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

TL;DR

This paper combines PALM and SOFI super-resolution microscopy techniques to achieve high temporal and spatial resolution imaging of live cell focal adhesions, enabling dynamic analysis and molecular parameter estimation.

Contribution

It introduces a novel PALM-SOFI framework that enhances live cell imaging by combining their strengths for improved resolution and functional analysis.

Findings

Achieved sub-10 second temporal resolution in live cell imaging.

Visualized focal adhesion dynamics with velocities around 190 nm/min.

Provided a quantitative framework for molecular parameter estimation.

Abstract

Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative 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 the fluorophore density and the photo-activation and photo-switching rates.