Akitoshi Toda

A high-speed atomic force microscope for studying biological macromolecules

The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450–650 kHz) and small spring constants (150–280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 × 100 pixel2 image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm).

A High-Speed Atomic Force Microscope for Studying Biological Macromolecules in Action

The atomic force microscope (AFM) is a powerful tool for imaging biological molecules on a substrate, in solution. However, there is no effective time axis with AFM; commercially available AFMs require minutes to capture an image, but many interesting biological processes occur at a much higher rate. Hence, what we can observe using the AFM is limited to stationary molecules, or those moving very slowly. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed various devices optimized for high-speed scanning. Combining these devices has produced an AFM that can capture a 100×100 pixel image within 80 ms, thus generating a movie consisting of many successive images of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica, in solution.

Batch Fabrication of Sharpened Silicon Nitride Tips

A novel sharpening technique for silicon nitride protrusions is reported. A silicon-rich nitride film, which contains more silicon than a stoichiometric silicon nitride film, shows low film stress and has been applied to micro mechanical devices. We found that the edge of the silicon nitride pattern on the silicon wafer became thinner and tapered by thermal oxidation, followed by the removal of the oxidized layer. By applying this technique for sharpening the apex of a beak-like cantilever of silicon nitride, the tip of the acute-angle triangular plate portion of the cantilever terminated into one and was sharpened with a radius of curvature of 17 nm. The small cantilevers integrated with the sharp protrusions for high speed atomic force microscopy in water were batch fabricated. The technique is useful for the fabrication of small cantilevers when low cost is required.

Power-Law Stress and Creep Relaxations of Single Cells Measured by Colloidal Probe Atomic Force Microscopy

We measured stress and creep relaxations of mouse fibroblast cells arranged and cultured on a microarray, by colloidal probe atomic force microscopy (AFM). A hydrophobic monolayer coating of perfluorodecyltrichlorosilane (FDTS) on the surface of colloidal silica beads significantly reduced the adhesion force of live cells, compared with untreated beads. The rheological behaviors of cells were estimated by averaging several relaxation curves of cells measured by the AFM. Longer-time tailing of both stress and creep relaxation curves followed single power-law behavior over a time scale of 60 s, with exponents in the range 0.1–0.4, varying with cells. The results were in good agreement with previous measurements of the frequency-domain rheology of cells using the force modulation mode.

Fabrication of Sharp Tetrahedral Probes with Platinum Coating

Metal-deposited probes with a 13 nm tip radius for conducting scanning probe microscopy (SPM) are obtained by sputter-coating a silicon cantilever with platinum on a single side. By optimizing the metal layered structure and the film stress, the silicon cantilever, whose spring constant and resonant frequency are 2 N/m and 70 kHz, respectively, is freestanding without bending. Besides the cantilever bending, curling of the probe apex was monitored and minimized in the series of the experiments conducted. The relatively soft probe apex was another part of the cantilever chip that was deformed by the metal film stress because the basic silicon tetrahedral probe was thin and inclined against the normal direction of the cantilever. In the final process, aluminum was deposited as a reflex coating on the back side of the silicon cantilever by vacuum evaporation because the light-reflectance was twice as high as that for platinum coating. The sharp probe apex, good conductivity of precious-metals and high reflection contribute to high-resolution conduction SPM measurements.

Silicon Tip Cantilevers for Force Microscopy in Water with Resonance of 20 kHz or Above

Silicon nitride cantilevers with a silicon probe have been developed for force microscopy of soft samples in water. The cantilevers were soft with a small spring constant of from 0.05 to 0.11 N/m and some showed resonance of 20 kHz or higher in water. The typical dimensions of a cantilever were 40 µm ×15 µm ×0.19 µm. Autofluorescent light emissions from the resulting cantilever and probe were studied. Autofluorescence of the silicon nitride cantilever was smaller than that of a conventional silicon nitride cantilever. Autofluorescence of the silicon probe was small and of negligible intensity for fluorescence optical microscopy. The cantilever probe less disturbs fluorescence optical microscopy than conventional soft cantilever probes.

Oxide‐sharpened Silicon Nitride Cantilever Probe

A small cantilever probe for high speed AFM imaging in water has been developed. It is a rectangular cantilever, so‐called beak‐like cantilever, made of silicon‐nitride with dimensions of about 10 μm × 2 μm × 0.13 μm. By applying oxide‐sharpening technique for silicon nitride probe apexes, the triangular apexes that were shaped by photo‐lithography, were further sharpened and terminated into one point. An average radius of curvature of 18.4 nm was achieved. This novel sharpening technique is useful for the fabrication of small cantilevers when low cost is required. The typical resonant frequency of the small cantilevers was 1.4 MHz in air and the estimated spring constant was 0.2 N/m. Immersing the cantilevers in water, the resonant frequency dropped by a factor of three to five.