In-vivo dynamics of chromatin and DNA-binding proteins studied by light sheet fluctuation microscopy

Proteins acting on DNA need to penetrate a dense network of chromatin and associated macromolecules in the cell nucleus to access their target sites. Intracellular mobility of proteins is characterized by diffusion coefficients of the order of 1-100 μm2/s, leading to millisecond time scales for movement on the submicrometer scale.
Typical microscopic methods used for characterizing intracellular protein mobility are, e.g., fluorescence photobleaching recovery (FRAP) and fluorescence correlation spectroscopy (FCS). Of these, FRAP can image protein mobility in entire two-dimensional sections of live cells, but is typically limited to the time resolution of confocal image series, some frames per second. FCS, on the other hand, has fast time resolution but so far has been limited to single-point measurements in the focus of a laser beam, or to techniques that utilize the inherent time structure of confocal scans. In my seminar I will show results from single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS), a new method that combines the fast time resolution of FCS with the ac- quisition of mobility data in parallel on an entire two-dimensional cross-section. This provides diffusion coefficients, flow velocities, concentrations and interactions as imaging parameters.