A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.
Methods
; 140-141: 212-222, 2018 05 01.
Article
en En
| MEDLINE
| ID: mdl-29454082
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Espectrometría de Fluorescencia
/
Procesamiento de Imagen Asistido por Computador
/
Microscopía Intravital
/
Modelos Químicos
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Methods
Asunto de la revista:
BIOQUIMICA
Año:
2018
Tipo del documento:
Article
País de afiliación:
Francia