Multi-Dimensional Time-Correlated Single Photon Counting

Abstract

Optical spectroscopy techniques have found a wide range of applications in biomedical imaging and sensing because they are non-destructive and deliver biochemically relevant information about the systems investigated (Mycek and Pogue, 2003; Lakowicz, 1999). Typical applications are one- and two-photon fluorescence laser scanning microscopy, fluorescence endoscopy, control of drug delivery in photodynamic therapy, dynamics of protein-dye complexes on the single molecule level, chlorophyll fluorescence dynamics, and diffuse optical tomography of thick tissue. Most of these techniques use the fluorescence of exogenous or endogenous fluorophores to obtain information about the systems investigated. In the majority of the applications the fluorescence intensity, fluctuations of the fluorescence intensity, or the fluorescence spectra are recorded. However, the fluorescence of organic fluorophores is not only characterised by its intensity or spectrum, it has also a characteristic fluorescence lifetime. The fluorescence lifetime is useful as a separation parameter to distinguish the fluorescence components of endogenous fluorophores in cells and tissues. These components often have poorly defined fluorescence spectra but can be distinguished by their fluorescence lifetime (König and Riemann, 2003; Schweitzer, 2001; Urayama and Mycek, 2003). Moreover, the fluorescence lifetime of a fluorophore depends on the local environment of the molecules. Because the lifetime is widely independent of the concentration its measurement is a direct approach to quenching and energy transfer effects (Lakowicz, 1999). Typical examples are the mapping of cell parameters such as pH, ion concentrations, oxygen saturation or the binding state to proteins, lipids, or DNA (Gerritsen et al.,1997; Knemeyer et al., 2002; Lakowicz, 1999; Rück et al., 2003; Sanders et al., 1995, Van Zandvoort et al., 2002)