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Article Dans Une Revue Methods Année : 2018

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses

Résumé

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power. (C) 2018 Elsevier Inc. All rights reserved.

Domaines

Immunologie
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Dates et versions

hal-02118001 , version 1 (02-05-2019)

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Ruixing Wang, Sophie Brustlein, Sebastien Mailfert, Roxane Fabre, Mathieu Fallet, et al.. A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses. Methods, 2018, 140, pp.212-222. ⟨10.1016/j.ymeth.2018.02.010⟩. ⟨hal-02118001⟩
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