A Precision Scanning Probe Microscope with Direct Access to the Three Dimensional Tip-Sample Interaction Force Vector

Abstract

Single-molecule imaging and force spectroscopy studies have produced rich insights into macromolecular structure and dynamics in biological settings and the atomic force microscope (AFM) has thus emerged as an important complementary apparatus in the structural biologist's toolkit. While conventional AFM has achieved sub-nm resolution imaging of membrane proteins in native membrane, atomic-scale tip-sample stability - which opens promising new avenues of AFM study - has previously only been achieved in highly isolated non-biological environments (i.e., cryogenic temperatures, ultra-high vacuum). We adapted techniques originally developed by the optical trapping microscopy community, and have constructed an ultra-stable AFM in which the tip and the sample positions are independently measured by, and stabilized with respect to, a pair of laser foci in three dimensions. Recently, we have extended ultra-stable AFM to common biological imaging conditions (tapping mode in aqueous buffer solution) and have exploited local observation of the three dimensional (3D) tip trajectories to yield 3D interaction force components in a direct manner. In this talk I will discuss these developments in the context of addressing central questions in membrane biophysics.