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.