Although total internal reflection fluorescence microscopy (TIRFM) allows imaging single actin filaments in vitro under conditions that prevent actin-filament bundling, it has insufficient resolution to zoom in to the finest details of F-actin and its interacting proteins. Not surprisingly, SMLM is employed to study cellular processes depending on the assembly of monomeric actin into approximately 6 nm-wide filaments, such as formation of membrane protrusions, cell migration, cytokinesis, endocytosis, vesicle trafficking and organelle homeostasis ( Isogai et al., 2015 van den Dries et al., 2013 Xu et al., 2012, 2013).
SMLM is receiving ever-increasing attention from biologists interested in capturing the distribution of individual molecules within the cell ( Endesfelder and Heilemann, 2014 Patterson et al., 2010). In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method.
Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible.
Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton.
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