Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure and assembly of macromolecular complexes. We are wrapping up a decade of studying the dynamics of water near proteins using the picosecond response of Tryptophan(Trp), an amino acid found in most proteins, to the electric field caused by water dipole motions. The overall subject of upconversion (sub-ps) studies of Trp was separately published by us in a book chapter, and the Trp-analog (unnatural amino acid) experiments we published showed concurrent relaxation and electronic ultrafast ionization may occur. 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 Light Microscopy Core and LCE, we are continually refining 'Decay-Associated Images' software to more rapidly extract profiles of NADH binding within isolated cardiac myocytes and/or tumor cells. This year we updated DAI software for large images. We continue to develop coupled lifetime and translational diffusion capabilities in time-resolved FCS for this and other projects. We used our spectroscopic tools to help develop a new imaging molecule for cellular(tissue) oxygen concentration detection- we modified myoglobin to contain a red fluorescent dye (e.g. Alexa594) or a red fluorescent protein tail (e.g. mCherry). They respond to the oxygen-binding of myoglobin by changing lifetime and brightness.
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