Browsing by Subject "fluorescence fluctuation"

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    Advancing Fluorescence Fluctuation Microscopy in Living Cells: From Non-stationary Signals to Ternary Protein Interactions
    (2015-10) Hur, Kwang Ho
    Fluorescence fluctuation spectroscopy (FFS) is a powerful method for quantifying protein interactions. By exploiting the brightness of fluorescence intensity fluctuations we are able to measure the stoichiometry of protein complexes. FFS is particularly valuable because it allows real-time measurements within living cells, where protein complex formation plays a crucial role in the regulation of cellular processes. However, intensity fluctuations are frequently altered by the cell environment in subtle and unanticipated ways, which can lead to failure of the available FFS analysis methods. This thesis demonstrates that measuring in very small volumes, such as yeast and E. coli cells, can introduce a significant bias into the measured brightness as a result of cumulative sample loss, or photodepletion. This loss leads to a non-stationary signal, which is incompatible with the implicit assumption of a stationary process in conventional FFS theory. We addressed this issue by introducing a new analysis approach that serves as a foundation for extending FFS to non-stationary signals. FFS measurements in cells are also currently limited to the study of binary interactions involving two different proteins. However, most cellular processes are mediated by protein complexes consisting of more than two different proteins. Observation of pairwise interactions is not sufficient to unequivocally determine the binding interactions involving three or more proteins. To address this issue, we extended FFS beyond binary interactions by developing tricolor heterospecies partition analysis to characterize ternary protein systems. The method is based on brightness analysis of fluorescence fluctuations from three fluorescent proteins that serve as protein labels. We verified tricolor heterospecies partition analysis by experiments on well-characterized protein systems and introduced a graphical representation to visualize interactions in ternary protein systems.