Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry;it can provide unique insights into the structure and assembly of macromolecular complexes. This year, we pursued protein-protein association within living cells, ultrafast protein solvation, mitochondrial energetics and ultrafast microscopy development. We also continued studies of DNA using fluorescent nucleotide analogs that reveal disruptions in DNA structures, publishing accounts of analog base pairing effects. We continued and expanded our femtosecond upconversion studies of Trp in proteins and peptides to quantify early """"""""quasistatic self-quenching"""""""" processes. We found extremely rapid (10-100ps) decays are important in several proteins (crystallins, thioredoxin, GB1,etc.), as they detect previously silent conformers engaged in ultrafast charge transfer. Our earlier study of protein *solvation* on the 330fs-200ps time scale, using proteins such as Monellin, found QSSQ that others attributed to a class of unique water molecules that desorb from protein in 20ps. Local quenching is the dominant mechanism in all but a few cases we have studied. The QSSQ was found even in simple dipeptides (just published)(suggesting a general process underlies all protein QSSQ) and we suggest that in crystallins QSSQ is an important factor in preventing eye lens cataract formation. We contined collaborative studies with LCE into the status of a primary fuel of heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and in collaboration with Microscopy Core and LCE, we are refining 'Decay-Associated Images'of NADH binding within isolated cardiac myocytes. This year, we continued CARS (Coherent AntiStokes Raman Spectroscopy) microscope engineering to permit us to uniquely image the nonfluorescent oxidized form of NADH, NAD+ ( and lipids or cell water itself). We have used our 2-photon FCS (Fluorescence (Cross) Correlation Spectrometer) with imaging capabilities to study the transport and binding of fluorescent proteins in both stably and transiently transfected cells. FCCS is a useful tool for quantifying mobility and stoichiometry of dilute proteins either in solution or in a cell. The system was used to study the downregulating interactions of the HIV nef protein with important cell-surface proteins like CD4 and HLAA2 . We also employed FCS to study the mobility of very low levels of C-myc ,finding exogenous C-myc is much more mobile than native protein and dependent on DNA-binding cofactors. We built (and pursued patents on) light collection devices for multiphoton microscopy of tissue that salvage any light that is emitted by the sample but does not enter the pupil of the objective. We showed that deep in rat brain slices, GFP labeled myosin gave >7X brighter signals using our TED(Total Emission Detection (=""""""""Morelight"""""""") ) device. We prototyped a new version for in vivo multiphoton imaging. It has since been tested on live mice, leading to over 2.5X signal improvements from cells within the exposed brain. We began collaborative simulation of deep multiphoton imaging with NICHD to seek the theoretical limits of such devices, with or without phase compensation array devices, and a prototype Adaptive Optics microscopy testbed (using both interferometry/OCT and waveform sensors) is under construction. Most recently, we built a superresolution microscope employing the STED method (STimulated Emission Depletion), finding we could image at less than half the """"""""diffraction limit"""""""". Working with the InterInstitute Probe Development Center, we designed special dyes to further improve superresolved imaging.
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