Chemical Fixation Creates Nanoscale Clusters on the Cell Surface by Aggregating Membrane Proteins

Abstract

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a new method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

“…To overcome these problems, the spatial resolution and field of view (FOV) are sacrificed in a trade-off with image acquisition time [18], [19], [20]. The sample fixation by chemical reagents (e.g., paraformaldehyde (PFA)) or organic solvents has been used for suppressing the motion of biological specimen and preserving biomolecules [21], [22], but denatures cells by chemical reaction or dehydration [20], [21], [23].…”

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

“…To overcome these problems, the spatial resolution and field of view (FOV) are sacrificed in a trade-off with image acquisition time [18], [19], [20]. The sample fixation by chemical reagents (e.g., paraformaldehyde (PFA)) or organic solvents has been used for suppressing the motion of biological specimen and preserving biomolecules [21], [22], but denatures cells by chemical reaction or dehydration [20], [21], [23].…”

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

“…To overcome these problems, the spatial resolution and field of view (FOV) are sacrificed in a trade-off with image acquisition time [18], [19], [20]. The sample fixation by chemical reagents (e.g., paraformaldehyde (PFA)) or organic solvents has been used for suppressing the motion of biological specimen and preserving biomolecules [21], [22], but denatures cells by chemical reaction or dehydration [20], [21], [23].…”

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

“…To overcome these problems, the spatial resolution and field of view (FOV) are sacrificed in a trade-off with image acquisition time [18], [19], [20]. The sample fixation by chemical reagents (e.g., paraformaldehyde (PFA)) or organic solvents has been used for suppressing the motion of biological specimen and preserving biomolecules [21], [22], but denatures cells by chemical reaction or dehydration [20], [21], [23].…”