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Biophys. J. BioFAST: First Published February 16, 2007. doi:10.1529/biophysj.107.104497
© 2007 by the Biophysical Society.

A more recent version of this article appeared on April 15, 2007.
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BIOPHYSICAL LETTERS

Two-Color Far-field Fluorescence Nanoscopy

Gerald Donnert 1, Jan Keller 1, Christian Wurm 1, Silvio Rizzoli 1, Volker Westphal 1, Andreas Schoenle 1, Reinhard Jahn 1, Stefan Jakobs 1, Christian Eggeling 1 and Stefan W. Hell 1*

1 MPI BPC

* To whom correspondence should be addressed. E-mail: shell{at}gwdg.de.

Submitted on January 21, 2007
Revised on February 3, 2007
Accepted on 6 February 2007


  Abstract
We demonstrate two-color fluorescence microscopy with nanoscale spatial resolution by applying stimulated emission depletion (STED) on fluorophores differing in their absorption and emission spectra. Green and red emitting fluorophores are selectively excited and quenched using dedicated beam pairs. The STED 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 proteins 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.

Key Words: endosome, fluorescence, imaging, microscopy, mitochondria, resolution




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Copyright © 2007 by the Biophysical Society.