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Commonly used chemical fixation causes aggregation artifacts

Figure 1. A new technique for observing living cell membranes for AFM using porous silicon nitride membrane (MPM). (a) The appearance of MPM. (b) The gaps of MPM observed by AFM. (c) The MPM when placed in the chamber for observation. (d) Schematic of AFM observation of the cell surface using MPM. Credit: Kanazawa University

Kanazawa University researchers report in Communication biology that using common chemicals to fix live cell samples for microscopy studies causes membrane proteins to aggregate.

For histological examination of biological tissues, ie anatomical studies under the microscope, samples are usually fixed to prevent decay. Fixation is usually done by immersing or perfusing the sample in a chemical – aldehydes and alcohols are common fixatives. It has been speculated that membrane proteins that move to some extent on a cell membrane may form aggregates during fixation. Still, detailed cell surface studies with the nanometer-scale resolution are needed to gain definitive insights into this potential problem. Now, Takehiko Ichikawa and colleagues at Kanazawa University have conducted atomic force microscopy (AFM) studies on living mammalian cell surfaces. By comparing unfixed and fixed samples, they found that fixation does indeed lead to structural changes.

The researchers developed a method for using microporous silicon nitride membrane (MPM), used in transmission electron microscopy (Figure 1), for AFM imaging. Importantly, MPM can flatten the cell surface and prevent fluctuations by supporting the area outside the observation area. In AFM images of the surfaces of the cultured colon cancer cells on MPM, biomolecular structures on the cell membranes emerged as protrusions with a typical size of several nanometers (Figure 2 live cell surface).

Chemical fixation causes aggregation artifact

Figure 2. Observation of AFM cell surface before and after treatment with glutaraldehyde, paraformaldehyde and methanol, respectively. The left image is an AFM image, the middle image is an AFM image with the protrusions framed, and the right image is an elevation profile along the dotted line in the left image. Credit: Kanazawa University

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

The study shows that the observed aggregates are artifacts resulting from the fixation process. This should prompt caution in the community of researchers working with chemical fixatives. Citing Ichikawa and colleagues, “Researchers observing nanoscale clusters should also be careful when interpreting their experimental results when using fixed cells. We recommend that researchers use live cells as much as possible to avoid the effect of fixation when investigating clusters at the nanoscale.”

The general principle behind atomic force microscopy (AFM) is to have a very small tip scan the surface of a sample. During this horizontal (xy) scan, the tip, which is attached to a small cantilever, follows the vertical (z) profile of the sample, creating a force on the cantilever that can be measured. The magnitude of the force at the xy position can be related to the z value; the xyz data generated during a scan then results in an elevation map that provides structural information about the sample under investigation. AFM is not affected by the diffraction limit due to the use of light or electron beams and can observe the intact surface topography with high resolution.

Mapping Moving Features in Fast Atomic Force Microscopy

More information:
Takehiko Ichikawa et al, Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins, Communication biology (2022). DOI: 10.1038/s42003-022-03437-2

Provided by Kanazawa University

Quote: Commonly used chemical fixation causing aggregation artifact (2022, Aug 10) retrieved Aug 10, 2022 from https://phys.org/news/2022-08-commonly-chemical-fixation-aggregation-artifact.html

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