Kanazawa University NanoLSI Podcast: Chemical fixation causes aggregation artefact

Kanazawa University NanoLSI Podcast: Chemical fixation causes aggregation artefact

Transcript of this podcast

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takehiko Ichikawa and his co-researchers at the Kanazawa University NanoLSI. 

The research described in this podcast was published in the journal Communications Biology, in May 2022.

Learn more about their research here: WPI Kanazawa Nano Life Science Institute

https://nanolsi.kanazawa-u.ac.jp/en/research/researchers/

Original article 

Takehiko Ichikawa, Dong Wang, Keisuke Miyazawa, Kazuki Miyata, Masanobu Oshima, and Takeshi Fukuma.  Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins, Communications Biology 5, 487 (2022).

DOI: 10.1038/s42003-022-03437-2

URL: https://www.nature.com/articles/s42003-022-03437-2

Chemical fixation causes aggregation artefact

Researchers at Kanazawa University report in Communications Biology that using common chemicals for fixing living cell samples for microscopy studies causes membrane proteins to aggregate.

For histological investigations of biological tissues, i.e. anatomical studies under the microscope, samples are usually fixated to prevent them from decaying. 

Fixation is typically done by immersing or perfusing the sample in a chemical — aldehydes and alcohols are common fixatives. It has been speculated that membrane proteins moving to some extent on a cell membrane can form aggregates during fixation. 

Yet, detailed cell surface studies with nanometer-scale resolution are necessary to obtain definitive insights into this potential issue. 

Now, Takehiko Ichikawa and colleagues at the NanoLSI at Kanazawa University have performed atomic force microscopy (AFM) studies of living mammalian cell surfaces. 

By comparing non-fixated and fixated samples, they found that fixation indeed leads to structural changes.

The researchers developed a method of using microporous silicon nitride membranes (MPM)—that are widely used in transmission electron microscopy—for AFM imaging. 

Importantly, microporous silicon nitride membranes can make the cell surface flat and prevent fluctuations by supporting the area outside the observation area. 

 In AFM images of the surfaces of the cultured colon cancer cells on microporous silicon nitride membranes, biomolecular structures on the cell membranes showed up as protrusions, with a typical size of a few nanometers. 

When the cells were treated with commonly used fixatives such as paraformaldehyde, glutaraldehyde, and methanol, a few nanometer structures disappeared, and only large protrusions with diameters ranging from 20 to 100 nanometers were observed (Figure 2). The researchers performed several fluorescence experiments and concluded that large protrusions observed after fixation were formed by the aggregation of membrane proteins.

The study demonstrates that the observed aggregates are artefacts resulting from the fixation process. This should call for caution among the community of researchers working with chemical fixatives. Quoting Ichikawa and colleagues: “Researchers who observe nanoscale clusters also should be careful in interpreting their experimental results when using fixed cells. We recommend that researchers use living cells as much as possible to avoid the effect of fixation when investigating nanoscale clusters […].”

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