A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.
- Resource Type
- Article
- Authors
- Wang, Ruixing; Brustlein, Sophie; Mailfert, Sébastien; Fabre, Roxane; Fallet, Mathieu; Sivankutty, Siddharth; Rigneault, Hervé; Marguet, Didier
- Source
- Methods. May2018, Vol. 140, p212-222. 11p.
- Subject
- *UNBIASED estimation (Statistics)
*STIMULATED emission
*FLUORESCENCE spectroscopy
*GAUSSIAN processes
*DIFFUSION
- Language
- ISSN
- 1046-2023
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. [ABSTRACT FROM AUTHOR]