Time-Domain Lifetime Measurements

Abstract

Time-resolved measurements are widely used in fluorescence spectroscopy, particularly for studies of biological macromolecules and increasingly for cellular imaging. Time-resolved measurements contain more information than is available from the steady-state data. For instance, consider a protein that contains two tryptophan residues, each with a distinct lifetime. Because of spectral overlap of the absorption and emission, it is not usually possible to resolve the emission from the two residues from the steady-state data. However, the time-resolved data may reveal two decay times, which can be used to resolve the emission spectra and relative intensities of the two tryptophan residues. The time-resolved measurements can reveal how each of the tryptophan residues in the protein is affected by the interactions with its substrate or other macromolecules. Is one of the tryptophan residues close to the binding site? Is a tryp-tophan residue in a distal domain affected by substrate binding to another domain? Such questions can be answered if one measures the decay times associated with each of the tryptophan residues.