The atomic force microscope (AFM) is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years, but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. The advantages of both methods may be exploited by combining and synchronizing them. In this paper, the biological applications of AFM, fluorescence, and their combinations are briefly reviewed, and the assembly and utilization of a spatially and temporally synchronized AFM and total internal reflection fluorescence microscope are described. The application of the method is demonstrated on a fluorescently labeled cell culture.