and showed an overall 'rounding' of the surface with vague, ill-defined structures. Nuclear areas of these samples were difficult to image. Viscoelasticity measurements also exhibited large differences. Dehydrated cells were much harder and showed a uniform indentation profile over the whole cell that was independent of fixation. Indentation on hydrated cells was large and depended on the height of the measuring spot, the submembranous structure and, to a lesser extent, on fixation. To calculate an overall 'cellular' viscoelasticity, different methods were tested on these samples. Indentations of multiple, randomly chosen points, covering the whole cell, were measured and averaged to yield a mean indentation score. We avoided the thin and shadowed areas since it was shown that these regions were less suited for measuring. Using this design, large viscoelasticity differences were found, on which the influence of the external parameters could be shown. In another set-up, layered imaging was tried. However, long data acquisition times caused cellular activation and rearrangement, making this scanning mode unsatisfactory.

In plain English

This was a laboratory pilot study of microscope technique. It did not study asbestos exposure or people. The authors used an atomic force microscope, which scans and presses a fine tip into a surface, on cultured malignant mesothelioma cells from a cell line (UIA-MM 8) first taken from pleural fluid. Some cells were fixed in 1%, 2% or 4% formaldehyde for 1, 5 or 10 minutes, then air dried or kept wet in culture medium; unfixed cells were scanned wet in medium. Air-dried cells were much harder. Their indentation slopes were 1.4 to 2.9 nanometres per nanonewton, compared with up to 266 for unfixed wet cells; a higher number means a softer cell. In dried cells, strong fixation made cytoskeleton fibres clearly visible and caused loss of nuclear structure. Fixation changed the look of wet cells little but made them stiffer, and spots over cytoskeleton fibres were stiffer. A slower 'layered imaging' method failed because the cells reacted to the long scans. The authors concluded that their averaging method showed the effects of fixation and environment, but that more work was needed before any quantification.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory pilot study of atomic force microscopy methods on cultured cells (in vitro)
• Who was studied: Cultured malignant mesothelioma cells from a cell line (UIA-MM 8) originally isolated from pleural fluid. Whole-cell averages used about 10 to 12 measuring spots per cell. The number of cells examined is not stated.
• Exposure: No asbestos exposure was studied. Cells were fixed in 1%, 2% or 4% formaldehyde for 1, 5 or 10 minutes and then either kept in medium for wet scanning or air dried for dry scanning. Unfixed cells were kept in the incubator and scanned in medium.
• Main results:
  • Average indentation slopes ranged from 1.4 nanometres per nanonewton for dehydrated (air-dried) cells to 266 for unfixed, hydrated cells. Larger slopes mean more indentation, that is, softer cells.
  • In dehydrated cells, indentation was very small (slopes 1.4 to 2.9) and was similar across cytoplasm, nucleus, cytoskeleton and nucleolus, whatever the height of the measuring spot.
  • In unfixed, hydrated cells, indentation near the cell border (slope 26) was smaller than towards the centre (slopes 125 and 266).
  • In an unfixed, hydrated cell, the slope on top of a cytoskeleton fibre was 66, compared with 95 right next to it. The authors read this as the cytoskeleton being more rigid.
  • Fixation made hydrated cells harder. Slopes were 187 for unfixed cells, 145 for cells fixed in 1% formaldehyde for 10 minutes, and 53 for cells fixed in 4% formaldehyde for 10 minutes.
  • Whole-cell average slopes, as means plus or minus standard deviation, per the Figure 12 legend. With randomly selected spots: 256 plus or minus 122 for unfixed cells, 226 plus or minus 21 for 1% formaldehyde for 10 minutes, and 59 plus or minus 17 for 4% formaldehyde for 10 minutes. With preselected spots of similar height: 183 plus or minus 69, 168 plus or minus 41, and 47 plus or minus 9, respectively.
  • In appearance, dehydrated cells showed a rougher cytoplasm with cytoskeleton fibres clearly visible in the direction of growth, more so with stronger fixation. Hydrated cells changed little with fixation.
  • Layered imaging failed. During prolonged scanning, unfixed cells became activated and rounded up, and fixed hydrated cells detached easily.
• The authors' conclusion: The authors concluded that averaging indentations over a cell clearly showed how viscoelasticity depends on fixative and environment, and on structures under the membrane such as the cytoskeleton. They stressed that further work on scanning conditions and preparation is needed before any quantification.
• Limitations the authors state:
  • Nuclear areas of hydrated cells could not be measured because the cells were too tall for the scanner.
  • The effect of tip size was not considered.
  • Each cellular spot could be measured only once, because the cells moved sideways.
  • The set-up did not allow ideal temperature conditions.
  • Air drying was chosen for practicality over methods that might have reduced shrinkage and other artefacts.
  • The authors say further work is needed before any quantification.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Malignant Mesothelioma.

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