Simultaneous single molecule atomic force and fluorescence lifetime imaging

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.

Tip induced fluorescence quenching for nanometer optical and topographical resolution

“…The AFM collects information about the sample topography whereas the optical microscope records fluorescence intensity and fluorescence lifetime while scanning the sample. The alignment of the AFM probe and the microscope objective in combination with the synchronization of the two microscopes allows for the acquisition of temporally and spatially correlated topographical and fluorescence data (Schulz et al, 2010). In the combined measurements, the AFM tip, which is in direct contact with the sample surface, alters the optical response of the sample fluorophores.…”

“…It is worth noting that the topography image and the quenching image were not translated to match each other. This is possible because the two microscopes are synchronized, and the data is sorted into pixels according to the time at which it was acquired (Schulz et al, 2010). Since the tip records the topography at the molecule's position and changes its fluorescence at the same time, these features appear in the same position in both images.…”

“…The setup, synchronization, and alignment have been described in detail (Schulz et al, 2010). Pulsed diode lasers were used for excitation at 640 nm and 470 nm.…”

AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells

“…The combined AFM-CLSM setup we used consists of a sample scanning AFM (MFP-3D Bio, Asylum Research, CA) and a single molecule sensitive confocal fluorescence microscope (Microtime 200, PicoQuant, Germany), equipped with 470 nm and 640 nm lasers for excitation, a high-end 100× 1.45 NA oil immersion objective (Olympus, San Diego CA), and two single photon counting modules for detection [24]. The whole optical setup is built on an inverted microscope (IX71, Olympus), so that it can combine with the AFM scanner and head.…”

“…The ability to move the sample and objective independently allows for precise alignment of the AFM probe and laser focus with an accuracy down to a few nanometers [24]. This enables direct correlation of the point of indentation and the sub-cellular structures in the FLIM image.…”