Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED-FCS.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
04 2019
04 2019
Historique:
received:
18
09
2018
accepted:
04
01
2019
pubmed:
8
3
2019
medline:
17
4
2019
entrez:
8
3
2019
Statut:
ppublish
Résumé
Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.
Identifiants
pubmed: 30842616
doi: 10.1038/s41596-019-0127-9
pii: 10.1038/s41596-019-0127-9
doi:
Substances chimiques
Fluorescent Dyes
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1054-1083Subventions
Organisme : Medical Research Council
ID : MR/S005382/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/K01577X/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_PC_15060
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UU_00008/9
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_PC_16082
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UU_12010/9
Pays : United Kingdom
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