Combined use of AFM and soft X-ray microscopy to reveal fibres' internalization in mesothelial cells

Our plain-English summary of Combined use of AFM and soft X-ray microscopy to reveal fibres' internalization in mesothelial cells, by A. Gianoncelli, G. Kourousias, F. Cammisuli, et al (The Analyst, 2017).

The study

• Authors: A. Gianoncelli, G. Kourousias, F. Cammisuli, D. Cassese, C. Rizzardi, O. Radillo, M. Lazzarino, L. Pascolo
• Journal: The Analyst
• Published: 2017;142(11):1982-1992
• Cited by: 2 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Nanotoxicology and nanomedicine investigations often require the probing of nano-objects such as fibres and particles in biological samples and cells, whilst internalization and intracellular destiny are the main issues for in vitro cellular studies. Various high resolution microscopy techniques are well suited for providing this highly sought-after information. However, sample preparation, nanomaterial composition and sectioning challenges make it often difficult to establish whether the fibres or particles have been internalized or they are simply overlaying or underlying the biological matter. In this paper we suggest a novel suitable combination of two different microscopic techniques to reveal in intact cells the uptake of asbestos fibres by mesothelial cells. After exposure to asbestos fibres and fixation, cells were first analysed under the AFM instrument and then imaged under the TwinMic soft X-ray microscope at Elettra Sincrotrone. The suggested approach combines standard soft X-ray microscopy imaging and AFM microscopy, with a common non-invasive sample preparation protocol which drastically reduces the experimental uncertainty and provides a quick and definitive answer to the nanoparticle cellular and tissue uptake.

In plain English

This was a laboratory methods paper, not a study of people. The authors tested whether two microscope techniques used together could show if asbestos fibres had entered cells or were only lying on top of them. Human mesothelial cells from a laboratory cell line (MeT5A) were exposed for 24 hours to crocidolite asbestos fibres at 5 µg per ml, then fixed and examined first by atomic force microscopy (AFM), which maps the cell surface, and then by soft X-ray microscopy, which shows all fibres but not their depth. In the X-ray images all fibres looked equally sharp. In the AFM images some looked sharp, some blurred and some could not be seen. The authors read these as fibres outside the cell, fibres just under the cell membrane, and fibres fully inside the cell. They concluded that only the combination of the two techniques could tell fibres inside the cell from fibres outside it. They described the work as a proof of principle and said it was not yet fully quantitative.

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 (in vitro) methods study presented as a proof of principle, imaging cultured cells with atomic force microscopy and soft X-ray microscopy
• Who was studied: Human mesothelial cells from a cell line (MeT5A, from ATCC), grown on silicon nitride membranes. The examples shown were several isolated cells and a single layer of cells. Imaging was done at the Elettra synchrotron facility and IOM-CNR in Trieste, Italy. The number of cells analysed in total is not stated.
• Exposure: Crocidolite asbestos (UICC standard fibres), added to the culture medium at a final concentration of 5 µg per ml for 24 hours
• Main results:
  • In the X-ray absorption and phase contrast images, all fibres had the same sharpness. In the AFM maps, some fibres had sharp edges and others appeared blurred.
  • The authors interpreted fibres that appeared wider and blurred in AFM than in the X-ray image as lying just under the cell membrane, which they called a "blanket effect".
  • In the cell monolayer, most of the smaller fibres were hardly detectable by AFM, which the authors took as indicating that these fibres were inside the cells.
  • Some fibres seen by X-ray microscopy could not be detected by AFM at all, which the authors interpreted as completely internalized fibres.
• The authors' conclusion: The authors concluded that AFM and soft X-ray microscopy are each useful for revealing fibres in cells, but only their combination can tell intracellular fibres from extracellular ones. They stated that the method could be extended to other cell types, fibres and particles.
• Limitations the authors state:
  • The authors described the work as a proof of principle. To be fully quantitative, peak widths would need to be measured, corrected for probe and tip size, and compared.
  • Because cells dehydrate during fixation and the mineral fibres are dense, fibres inside the cell can sometimes still produce a signal in AFM.
  • Very small fibres forming a bundle may not be distinguishable from the cell's own surface features by AFM.
  • Soft X-ray microscopy alone gives a flat two-dimensional image and cannot show how deep a fibre lies.
  • Aligning the images from the two microscopes to a common scale and position was difficult.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelial Cells.

Related studies

• Iron-related toxicity of single-walled carbon nanotubes and crocidolite fibres in human mesothelial cells investigated by Synchrotron XRF microscopy — F. Cammisuli, S. Giordani, et al. (2018)
• Synchrotron soft X-ray imaging and fluorescence microscopy reveal novel features of asbestos body morphology and composition in human lung tissues — L. Pascolo, A. Gianoncelli, et al. (2011)
• The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near-field optical microscopy — L. Andolfi, E. Trevisan, et al. (2013)
• Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells — P. Munson, Y.W. Lam, et al. (2018)
• Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice — L. Pascolo, G. Zabucchi, et al. (2016)
• Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells — B. Weyn, W. Kalle, et al. (1998)

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