Immobilizing and imaging microtubules by atomic force microscopy

Microtubules isolated from pig brains have been immobilized on an inorganic substrate for use in AFM studies. The method employs 4-aminobutyldimethylmethoxysilane and glutaraldehyde to activate a silicon wafer for binding the biopolymer. The covalent bond ensures the positional stability of the tubules on the substrate, and allows reproducible scanning probe experiments. Microtubules have been imaged both by atomic force and scanning tunneling microscopy, yielding results very similar to electron microscopy. The average apparent height of the tubules is smaller than observed with transmission electron microscopy (25 nm) and is smaller in buffer solution (10 nm) than in air (15 nm). The biopolymer surface is softer under buffer than in air. The highest resolution was obtained with the tapping mode where surface features as small as 10 nm in X and Y have been resolved. Gold-coated tubules bound on silicon have been successfully imaged by STM, while images of uncertain origin were generated for tubules deposited on graphite. It is shown that artefacts imaged on a blank graphite surface can easily be confounded with collapsed tubules.