Fluorescence microscopy techniques have been widely adopted
in
biology for their ability to visualize the structure and dynamics
of a wide range of cellular and subcellular processes. The specificity
and sensitivity that these techniques afford have made them primary
tools in the characterization of protein localizations within cells.
Many of the fluorescence microscopy techniques require cells to be
fixed via chemical or alternative methods before being imaged. However,
some fixation methods have been found to induce the redistribution
of particular proteins in the cell, resulting in artifacts in the
characterization of protein localizations and functions under physiological
conditions. Here, we review the ability of commonly used cell fixation
methods to faithfully preserve the localizations of proteins that
bind to chromatin, undergo liquid–liquid phase separation (LLPS),
and are involved in the formation of various membrane-bound organelles.
We also review the mechanisms underlying various fixation artifacts
and discuss potential alternative fixation methods to minimize the
artifacts while investigating different proteins and cellular structures.
Overall, fixed-cell fluorescence microscopy is a very powerful tool
in biomedical research; however, each experiment demands the careful
selection of an appropriate fixation method to avoid potential artifacts
and may benefit from live-cell imaging validation.