High-speed atomic force microscopy (HS AFM) in 'contact' mode was used to image at video rate the surfaces of both calcium hydroxyapatite samples, often used as artificial dental enamel in such experiments, and polished actual bovine dental enamel in both neutral and acidic aqueous environments. The image in each frame of the video of the sample was a few micrometers square, and the high-speed scan window was panned across the sample in real time to examine larger areas. Conventional AFM images of the same regions of the sample were also recorded before and after high-speed imaging. The ability of HS AFM to follow processes occurring in liquid on the timescale of a few seconds was employed to study the dissolution process of both hydroxyapatite and bovine enamel under acidic conditions. Buffered citric acid at pH values between 3.0 and 4.0 was observed to dissolve the surface layers of these samples. The movies recorded showed rapid dissolution of the bovine enamel in particular, which proceeded until the relatively small amount of acid available had been exhausted. A comparison was made with enamel samples that had been treated in fluoride solution (1 h in 300 ppm NaF, pH 7) prior to addition of the acid; the speed of dissolution for these samples was much less than that of the untreated samples. The HS AFM used an in-house designed and constructed high-speed flexure scan stage employing a push-pull piezo actuator arrangement. The HS AFM is able to follow the large changes in height (on the micrometer scale) that occur during the dissolution process.

Scanning probe microscopy (SPM) is the main technique for both the characterization and to a much lesser extent the generation of nanostructures. SPM is a family of microscopy techniques with atomic force microscopy (AFM), scanning tunneling microscopy (STM), and near-field scanning optical microscopy (NSOM) as the most frequently used of these microscopies. Conventional versions of these three microscopes typically require over one minute and up to 10s of minutes to record one image, depending on the nature of the specimen and image size. Improvements in speed are very important for both imaging and fabrication, and high-speed versions of AFM [1,2], the STM [3], and the NSOM [4] have been demonstrated.

The second new high-speed technique presented here is a non-contact force microscopy. It is recognized that non-contact imaging in liquid is of importance in biomolecular systems in order to cause the least distortion of the sample in an environment that is close to the natural state. Non-contact FM AFM techniques [4] have recently made a major step forward to attain these capabilities. The further challenge of high speed must be met in order to achieve the goal of following many biomolecular processes in situ. In the work presented here, we show a newly developed non-contact transverse dynamic force microscopy (TDFM) technique with the potential for high-speed imaging. TDFM has for many years been known to offer high-resolution imaging on delicate biological specimens in air or liquid in a non-contact mode, and these recent development, reported here, are a proof of concept of its capability as a high-speed imaging technique with the potential to image at video rate and beyond in a non-contact mode. TDFM approach curves show features associated with the ordering of water molecules above the sample. A demonstration of the 3D imaging of the water structure above a sample surface using the new version of TDFM will be presented. The visualization of local water structure in biomolecular systems is a dream for the understanding of structure formation and processes at the molecular level.

High-speed AFM imaging is important for many applications not least in biology where the ability to follow processes occurring in the millisecond regime at the molecular level is a major goal. High-speed imaging also allows greater areas of specimen to be examined in a given time, and it allows the patterning of surfaces over useful areas on a practical timescale for the creation of nanostructures. In recent years, Ando et al. [1] reported major achievements in high-speed tapping mode AFM. Here we will briefly present two alternative AFM techniques for achieving high-speed imaging.

Gas vesicles encoded by gyp genes provide buoyancy in many prokaryotes. In a recent Trends in Microbiology article entitled 'Gas vesicles in actinomycetes: old buoys in novel habitats?' van Keulen et al. documented the occurrence of gyp genes in soil-inhabiting actinomycetes but questioned whether any of them produce gas vesicles. We suggest that the protein encoded by gvpA in actinomycetes might be incompatible with the structure of the standard gas vesicle. Perhaps it has another role associated with the air-water interface.

The gas vesicles of the cyanobacterium Anabaena flos-aquae contain two main proteins: GvpA, which forms the ribs of the hollow cylindrical shell, and GvpC, which occurs on the outer surface. Analysis by MALDI-TOF MS shows that after incubating Anabaena gas vesicles in trypsin, GvpA was cleaved only at sites near the N-terminus, whereas GvpC was cleaved at most of its potential tryptic sites. Many of the resulting tryptic peptides from GvpC remained attached to the underlying GvpA shell: the pattern of attachment indicated that there are binding sites to GvpA at both ends of the 33-residue repeats (33RRs) in GvpC, although one of the tryptic peptides within the 33RR did not remain attached. Tryptic peptides near the two ends of the GvpC molecule were also lost. The mean critical collapse pressure of Anabaena gas vesicles decreased from 0.63 MPa to 0.20 MPa when GvpC was removed with urea or fully digested with trypsin; partial digestion resulted in partial decrease in critical pressure.