Particle Image Cross Correlation Spectroscopy (PICCS)

Abstract

In vivo studies of the dynamics of single molecules and particles have produced a wealth of biological insights. The diffusion behavior of a membrane receptor, for example, reveals the structure of the plasma membrane. In recent years we have developed an analysis technique to quantify single molecule translational movement on a nm length and ms time scale (Particle Image Correlation Spectroscopy (PICS), Semrau, Schmidt., Biophys. J., 2007).The insight gained from experiments on a single molecular species is however limited. No biomolecule operates on its own and often it is the very interaction between different types of molecules which is biologically most relevant. To that end we further developed PICS for experiments with two differently labeled molecular species. Particle Image Cross-Correlation Spectroscopy (PICCS) allows us to unambiguously measure molecule colocalization, even at large molecule densities and down to a length scale of 10 nm. To demonstrate the method's power we studied the intracellular transport of the morphogen Dpp enclosed in endosomes. Dpp establishes a gradient in the wing imaginal disk of fruit fly larvae, providing positional information to cells. Using PICCS we found that 52% of apical Dpp is in early endosomes and that early endosomes contain 1.9 times as much Dpp as other endosomes. Our data suggests that Dpp resides shorter in early endosomes compared to late/recycling endosomes.PICCS makes it also possible to push the limits of the time scales on which molecular movement can be measured. By labeling one molecule with two spectrally resolvable fluorophores we can follow the dynamics of the molecule on a 100 μs time scale.To summarize, PICCS opens up a whole new range of in vivo single molecule experiments: Molecular correlations and dynamics can be measured with unprecedented accuracy and temporal resolution.