Fluorescence Lifetime Imaging (FLlM) of Green Fluorescent Fusion Proteins in Living Cells

Abstract

Fluorescence microscopy is an exceptional tool for looking inside cells and tissues. Recent advances in fluorescence microscopy, including improved optics, sensitive fluorescent dyes, and high-sensitivity cameras, coupled with technological advances in computers and sophisticated software now permit quantitative measurement and noninvasive acquisition of spectroscopic information from a single living cell (1–4). Because of the specificity inherent in current fluorescence labeling techniques, and the sensitivity in fluorescence microscopic techniques, it is possible to detect very small amounts of proteins with very high sensitivity and precision. The enormous advantage of fluorescence is that most fluorophores used in biology are excited by absorption, and emit light by fluorescence, all within 100 ns. This time-scale coincides with the time-scale of molecular interactions in biological systems under physiological conditions. As a consequence, changes in an active biological system may be followed, theoretically by monitoring the fluorescence lifetime, i.e., the duration of the excited state. To calculate the lifetime, multiple images are collected within ns (a considerable technical challenge) then the decay of fluorescent intensity vs time fit to one or more exponentials. The lifetime of a fluorophore can be used to monitor functional and structural aspects of living specimens.