University of Minnesota
School of Physics & Astronomy

Condensed Matter Seminar

Thursday, September 13th 2007
1:25 pm:
Condensed Matter Seminar in 210 Physics
Speaker: Joe Skinner, University of Minnesota
Subject: Fluorescence Fluctuation Spectroscopy Extended Beyond Diffusion: Flow and Immobilization
This talk is also his final Ph.D. Thesis

Fluorescence fluctuation spectroscopy (FFS) extracts information about the transport and assembly of proteins from the signal fluctuations of fluorescently labeled biomolecules diffusing through a small optical observation volume. FFS was mainly developed for studies in aqueous solution, but has found an increasing number of applications measuring diffusion and protein association directly inside living cells. However, unlike in solution, proteins in cells are not only undergoing random diffusion, but also exist in immobile form when bound to large structures, such as the chromosome. The presence of immobile fluorophores in the observation volume presents new challenges for FFS that need to be addressed to allow a quantitative interpretation of cellular experiments. Two extreme cases of immobilization exist. The first is very sparse immobilization requiring the ability to detect single immobile molecules. The second is uniform distribution of immobilized molecules creating background fluorescence which biases the interpretation of fluctuation data. I present new approaches that extend FFS in order to quantify systems which contain these two types of immobilization. The first approach, based on periodic scanning of the laser beam, is also capable of characterizing hydrodynamic flow, which will be experimentally demonstrated. In addition, I will show that immobilization, at both extremes, can be quantitatively differentiated from mobile molecules by the modified FFS techniques. Finally, I will demonstrate the robustness of the methods by applying them to measure proteins in living cells.

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