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Methods. 2015 Oct 15;88:67-75. doi: 10.1016/j.ymeth.2015.06.017. Epub 2015 Jun 27.

A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics.

Author information

1
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
2
Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
3
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom; Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom. Electronic address: christian.eggeling@rdm.ox.ac.uk.

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.

KEYWORDS:

Fluorescence correlation spectroscopy; Lipid diffusion; Plasma membrane dynamics; Super-resolution STED microscopy; Time-gated single-photon counting

PMID:
26123184
PMCID:
PMC4641872
DOI:
10.1016/j.ymeth.2015.06.017
[Indexed for MEDLINE]
Free PMC Article

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