PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins.

Daniela Leyton-Puig,Katarzyna M Kedziora,Kees Jalink,Metello Innocenti,Bram Van Den Broek,Tadamoto Isogai

doi:10.1242/bio.019570

Daniela Leyton-Puig, Katarzyna M Kedziora + Show 4 more

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https://doi.org/10.1242/bio.019570

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Abstract

ABSTRACTSuper-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. 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. 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. 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.

Highlights

Single-molecule-based localisation microscopy (SMLM) has circumvented the resolution limit of light microscopy, which prevents resolving details smaller than about half the wavelength of light (Hell, 2009)
Proper PFA fixation enables high-quality SMLM imaging of the actin cytoskeleton We initially set out to improve a fixation protocol that employs PFA dissolved in phosphate buffered saline (PBS) (PFA-PBS) and preserves densely packed F-actin bundles but not thin and short actin filaments (Bachmann et al, 2015; Whelan and Bell, 2015)
We systematically varied fixation time and temperature, and obtained the best images when the specimen was fixed for 10 min with all washing buffers and PFA kept at 37°C

Summary

Introduction

Single-molecule-based localisation microscopy (SMLM) has circumvented the resolution limit of light microscopy, which prevents resolving details smaller than about half the wavelength of light (Hell, 2009). Over the last ten years, several SMLM variants have been developed, the most prominent being photoactivated localization microscopy (PALM) (Betzig et al, 2006; Gould et al, 2009), (direct) stochastic optical reconstruction microscopy (STORM and dSTORM) (Rust et al, 2006; Heilemann et al, 2008) and groundstate depletion and single-molecule return (GSDIM) (Folling et al, 2008). Depending on the photoactivation or the photoswitching method, SMLM Received 25 May 2016; Accepted 1 June 2016 techniques can be divided in ‘targeted switching and readout’ and ‘stochastic switching and readout’ (Hell, 2009; van de Linde et al, 2011)

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