A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics
Author(s)
Clausen, Mathias P
Sezgin, Erdinc
Bernardino de la Serna, Jorge
Waithe, Dominic
Lagerholm, B Christoffer
Type
Journal Article
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.
Date Issued
2015-10-15
Date Acceptance
2015-06-24
Citation
Methods, 2015, 88, pp.67-75
ISSN
1046-2023
Publisher
Elsevier
Start Page
67
End Page
75
Journal / Book Title
Methods
Volume
88
Copyright Statement
2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
(http://creativecommons.org/licenses/by/4.0/).
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000364607800009&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Subjects
Science & Technology
Life Sciences & Biomedicine
Biochemical Research Methods
Biochemistry & Molecular Biology
Plasma membrane dynamics
Super-resolution STED microscopy
Fluorescence correlation spectroscopy
Lipid diffusion
Time-gated single-photon counting
FLUORESCENCE CORRELATION SPECTROSCOPY
EMISSION DEPLETION MICROSCOPY
SINGLE-MOLECULE SENSITIVITY
LASER-SCANNING MICROSCOPE
PLASMA-MEMBRANE
STIMULATED-EMISSION
CELL-MEMBRANES
DIFFUSION LAWS
LIVING CELLS
FLUCTUATION SPECTROSCOPY
Publication Status
Published
Date Publish Online
2015-06-27