High-Speed Atomic Force Microscopy: Integration with Optical Microscopy and High-Speed Force Spectroscopy

Abstract

High-speed atomic force microscopy (HS-AFM, [1]) offers unique novel possibilities to study single molecule dynamics [2,3]. Here we present two HS-AFM developments, first the integration of optical microscopy into HS-AFM for imaging membrane proteins on cells, and second high-speed force spectroscopy (HS-FS) for fast protein unfolding studies:HS-AFM is a powerful tool for studying structure and dynamics of proteins. So far, however, HS-AFM was restricted to well-controlled molecular systems. Here, we integrate optical microscopy (OM) into HS-AFM, allowing bright field and fluorescence microscopy, without loss of HS-AFM performance. This hybrid HS-AFM/OM setup [4] allows positioning the HS-AFM tip on an optically identified zone of interest on cells. We present movies at 960ms per frame displaying aquaporin-0 array and single molecule dynamics on intact eye lens cells. This hybrid setup allows HS-AFM imaging on cells ∼1000 times faster than conventional AFM/OM setups, and first time visualization of unlabeled membrane proteins on a eukaryotic cell under physiological conditions.The mechanical unfolding of muscle protein titin by AFM is a landmark experiment in single molecule biophysics. Molecular dynamics simulations offered an atomic level mechanistic description of the process. However, experiment and simulation differ in pulling velocity by orders of magnitude. We have developed HS-FS [5] to unfold titin at velocities reached by simulation (∼4 mm/s). Our results show that an intermediate state in a small βeta-strand pair dynamically unfolds and refolds, buffering pulling forces up to ∼100pN. Furthermore, our data indicates that the distance to the transition state of domain unfolding is much larger than previously estimated, but in better agreement with atomistic predictions.[1] Ando, et al., PNAS,2001,98(22):12468-12472.[2] Kodera, et al., Nature,2010,468(7320):72-76.[3] Casuso, et al., Nature Nanotechnology,2012,7(8):525-529.[4] Colom, et al., Nature Communications,2013,4:DOI:10.1038/ncomms3155.[5] Rico, et al., submitted,2013.