High-speed atomic force microscope combined with single-molecule fluorescence microscope.

High-speed atomic force microscopy (HS-AFM) and total internal reflection fluorescence microscopy (TIRFM) have mutually complementary capabilities. Here, we report techniques to combine these microscopy systems so that both microscopy capabilities can be simultaneously used in the full extent. To combine the two systems, we have developed a tip-scan type HS-AFM instrument equipped with a device by which the laser beam from the optical lever detector can track the cantilever motion in the X- and Y-directions. This stand-alone HS-AFM system is mounted on an inverted optical microscope stage with a wide-area scanner. The capability of this combined system is demonstrated by simultaneous HS-AFM∕TIRFM imaging of chitinase A moving on a chitin crystalline fiber and myosin V walking on an actin filament.