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The atomic force microscope (AFM) and optical tweezers are tools that allow single biomolecules to be imaged and manipulated. Progress in instrumentation, sample preparation, and image acquisition conditions make novel applications of these tools possible. Biological membranes can be imaged in their native state at a lateral resolution of 0.4–1 nm and a vertical resolution of 0.1–0.2 nm. Function-related conformational changes are resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to observe single proteins directly allows the interaction of proteins forming functional assemblies to be assessed. Single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities to analyze intra- and intermolecular forces. Optical tweezers expand the range of measurable forces to those produced by molecular motors. Combined with fluorescence measurements, optical tools give insights into fundamental biological processes such as the molecular conversion of chemical into mechanical energy.