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

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 nmmin (-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.

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Hendrik Georges Deschout, Lely Feletti, Theo Lasser, Marcel Leutenegger, Tomas Lukes, Aleksandra Radenovic, Azat Sharipov, Daniel Pawel Szlag
Nature Publishing Group, 2016
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https://infoscience.epfl.ch/record/223732?ln=en

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Super-resolution microscopy

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffracti

Live-cell imaging

Live-cell imaging is the study of living cells using time-lapse microscopy. It is used by scientists to obtain a better understanding of biological function through the study of cellular dynamics. L

Molecular modelling

Molecular modelling encompasses all methods, theoretical and computational, used to model or mimic the behaviour of molecules. The methods are used in the fields of computational chemistry, drug de

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

Hendrik Georges Deschout, Lely Feletti, Theo Lasser, Tomas Lukes, Aleksandra Radenovic, Azat Sharipov

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM–SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

Spie-Int Soc Optical Engineering2017

FALCON: fast and unbiased reconstruction of high-density super-resolution microscopy data

Lina Carlini, Seamus John Holden, Hagay Kirshner, Suliana Manley, Michaël Unser, Cédric René Jean Vonesch

Super resolution microscopy such as STORM and (F) PALM is now a well known method for biological studies at the nanometer scale. However, conventional imaging schemes based on sparse activation of photo-switchable fluorescent probes have inherently slow temporal resolution which is a serious limitation when investigating live-cell dynamics. Here, we present an algorithm for high-density super-resolution microscopy which combines a sparsity-promoting formulation with a Taylor series approximation of the PSF. Our algorithm is designed to provide unbiased localization on continuous space and high recall rates for high-density imaging, and to have orders-of-magnitude shorter run times compared to previous high-density algorithms. We validated our algorithm on both simulated and experimental data, and demonstrated live-cell imaging with temporal resolution of 2.5 seconds by recovering fast ER dynamics.

Nature Publishing Group2014

Spectral cross-cumulants for multicolor super-resolved SOFI imaging

Adrien Charles-François Raymond Descloux, Kristin Stefanie Grussmayer, Theo Lasser, Marcel Leutenegger, Tomas Lukes, Aleksandra Radenovic

Super-resolution opticalfluctuation imaging provides a resolution beyond the diffraction limitby analysing stochasticfluorescencefluctuations with higher-order statistics. Usingnthorderspatio-temporal cross-cumulants the spatial resolution and the sampling can be increased upton-fold in all spatial dimensions. In this study, we extend the cumulant analysis into thespectral domain and propose a multicolor super-resolution scheme. The simultaneousacquisition of two spectral channels followed by spectral cross-cumulant analysis andunmixing increases the spectral sampling. The number of discriminablefluorophore species isthus not limited to the number of physical detection channels. Using two color channels, wedemonstrate spectral unmixing of threefluorophore species in simulations and experimentsinfixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for anoptimized spectralfilter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conservingthe spatial super-resolution property.

2020