Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions.

Hendrik Deschout,Lely Feletti,Azat Sharipov,Tomas Lukes,Aleksandra Radenovic,Theo Lasser,Marcel Leutenegger,Peter Dedecker,Wim Vandenberg,Daniel Szlag,Johan Hofkens

doi:10.1038/ncomms13693

Abstract

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Highlights

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy
Focal adhesions are dynamic entities demanding fast live-cell imaging. This has been further investigated by using photoactivated localization microscopy (PALM) to image the dynamic behaviour of paxillin[6], but elucidating the full cell adhesion process remains a challenging task for Single-molecule localization microscopy (SMLM)
Our results indicate that PALM and super-resolution optical fluctuation imaging (SOFI) are complementary techniques for the observation of focal adhesions in living cells

Summary

Introduction

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. We address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. These protein assemblies contain transmembrane receptors, such as integrins, binding to the extracellular matrix and recruiting other proteins inside the cytoplasm, such as talin and paxillin This entails the formation of small structures with an extent in the order of 100 nm, which either disassemble after a few seconds, or mature into larger focal adhesions that remain stable typically for tens of minutes. Undercounting errors can appear as well, by merging localizations of separate fluorophores in high-density samples, and due to incomplete maturation and limited detection efficiency[16]

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Results

Conclusion