Simultaneous single molecule atomic force and fluorescence lifetime imaging

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.