Single molecule microscopy

Our group is interested in detecting and tracking of single receptor molecules within the plasma membrane of living cells. Single molecule microscopy provides a deep insight into individual molecule paths by identifying single signals and tracing them time-dependently with a high temporal and spatial resolution.
The microscope setup consists of an inverted wide-field epi-fluorescence microscope. For excitation of the fluorescently labeled single molecules, a diode-pumped solid-state laser is used. The signals are detected by an EMCCD camera system, which facilitates a very high temporal resolution and a low signal-to-noise-ratio. A high spatial resolution can be achieved by localizing the single molecules by approximation of a two-dimensional Gaussian function to the intensity profile of each particle. Afterwards, the localized particles can be connected to trajectories.
In that way, single molecule trajectories can be captured and the two-dimensional diffusion characteristics can be analyzed. Evaluating the frequency distribution of the jump distances and the trajectory length, conclusions can be drawn concerning the correlation between receptor state (activated/non-activated) and the diffusion behaviour of the receptor. Furthermore, information about the membrane organization in respect of the regarding protein type can be gathered.