Nanoscope 3 afm

Freeze etching of early enamel was reported by Robinson et al. (1981 (link)). Briefly, early enamel was carefully frozen in liquid nitrogen (−198°C) and fractured under vacuum using a histological knife. The knife was then repositioned over the fractured surface and its temperature lowered to sublime ice from the tissue on to the knife blade. This left a fractured tissue surface unencumbered by ice. The fractured frozen surface was then shadowed, under vacuum, with gold or aluminum. Tissue was dissolved away and the metal replica examined using TEM.
AFM was carried out as described previously (Kirkham et al., 2001 (link); Robinson et al., 2004 (link)) using a Nanoscope III AFM (Digital Instruments) equipped with a 16E16-μm scanner and 25 μm silicon nitride cantilevers. Images were obtained in oscillating mode at 0.2 Hz below resonance with drive amplitudes in the range 300–950 mV. Measurements of crystal width and height were made using the software provided.

Sso10a-DNA complexes were formed by incubating 100 ng of nicked pRD24 with varying amounts of Sso10a proteins in 10 μl AFM buffer [40 mM Hepes (pH 7.5) and 25 mM NaCl] for 10 min at room temperature (~23 °C). After incubation, this mixture was diluted 10-fold in 1 mM MgCl₂ and directly deposited onto freshly cleaved mica. After 40 s, the mica disc was rinsed with HPLC water and dried with nitrogen gas. The AFM images were collected on a NanoScopeIII AFM (Digital Instruments, Santa Barbara, CA) using micro cantilevers (Olympus MCL-AC240TS-W2, resonance frequency 70 kHz, spring constant 2 N/m). Images were obtained using tapping mode in air at 2 Hz and flattened using Nanoscope software (Veeco Instruments).