Photonic Force Microscopy: A New Tool Providing New Methods to Study Membranes at the Molecular Level

The PFM is a three-dimensional scanning probe microscope based on optical tweezers. It evolved from AFM techniques by replacing the mechanical cantilever of the AFM with optical tweezers and the cantilever tip by a trapped bead. Various detection systems measure the three-dimensional position of the bead with a spatial and temporal resolution in the nanometer and microsecond range, respectively. Due to the small trapping force constants, thermal position fluctuations of the probe are relatively large in comparison to the thermal motion of an AFM cantilever. However, these position fluctuations provide information about the local environment and specific interactions of the probe with molecules such as membrane proteins. Within the few last years, the instrument turned out to be a powerful tool to study properties of the plasma membrane of intact cells at the nanometer scale. For instance, the diffusion of membrane components could be observed over minutes at high spatial and temporal resolutions. For the first time, the diffusion coefficient measured locally in the plasma membrane of an intact cell agreed well with previous measurements for lipid model membranes, thus providing new ways to characterize membrane structures with unknown properties, such as lipid rafts. Furthermore, the technique can be used to determine the elasticity of the lipid bilayer and the binding properties of membrane components to the cytoskeleton. Introduction The Atomic Force Microscope (AFM) [1] allows to study nonconducting materials under ambient conditions and/or in solution. It was considered to be the ideal tool for physical studies of live biological specimens [2], soon after its invention living cells were imaged [3] and dynamic processes occurring at the cell membrane were examined [4]. However, the rough surface of a cell often prevents the tip of a mechanical cantilever from following fine topographic details. Furthermore, forces of several tens of piconewtons are necessary for stable imaging which can have the detrimental effect of deforming soft cellular structures, such as the plasma membrane. Only stiff structures, such as the cytoskeleton, are visible when imaging, whilst imaging inside cells is impossible due to the mechanical connection to the imaging tip. Therefore, a scanning probe microscope without a mechanical connection to the tip and working with extremely small loading forces is desirable. Such an instrument, the Photonic Force Microscope (PFM), has been developed at the European Molecular Biology Laboratory (EMBL) in Heidelberg during the last few years. Experimental Details