Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

Per Niklas Hedde, René M. Dörlich, Rosmarie Blomley, Dietmar Gradl, Emmanuel Oppong, Andrew C.B. Cato and G. Ulrich Nienhaus ()
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Per Niklas Hedde: Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology
René M. Dörlich: Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology
Rosmarie Blomley: Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology
Dietmar Gradl: Zoological Institute, Karlsruhe Institute of Technology
Emmanuel Oppong: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology
Andrew C.B. Cato: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology
G. Ulrich Nienhaus: Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology

Nature Communications, 2013, vol. 4, issue 1, 1-8

Abstract: Abstract Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor–ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from raster-scanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10–100 times higher fluorophore concentrations.

Date: 2013
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DOI: 10.1038/ncomms3093

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