Time Resolved Fluorescence Spectroscopy

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure and assembly of macromolecular complexes. Having fully concluded prior work 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, We continued multiple collaborative studies 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, we are continually refining 'Decay-Associated Images' software to more rapidly extract profiles of NADH binding within isolated tissue and/or tumor cells. This year we updated DAI software for special emphasis on ** statistical confidence ** in large images. This is key to making FLIM reproducible. 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 prototype new imaging molecules for cellular(tissue) oxygen and reactive oxygen radical concentration detection- modified myoglobin to abut a fluorescent protein tail (e.g. "mCherry" or "EYFP" or "mScarlet"). They respond to the oxygen-binding or radical chemistry of myoglobin by changing lifetime and brightness in distinct ways. We also collaborated on ultrafast studies to aid design and exploitation of fluorescent aptamers, a new window into cellular RNA. An aptamer is a (usually RNA) string that folds up to enclose, restrict and protect a floppy, non-fluorescent molecule that becomes fluorescent in the perfect groove. Fluorescent lifetimes (both picosecond and nanosecond timing) reveal the quality of the "lock and key" that groove provides. We have studied the floppy fluorophores both in solution and in various aptamers, quantifying the processes that brighten them in the latter environment. We also study nucleic acid structure and motion when interacting with SARS nucleocapsid and HIV TAR. We are beginning to also study quantum aspects of closely coupled dyes, with an eye to future communications and superresolution..