title: Atomic force microscopy imaging of macromolecular complexes.
abstract: This chapter reviews amplitude modulation (AM) AFM in air and its applications to high-resolution imaging and interpretation of macromolecular complexes. We discuss single DNA molecular imaging and DNA-protein interactions, such as those with topoisomerases and RNA polymerase. We show how relative humidity can have a major influence on resolution and contrast and how it can also affect conformational switching of supercoiled DNA. Four regimes of AFM tip-sample interaction in air are defined and described, and relate to water perturbation and/or intermittent mechanical contact of the tip with either the molecular sample or the surface. Precise control and understanding of the AFM operational parameters is shown to allow the user to switch between these different regimes: an interpretation of the origins of topographical contrast is given for each regime. Perpetual water contact is shown to lead to a high-resolution mode of operation, which we term SASS (small amplitude small set-point) imaging, and which maximizes resolution while greatly decreasing tip and sample wear and any noise due to perturbation of the surface water. Thus, this chapter provides sufficient information to reliably control the AFM in the AM AFM mode of operation in order to image both heterogeneous samples and single macromolecules including complexes, with high resolution and with reproducibility. A brief introduction to AFM, its versatility and applications to biology is also given while providing references to key work and general reviews in the field.
authors.affiliation: Department of Oral Biology, University of Leeds, Leeds, UK.
authors.forename: Sergio
authors.initials: S
authors.lastname: Santos
authors.forename: Daniel
authors.initials: D
authors.lastname: Billingsley
authors.forename: Neil
authors.initials: N
authors.lastname: Thomson
mesh_terms: D000538:Aluminum Silicates; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D006813:Humidity; D057927:Hydrophobic and Hydrophilic Interactions; D046911:Macromolecular Substances / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008858:Microscopy, Phase-Contrast; D057054:Molecular Imaging / Q000379:methods*; D009690:Nucleic Acid Conformation; D011312:Pressure; D014158:Transcription, Genetic; D014867:Water
chemical_list: D000538:Aluminum Silicates; D004278:DNA, Superhelical; D046911:Macromolecular Substances; D014867:Water; C011934:mica