Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED-FCS.

Nature Protocols
Erdinc SezginChristian Eggeling

Abstract

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.

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Citations

Jul 5, 2019·The Journal of Biological Chemistry·Kerstin PinkwartErdinc Sezgin
Mar 13, 2020·APL Bioengineering·Peter J Butler
Apr 5, 2020·Viruses·Pablo CarravillaChristian Eggeling
Sep 13, 2019·Optics Express·Aurélien BarbotinMartin J Booth
Dec 29, 2020·Frontiers in Chemistry·Yousef BagheriMingxu You
Nov 17, 2020·Journal of Physics D: Applied Physics·Falk SchneiderIztok Urbančič
Dec 18, 2020·Frontiers in Cell and Developmental Biology·Hong-Yin WangIlya Levental
May 1, 2021·Cells·Veronika PfannenstillMarco Fritzsche
Aug 7, 2021·Biosensors·Madhurima ChattopadhyayLukasz Piatkowski
Jul 21, 2020·ACS Photonics·Aurélien BarbotinMartin Booth
Feb 7, 2020·The Journal of Physical Chemistry. B·Daniel BeckersErdinc Sezgin

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Methods Mentioned

BETA
imaging techniques
fluorescence microscopy
optical microscopy
super-resolution microscopy
FCS
transfections
transfection
transfect
Fluorescence

Software Mentioned

SPT
STED
Leica
point
GPI
Fiji
/ Fiji
pSTED
FoCuS
fcs

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