Simultaneous single molecule atomic force and fluorescence lifetime imaging

Olaf Schulz, Felix Koberling, Deron Walters, Marcelle Koenig, Jacob Viani, Robert Ros

Research output: Chapter in Book/Report/Conference proceedingConference contribution

10 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationSingle Molecule Spectroscopy and Imaging III
DOIs
StatePublished - May 6 2010
EventSingle Molecule Spectroscopy and Imaging III - San Francisco, CA, United States
Duration: Jan 23 2010Jan 25 2010

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume7571
ISSN (Print)1605-7422

Other

OtherSingle Molecule Spectroscopy and Imaging III
CountryUnited States
CitySan Francisco, CA
Period1/23/101/25/10

Keywords

  • AFM
  • Confocal
  • DNA
  • FLIM
  • Quenching

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

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  • Cite this

    Schulz, O., Koberling, F., Walters, D., Koenig, M., Viani, J., & Ros, R. (2010). Simultaneous single molecule atomic force and fluorescence lifetime imaging. In Single Molecule Spectroscopy and Imaging III [757109] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 7571). https://doi.org/10.1117/12.842502