Chapter 36 Phase-Sensitive Detection Methods for Resolving Fluorescence Emission Signals and Directly Quantifying Lifetime

Abstract

This chapter discusses phase-sensitive detection methods for resolving fluorescence emission signals and directly quantifying lifetime. A new flow cytometry (FCM) approach, based on phase-resolved fluorescence spectroscopy methods that provides unique capabilities for separating signals from multiple overlapping emissions in fluorochrome-labeled cells as they pass across a modulated excitation source, has been developed. The measurement of fluorescence lifetime also is of importance because it provides additional information about fluorochrome/cell interactions. An important advantage of lifetime measurements is that lifetimes in some cases can be considered as absolute quantities. However, the lifetime of dye molecules bound to cellular macromolecules can be influenced by physical and chemical factors near the binding site, such as solvent polarity, cations, pH, energy transfer, excited-state reactions, and quenching. Spectroscopic methods also have been developed to record fluorescence emission spectra on each cell in real time and to resolve multiple emission spectra signals using computational methods. If fluorochromes cannot be resolved by these means, but have separated excitation spectra, multiple excitation sources can be employed to sequentially excite cells and spatially resolve the spectrally overlapping fluorescence signals.