Kazunari MOURI

RIKEN Center for Biosystems Dynamics Research

Development of imaging and image processing techniques for three-dimensional single molecule tracking

Enzyme reactions in cells have been measured invasively, or recently measured by FRET probes in living cells. However, it is difficult to estimate the concentration of enzymes or substrates in cells, and semi-quantitative analyses have been done on these experiments. In our study, we developed a new FCS method with a conventional confocal microscope. We carefully selected the scanning conditions and applied signal processing methods, and succeeded in getting the time-series data of the number of photons at a detector, which is comparable to those with FCS. We quantified physical parameters, such as the diffusion coefficient and the concentration of proteins using some image processing techniques. Applying this, we can perform biochemical experiments in living cells. For example, we applied this method to MAPK signal transduction processes, and estimated the enzyme reaction rate constants of nuclear pore proteins which transport ERK proteins from cytoplasm to nucleoplasm. Furthermore, we are developing a 3D single molecule imaging system and image processing methods for it. Our goal is to achieve quantitative analyses of single molecule enzyme reaction steps.