Scanning tunneling microscopy of biopolymers: Conditions for microtubule stabilization

Applications of scanning tunneling microscopy (STM) to biological materials have been limited by poor conductivity and instability of biomolecules. We have used STM to probe three different biopolymer materials in air on graphite surfaces: collagen protein, phospholipid membranes, and microtubule protein assemblies. In the case of microtubules, we evaluated parameters for fixation and stabilization for STM imaging and found conditions which resulted in stable, reproducible images. STM of collagen showed linear strands ~ 1.5 nanometers (nm) in diam. Periodicity of ~ 3 nm was accentuated by “spikes” which appear to match pyrrolidine ring structures in amino acids proline and hydroxyproline. STM of Langmuir diphosphatidyl ethanolamine phospholipid membranes showed a lattice periodicity (0.4 to 0.5 nm) which appears to match the spacing of phospholipid molecules in membranes. Preparation of microtubules for STM which consistently resulted in reproducible and stable images includes a specific salt and magnesium containing buffer, glutaraldehyde fixation and 0.8 M glycerol. Freeze drying was also utilized, however stable images were also obtained without freeze drying. STM imaging of microtubules in several magnifications showed linear structures of appropriate (≈ 25 nm) width comprised of 4 nm wide protofilaments. Microtubules often appeared “buckled” and flattened and shaded images (not shown) demonstrated individual “tubulin” subunits. Evidence of tip interaction with the biopolymers was seen with collagen and phospholipid membranes. We feel STM and related technologies have great promise for the study of biomolecular surfaces.