Imaging two-dimensional arrays of soluble proteins by atomic force microscopy in contact mode using a sharp supertip

A sharp tip with high aspect ratio is required for imaging biological macromolecules by atomic force microscopy (AFM). A tip with the end radius of curvature less than 3 nm has been reproducibly fabricated by means of electron beam deposition (EBD) in a field-emission scanning electron microscope. Two-dimensional protein arrays of ferritin and catalase, prepared at air/water interface and transferred onto silicon wafer, could be imaged both in air and in water by AFM using this sharp EBD-tip in contact mode. The negative staining preparation conventionally used in the transmission electron microscopy of protein was applied and shown to be quite effective in fixing the protein arrays for the AFM imaging in air. Individual molecules of ferritin and catalase were visible in the two-dimensional arrays. Also, imaging in water of these protein arrays presented molecular images clearer than in air, due probably to the absence of the adhesion force and the resulting weak lateral force during scanning. These images convince us of the capability of this supertip for AFM studies of biological molecules under aqueous conditions.