Visualization of Hydrophobic-, Hydrophilic-, and Charged Areas of Protein by Photochromic Cantilevers.

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Atomic force microscope (AFM) was first invented as an imaging tool for nanometer worlds. However, it can be used also as a high-sensitive force sensor, which allows us to obtain a 2D map of forces exerted between a cantilever probe and sample surface. Adhesive, repulsive, rupture, or non-contact forces between an AFM probe and sample can tell us physicochemical properties of the sample such as hydrophobicity, hydrophilicity and electric charges, depending on the surface property of the probe used. To specify the physicochemical property of sample clearly it is best to scan the sample with probes having different properties. Yet, it is quite difficult to scan the same area of sample after changing cantilevers. This has been a main reason why AFM has not been successfully used for mapping property of protein surface. To detour this problem we developed an alternative method. Instead of changing cantilevers, we change the surface property of a photochromic probe by irradiating near-UV li … More ght. We synthesized a photochromic dye, vinyl malachite green (VMG) whose structure can change from hydrophobic to charged form on irradiation. This dye was attached to the tip of cantilever probes covalently. We also invented a scanning method to map rupture forces within a time shorter than an ordinary force-curve method. Although it was shorter, it still took 30 min, resulting in suffering from disturbance by mechanical and electrical drifts of the AFM.After confirming the usefulness of the photochromic probe in identifying physicochemical property of sample, we invented an additional scanning method to obtain non-contact force map and topography simultaneously. This method shortened the scan time to 5 min (although it is not short enough yet). The positive charges on a basic protein, lysozyme, was successfully visualized by this method. Parallel to these studies, we have developed a high-speed AFM.It can ultimately solve the problem that the observation of force map requires much longer time than topography. The frame rate for topography observation reached 2.5 frames/sec, about 250-times faster than ordinary AFM apparatus. Although we have not tried to use this new AFM for force mapping, its speed promises a few seconds of force mapping, and therefore in the near future we may be able to obtain much better resolution of force map on protein surfaces. Less