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Originally published as Biophys J. BioFAST on February 16, 2007.
doi:10.1529/biophysj.107.104497
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Biophysical Journal 92:L67-L69 (2007)
© 2007 The Biophysical Society

Two-Color Far-Field Fluorescence Nanoscopy

Gerald Donnert *, Jan Keller *, Christian A. Wurm *, Silvio O. Rizzoli {dagger}, Volker Westphal *, Andreas Schönle *, Reinhard Jahn {dagger}, Stefan Jakobs *, Christian Eggeling * and Stefan W. Hell *

Max Planck Institute for Biophysical Chemistry, Departments of * NanoBiophotonics and {dagger} Neurobiology, 37070 Göttingen, Germany

Correspondence: Address reprint requests and inquiries to Stefan W. Hell, E-mail: hell{at}4pi.de.

We demonstrate two-color fluorescence microscopy with nanoscale spatial resolution by applying stimulated emission depletion on fluorophores differing in their absorption and emission spectra. Green- and red-emitting fluorophores are selectively excited and quenched using dedicated beam pairs. The stimulated emission depletion beams deliver a lateral resolution of <30 nm and 65 nm for the green and the red color channel, respectively. The ~5 nm alignment accuracy of the two images establishes the precision with which differently labeled proteins are correlated in space. Colocalized nanoscopy is demonstrated with endosomal protein patterns and by resolving nanoclusters of a mitochondrial outer membrane protein, Tom20, in relation with the F1F0ATP synthase. The joint improvement of resolution and colocalization demonstrates the emerging potential of far-field fluorescence nanoscopy to study the spatial organization of macromolecules in cells.




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