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

Title: A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics
Authors: Clausen, MP
Sezgin, E
De la Serna, JB
Waithe, D
Lagerholm, BC
Eggeling, C
Item 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.
Issue Date: 15-Oct-2015
Date of Acceptance: 24-Jun-2015
URI: http://hdl.handle.net/10044/1/69269
DOI: https://doi.org/10.1016/j.ymeth.2015.06.017
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/).
Keywords: 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
Diffusion
Lipid Bilayers
Microscopy, Fluorescence
Spectrometry, Fluorescence
1103 Clinical Sciences
Publication Status: Published
Online Publication Date: 2015-06-27
Appears in Collections:National Heart and Lung Institute
Airway Disease
Faculty of Medicine



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Creative Commonsx