The fluorescence lifetime is the mean time a fluorophore remains in an excited state before emitting a photon (fluorescence) and returning to the initial ground state. The fluorescence lifetime of a fluorophore, either endogenous or synthetic, depends on the type of molecule, its conformation, and the way the molecule interacts with the surrounding microenvironment [15]. FLIM constructs a spatial distribution map of fluorescence lifetimes of a sample imaged by confocal, multiphoton, endoscopic, or wide-field microscopy. Importantly, FLIM can be used to measure molecular environmental parameters, and the metabolic state of cells and tissues via endogenous fluorescence. There are two major approaches used to measure the fluorescence lifetime of a sample: time and frequency domain. Typically, frequency-domain FLIM has superior signal-to-noise ratio for complex systems with multiple fluorophores and faster acquisition times, yet requires more complex analysis compared to time-correlated FLIM. Background and principles to these two different techniques are readily available [1].

NAD(P)H lifetime measurements are widely used for metabolic and redox imaging in vitro and in vivo [16, 17]. The fluorescence lifetime of NAD(P)H is resolved as a two-component system with the short (~0.3–4 ns) and long (~2.3 ns) lifetime represented as the free and protein-bound conformations, respectively [18]. NAD+ is not excited at 740 nm whereas NAD(P)H is. The ratio of NAD(P)H:NAD(P)+ has been used previously as a measure of the redox state of the cell. Bird and colleagues demonstrated that the free-to-bound ratio of NAD(P)H, represented by the ratio of the amplitude coefficients for the short and long lifetimes (i.e. α ₁/α ₂), is related to the NAD(P)H/NAD(P)+ ratio and can be used as an indicator for redox changes within the cell [17]. FAD is also examined routinely by FLIM for intracellular metabolic and redox analysis. While FAD can be found in a free and protein-bound conformation, the former has a significantly higher quantum yield with a lifetime of 2.91 ns. In contrast to NAD(P)H, only the oxidized form of FAD is fluorescent and is used to measure changes in the redox state of the cell in combination with NAD(P)H [19]. The fluorescence lifetime of NAD(P)H and FAD is a viable indicator of ischemia-reperfusion injury (IR) within the liver [2] and kidney [20] in vivo. The fluorescence lifetime changes associated with necrosis [14] and apoptosis [21] have also been described and can be used to measure tissue responses to drug treatments and disease states. To date, however, studies using FLIM analysis in the kidney are limited in number (see Note 3 ).

Metabolic alterations to the kidney are readily observable via MPM. Discerning the reason for these changes requires knowledge of some basic pathophysiological processes. The oxidized and reduced redox cofactor pairs FAD/FADH₂ and NAD+/NADH are becoming increasingly recognized as key metabolic indicators of the state of cellular metabolism associated with health and disease.

NADH and FADH₂ are formed during glycolysis, fatty acid oxidation, and the citric acid cycle, and are both energy-rich molecules due to the presence of an electron pair (Fig. 1). The kidney has minimal glycolytic capacity and relies primarily on oxidative phosphorylation to produce ATP, which occurs as a result of the transfer of electrons from NADH or FADH₂ to O₂ by a series of electron carriers located on the inner mitochondrial membrane. NADH is a substrate for complex I of the electron transport chain (ETC) which catalyzes the oxidation to NAD+ (Fig. 1a, b). Decreased O₂ supply causes reduced complex I activity and a failure to convert NADH to NAD+ (nonfluorescent). This “backing up” of NADH is observed by MPM (excitation ~740 nm) as increased fluorescence emission compared to basal conditions (Fig. 1c, d). Oxygen deprivation reduces ATP production and ATP-dependent processes, such as actin polymerization, causing plasma membrane disruption and alterations to ETC complexes. Oxygen re-presentation to damaged components of the ETC at reperfusion is thought to result in the production of ROS, mainly superoxide (O₂ ⁻) (Fig. 1e, f).

Fluorescence lifetime changes of NAD(P)H varying depending on the type of insult and temporal influences, and usually requires further investigation into the protein interactions of NAD(P)H such as the ratio of free-to-bound NAD(P)H (α ₁/α ₂). An increased ratio demonstrates either an increase in free NAD(P)H or decrease in protein-bound NAD(P)H. Binding of NAD(P)H occurs primarily on complex I of the ETC. An increased ratio is consistent with mitochondrial dysfunction and increased ROS production. FAD cannot be excluded when detecting fluorescence emission at 740 nm excitation, and further information can be provided by fluorescence lifetime differences of FAD detected by FLIM. Decreased lifetime fluorescence detected by FLIM likely indicates decreased protein-bound FAD to the succinate dehydrogenase enzyme complex of complex II of the ETC and, in addition, decreased free FAD due to decreased complex II utilization and re-presentation of FAD to the citric acid cycle (Fig. 1). Both explanations underlie impaired metabolism and/or mitochondrial dysfunction. Alternately, increased FAD lifetime may indicate enhanced metabolism and increased turnover of FADH₂ to FAD.

The specific use of endogenous and infused fluorophores is now described, using as an example our investigation of changes in renal pathophysiology from IR injury to the kidney, and the role of mitochondrial dysfunction. Measurements of NAD(P)H and FAD were compared with other measurements of oxidative stress in progressive IR injury in the kidney.