A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics.
Methods
; 88: 67-75, 2015 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-26123184
ABSTRACT
Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200 nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
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Bicamadas Lipídicas
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Microscopia de Fluorescência
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article