Fluorescence Lifetime Imaging Techniques—A Review on Principles, Applications and Clinical Relevance

Abstract

This article gives an overview of the most frequently used fluorescence-lifetime imaging (FLIM) techniques, their capabilities, and typical applications. Starting from a general introduction to fluorescence and phosphorescence lifetime, we will show that the fluorescence lifetime or, more accurately, the fluorescence decay function of a fluorophore is a direct indicator of the interaction with its molecular environment. FLIM is therefore more than a simple contrast technique in microscopy—it is a technique of molecular imaging. FLIM techniques can be classified into time-domain and frequency-domain techniques, analogue and photon counting techniques, and scanning and wide-field techniques. Starting from an overview of these general technical principles we will describe the features and peculiarities of the different FLIM techniques in use. An extended section is dedicated to TCSPC FLIM, addressing unique capabilities that make the technique especially interesting to FLIM of biological systems.