Scanning Tunneling Microscopy and low-temperature force microscopy of freeze-fractured samples

Abstract

Monolayer freeze-fracture combined with scanned probe microscopy (SPM) offers unique advantages for studies of biological structure. The freeze-fracture methodology incorporates rapid freezing approaches for sample preservation and stabilization, and the scanned probe microscopies, especially scanning tunneling microscopy (STM) and atomic force microscopy (AFM), allow high resolution examination of surface features including digital mapping, quantification, and display. In routine biological STM a sharp conductive probe is positioned with piezoelectric transducers close to a conductive surface. When electron tunneling begins, the probe is scanned while electronic feedback maintains constant current. Because the tunneling current is logarithmically sensitive to separation between the tip and the sample, the feedback signal can be calibrated to indicate height with sub-Angstrom sensitivity and precision. In routine biological AFM, the sample is scanned while the force between the tip and the sample is kept constant.There are two fundamental problems in STM examinations of biological systems. First, biological samples are soft and are often perturbed by the scanning probe; and second, they are not electrically conductive. Coating samples with metal replicas simultaneously circumvents both these difficulties, conferring sample stability and electrical conductivity. The STM can be used to measure sample heights quite accurately, and has been used to measure the thickness and changes in thickness of metal-coated purple membrane and the depth of surface features of freeze-fracture replicas of synthetic phospholipids.