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Proceedings Paper

Simultaneous single molecule atomic force and fluorescence lifetime imaging
Author(s): Olaf Schulz; Felix Koberling; Deron Walters; Marcelle Koenig; Jacob Viani; Robert Ros
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Paper Abstract

The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single molecule quenching on organic fluorophores.

Paper Details

Date Published: 25 February 2010
PDF: 10 pages
Proc. SPIE 7571, Single Molecule Spectroscopy and Imaging III, 757109 (25 February 2010); doi: 10.1117/12.842502
Show Author Affiliations
Olaf Schulz, Arizona State Univ. (United States)
Felix Koberling, PicoQuant GmbH (Germany)
Deron Walters, Asylum Research (United States)
Marcelle Koenig, PicoQuant GmbH (Germany)
Jacob Viani, Asylum Research (United States)
Robert Ros, Arizona State Univ. (United States)

Published in SPIE Proceedings Vol. 7571:
Single Molecule Spectroscopy and Imaging III
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)

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