Applications of Multiphoton Imaging Techniques To The Study Of Protein Interactions
Rosales, Tilman J.
Walker, Robert A
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Several recent improvements in microscopy have been driven by advances in ultrafast laser technology. The goal of the research described in this dissertation was to develop noninvasive, optically based methods to measure the mobility of macromolecules in biologically relevant systems. These methods exploit advances in ultrafast laser science and recent developments in multiphoton spectroscopy techniques. Each of the techniques described in this dissertation is validated and standardized using well characterized systems. We have explored the following techniques: First, 2-photon 2-color Fluorescence Cross Correlation Spectroscopy (FCCS) a powerful technique to measure dilute protein interactions in living cells. We have used FCCS to probe AR-Tif2 (Androgen Receptor - activating cofactor) interactions in the presence of casodex, an antagonist yielding decreased binding affinity. On a much faster timescale, exploring rotational rather than translational diffusion, we used molecular dynamics simulations of the model probe perylene to show that there is `room to wiggle' (sub-ps libration) within pure hexadecane. Third, combining picosecond and microsecond scales, we built a system to measure both rotational and translational motion in one experiment, using advanced Time Correlated Single Photon Counting (TCSPC) techniques. We have tested our ability to measure and link simultaneously the translational rates and decay rates of Alexa488 dye and other biologically relevant fluorophores. Next, exploiting non-linear vibrational spectroscopy, we have imaged the non-fluorescent molecule NAD+ in DPPC vesicles, the C-H stretch of lipids in vesicles and polystyrene beads, and the O-H stretch of water inside living cells (vs. O-D) to demonstrate the chemically selective imaging capabilities of Coherent Anti-Stokes Raman Spectroscopy (CARS) Microscopy. Most recently, we have built a STED (STimulated Emission Depletion) Microscope capable of extracting fluorescent images well below the diffraction limit. The STED microscope was tested using both 170nm fluorescent beads and a novel photochromic dye.