PMID- 26123184
OWN - NLM
STAT- MEDLINE
DCOM- 20160812
LR  - 20240324
IS  - 1095-9130 (Electronic)
IS  - 1046-2023 (Print)
IS  - 1046-2023 (Linking)
VI  - 88
DP  - 2015 Oct 15
TI  - A straightforward approach for gated STED-FCS to investigate lipid membrane 
      dynamics.
PG  - 67-75
LID - S1046-2023(15)30005-0 [pii]
LID - 10.1016/j.ymeth.2015.06.017 [doi]
AB  - 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.
CI  - Copyright (c) 2015 The Authors. Published by Elsevier Inc. All rights reserved.
FAU - Clausen, Mathias P
AU  - Clausen MP
AD  - MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University 
      of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
FAU - Sezgin, Erdinc
AU  - Sezgin E
AD  - MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University 
      of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
FAU - Bernardino de la Serna, Jorge
AU  - Bernardino de la Serna J
AD  - MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University 
      of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
FAU - Waithe, Dominic
AU  - Waithe D
AD  - Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of 
      Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
FAU - Lagerholm, B Christoffer
AU  - Lagerholm BC
AD  - Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of 
      Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
FAU - Eggeling, Christian
AU  - Eggeling C
AD  - 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.
LA  - eng
GR  - WT_/Wellcome Trust/United Kingdom
GR  - MC_UU_12010/9/MRC_/Medical Research Council/United Kingdom
GR  - MR/K01577X/1/MRC_/Medical Research Council/United Kingdom
GR  - BB_/Biotechnology and Biological Sciences Research Council/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20150627
PL  - United States
TA  - Methods
JT  - Methods (San Diego, Calif.)
JID - 9426302
RN  - 0 (Lipid Bilayers)
SB  - IM
MH  - Diffusion
MH  - Lipid Bilayers/*chemistry
MH  - Microscopy, Fluorescence/*methods
MH  - Spectrometry, Fluorescence/*methods
PMC - PMC4641872
OTO - NOTNLM
OT  - Fluorescence correlation spectroscopy
OT  - Lipid diffusion
OT  - Plasma membrane dynamics
OT  - Super-resolution STED microscopy
OT  - Time-gated single-photon counting
EDAT- 2015/07/01 06:00
MHDA- 2016/08/16 06:00
PMCR- 2015/10/15
CRDT- 2015/07/01 06:00
PHST- 2015/03/11 00:00 [received]
PHST- 2015/05/29 00:00 [revised]
PHST- 2015/06/24 00:00 [accepted]
PHST- 2015/07/01 06:00 [entrez]
PHST- 2015/07/01 06:00 [pubmed]
PHST- 2016/08/16 06:00 [medline]
PHST- 2015/10/15 00:00 [pmc-release]
AID - S1046-2023(15)30005-0 [pii]
AID - 10.1016/j.ymeth.2015.06.017 [doi]
PST - ppublish
SO  - Methods. 2015 Oct 15;88:67-75. doi: 10.1016/j.ymeth.2015.06.017. Epub 2015 Jun 
      27.