Fluorescence Lifetime Microscopy: Metabolic Signaling and Analysis
Rozhin Penjweini, Katie Link, Shureed Qazi, Christian Combs, Dan Sackett* and Jay R. Knutson
NHLBI and NICHD
The noninvasive monitoring of cellular metabolism has advanced significantly in recent years due to the ability of FLIM (Fluorescence Lifetime Microscopy) to distinguish free and bound states of key metabolites (and intrinsic fluorophores) NAD(P)H and FAD+. The consequent ability to quantify relative prevalence of OXPHOS vs. glycolytic fueling of cells is of considerable interest, not only in myocytes, but also in pathological (e.g., neoplastic or inflamed/activated) states of cells. We have joined these efforts by adding novel fluorescent protein - based FRET probes for both [O2] and ROS(reactive oxygen species). The former reports (through viewing the consequences of diffusion limited consumption) on the critical step in ATP production via OXPHOS, and the latter monitors species that signal cells to alter their OXPHOS/glycolytic switch. We also endeavor to make analysis of metabolic state more precise through globalDAI (Decay Associated Images) and thorough confidence interval testing, in order to yield absolute concentrations of the various free/bound species in tissue.