Fluorescence Lifetime Imaging Microscopy Quantitative Measurements
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 USA.
1. A Brief History of the Measurement of Fluorescence Lifetimes
Fluorescence describes the emission of light by an atom or molecule that follows the absorption of electromagnetic energy [1]. When a fluorescent molecule absorbs energy, it is driven into an excited state that persists for a brief time. The molecule then transitions back to the lower energy ground state by one of several possible pathways. Some of the pathways for de-excitation to the ground state are illustrated in the simplified Perrin-Jabłoński plot in Figure 1. The pathways include internal conversion ([math]ic[/math]), decay by fluorescence ([math]kf[/math]), quenching (loss of excitation energy without the emission of light, [math]knf[/math]), or intersystem crossing ([math]isc[/math]) to the triplet state followed by decay by phosphorescence ([math]kpf[/math]).The average time required for a population of fluorophores in the excited state to decay to the ground state is called the fluorescence lifetime, which is described by an exponential function (Figure 1):
[math] I(t)=I_{0}e^{-t/\tau}\tag 1[/math]
where [math]I(t)[/math] is the fluorescence impulse response at time [math]t[/math], [math]I_{0}[/math] is the initial intensity after the excitation pulse, and [math]\tau[/math]is the fluorescence lifetime.
