Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging.

Nano Letters
Falk SchneiderChristian Eggeling

Abstract

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED-FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method, line interleaved excitation scanning STED-FCS (LIESS-FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with s...Continue Reading

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Citations

Mar 8, 2019·Nature Protocols·Erdinc SezginChristian Eggeling
May 4, 2020·Biophysical Journal·Aurélien BarbotinErdinc Sezgin
Apr 17, 2021·Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy·Shawn Yoshida, Lydia Kisley
Aug 7, 2021·Biosensors·Madhurima ChattopadhyayLukasz Piatkowski

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Methods Mentioned

BETA
fluorescence correlation spectroscopy
FCS
transfection

Software Mentioned

scan
FCS
Matlab
sSTED
FoCuS
Imspector
LIESS

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