Abstract
The advent of high-speed atomic force microscopy (HS-AFM) over the recent years has opened up new horizons for the study of structure, function and dynamics of biological molecules. HS-AFM is capable of 1000 times faster imaging than conventional AFM. This circumstance uniquely enables the observation of the dynamics of all the molecules present in the imaging area. Over the last 10years, the HS-AFM has gone from a prototype-state technology that only a few labs in the world had access to (including ours) to an established commercialized technology that is present in tens of labs around the world. In this protocol chapter we share with the readers our practical know-how on high resolution HS-AFM imaging.
Highlights
The atomic force microscopy (AFM) [1] is a powerful tool for direct visualization of biological samples in an aqueous solution with sub-molecular resolution
AFM has rapidly emerged as an effective structural analysis tool, complementing atomic structure data acquired by other techniques such as X-ray crystallography, NMR and electron microscopy
The operating principle of the high-speed atomic force microscopy (HS-AFM) is based on the miniaturization of the moving components of the AFM to increase their velocity by 1000 times, in the interest of achieving reaction speeds of tens of microseconds
Summary
The atomic force microscopy (AFM) [1] is a powerful tool for direct visualization of biological samples in an aqueous solution with sub-molecular resolution. It allows non-invasive imaging in physiological conditions of unlabeled biological samples, such as nucleic acids [2–4] and cell membrane proteins [5, 6]. We describe in detail our HS-AFM imaging procedure for high resolution and high speed imaging, applicable to any version of the Ando-type HS-AFM system “SS-NEX” Such procedure is based on our experience imaging individual molecules [10, 16] or big macromolecular assemblies [17, 18]. Glass-rod (small glass cylinder of 1.5 mm in diameter and 2 mm in height purchased from RIBM, Japan or SCHOTT, Switzerland), colorless nail polish (nitrocellulose dissolved in butyl acetate or ethyl acetate), mica sheet, puncher purchased from RIBM, cyanoacrylate glue (Super Glue®) or two components epoxy adhesive (Araldite®), adhesive tape (Scotch®), cellulose wipers (Kimwipes®), humid hood, microfuge tube of 1 ml
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