Difference between revisions of "Fluorescence Lifetime Imaging Microscopy Quantitative Measurements"
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| − | a | + | <span style="font-family:Georgia;">[[Richard N. Day]] |
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| + | <span style="font-family:Georgia;">Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 USA. | ||
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| + | ---- | ||
| + | '''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 (kf), quenching (loss of excitation energy without the emission of light, knf), or intersystem crossing (isc) to the triplet state followed by decay by phosphorescence (kpf).The average time required for a population of fluorophores in the excited state to decay to the ground state is called the fluorescence lifetime, whichis described by an exponential function (Figure 1): | ||
| + | I(t) = I0e – t/τ (Eq 1) | ||
| + | where I(t) is the fluorescence impulse response at time t, I0 is the initial intensity after the excitation pulse, and τ is the fluorescence lifetime. | ||
Revision as of 06:35, 16 February 2017
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 (kf), quenching (loss of excitation energy without the emission of light, knf), or intersystem crossing (isc) to the triplet state followed by decay by phosphorescence (kpf).The average time required for a population of fluorophores in the excited state to decay to the ground state is called the fluorescence lifetime, whichis described by an exponential function (Figure 1): I(t) = I0e – t/τ (Eq 1) where I(t) is the fluorescence impulse response at time t, I0 is the initial intensity after the excitation pulse, and τ is the fluorescence lifetime.
