Abstract

Local probe techniques (LPTs) extend our sense of touching into the micro- and nano-world and in this way provide complementary new insight into the world we see with other microscopic techniques. Touching things is an essential prerequisite to manipulate them and the ability to feel and manipulate single molecules or atoms for sure marks another revolutionizing step in science. Experiments at the nanometer scale provide a complete new view on molecular processes hidden before in ensemble averages. As never all components of an ensemble can be forced into the same state, information about the time behavior of the individual components is lost. With LPTs we have a powerful tool to study functional cycles of single molecules and to get new insight into their structure and function relation. Especially one of these LPTs, the atomic force microscope (AFM) in its force spectroscopy mode, has meanwhile become a widely used tool to study mechanical properties of single molecules. With a further improved AFM we could elucidate the structural basis of the elastic properties of spectrin repeats. Three-dimensional analysis of thermal position fluctuations also can reveal mechanical properties of single molecular structures. We developed a new local probe instrument, the photonic force microscope (PFM), to make this possible. Thermally excited position fluctuations of an optically trapped bead tethered to a microtubule or actin filament by a single molecular motor can be recorded. The position fluctuations transform into three-dimensional energy profiles using Boltzmann’s equation, with a resolution of one tenth of the thermal energy. From such profiles, force versus extension or stiffness versus extension profiles can be calculated along arbitrary paths in three dimensions. The changes in these profiles within the functional cycle of the molecular motor provide new insight how thermal and chemical energies drive its movement.