Dresden 2009 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 20: DNA, RNA and Chromatin
BP 20.3: Vortrag
Donnerstag, 26. März 2009, 14:45–15:00, HÜL 186
Images of Intracellular Kinetics Reveal Accellerated DNA Hybridization — •Ingmar Schön and Dieter Braun — Systems Biophysics, LMU München, Germany
Molecular crowding affects the diffusion properties and the free energies of molecules in densely packed environments. Its impact on reaction kinetics in the relevant context of living cells is still elusive, mainly due to the difficulty of capturing fast kinetics in vivo. In this talk, we show spatially resolved measurements of DNA hybridization kinetics in single living cells. HeLa cells were transfected with a FRET labeled dsDNA probe by lipofection. We characterize the reaction kinetics at each image pixel with a kinetic range of 10−5… 100s by combining laser-driven temperature oscillations, stroboscopic illumination, fluorescence imaging, and frequency-based relaxation analysis. Within individual cells and between different cells, the time constant of the reaction varied according to different DNA concentrations. A quantitative analysis of the concentration dependence revealed that the association rate was considerably enhanced compared to free solution, likely due to molecular crowding effects inside the cell. The imaging modality of our technique facilitates the parallel measurement of different cellular compartments such as the cytoplasm, the nucleoplasm or even the nucleoli. In general, our technique which we call TOOL (Temperature Oscillation Optical Lock-in) microscopy opens up the possibility to map cellular differences in the reaction environment on the micrometer scale and provides quantitative data about intermolecular kinetics for systems biology.