SGER - Atomic Force Microscopy System with Single Molecule Fluorescence Capabilites

Arizona State University, Scottsdale AZ

Investigators

Abstract

Single molecule studies can provide unique information on the structure and dynamics of biological molecules and cellular complexes that is impossible to obtain using traditional structural techniques. These studies, to be performed at conditions close to physiological environments, open the way for looking directly at the function of individual biological molecules and their complexes. Scanning probe microscopy and atomic force microscope (AFM) in particular has proven to be a very useful technique permitting static and dynamic studies of molecules at nanometer range resolution. However, the requirement for the sample to be bound to the surface restricts considerably the dynamical studies. A breakthrough has recently been made in the real-time observation of dynamics of molecular complexes at the single-molecule level using single-photon sensitive fluorescence microscopes. However, the resolution of this technique is limited by the wavelength of the light and does not reveal structural details of molecules. The combination of AFM and a single molecule fluorescence (SMF) microscope into one instrument would enable us to identify structural details of biological systems at the molecular level by AFM and to use the SMF microscope to observe directly the dynamics of an experimental system at a the single molecule level. Importantly, the dynamics can be performed on molecular systems structurally characterized by AFM and localized on a comparatively large surface area. Therefore, the major objective of this proposal is to build AFM/SMF system based on the currently available Bioscope AFM/fluorescence microscope instrument. The sensitivity of the fluorescent system will be tested using specially designed samples. In addition, potential pitfalls of integrated AFM will be identified and analyzed for solutions.