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 pN͞nm), 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 pixel 2 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.jp͞phys͞biophys͞bmvmovie.htm). One of the advantages of the atomic force microscope (AFM) (1) is its capacity to image individual biomolecules in, say, a buffered solution containing ions at physiological concentrations (2, 3). Such capacity suggests that the instrument can be used to record the dynamic behavior of such molecules. In practice, however, only very slow processes can be recorded (2, 4-6), because commercially available AFMs require minutes to form an acceptable image, and many interesting biological processes occur at much higher rates. To understand, and overcome, the factors that limit the scanning rate of an AFM, we begin by considering relations between the characteristics of the constituting components.We consider only the ''tapping mode'' of AFM operation (Digital Instruments, Santa Barbara, CA). This is the mode suitable for imaging biological macromolecules, because vertical oscillation of the cantilever at (or near to) its resonance frequency reduces lateral forces between the tip and the sample (7). The oscillating tip briefly taps the surface at the bottom of each swing, resulting in a decrease in oscillation amplitude. During the x-y scan of the sample stage a feedback loop (see below) keeps this decrease (and hence the tapping force) constant; this is necessary for minimizing the deformation of soft samples. The error signal-the difference between a preset signal and the rms amplitude of the cantilever-is fed into a proportional-integraldifferential (PID) feedback circuit. The PID output is amplified and then sent to the z-piezo actuator; this is repeated until the error signal returns to zero. For the three-dimentional movement of the sample stage to follow the sample topography accurately, the bandwidth of the feedback loop should be comparable to, or larger than, the frequency determined by the x-y scan velocity and the apparent width of the features on the surface. To increase the imaging bandwidth, all elements in the feedback loop have to be optimi...

Visualization of single actin filaments by fluorescence microscopy led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level. The ability to manipulate actin filaments with a microneedle or an optical trap combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosins. To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to previously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface, our method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of many types of enzymes and biomolecules.

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The rotation of the ␥-subunit has been included in the binding-change mechanism of ATP synthesis͞ hydrolysis by the proton ATP synthase (F O F 1 ). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A f luorescently labeled actin filament connected to the ␥-subunit of the F 1 sector rotated on addition of ATP.This progress enabled us to analyze the ␥M23K (the ␥-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F 1 sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the ␥M23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.