Characterization of mitochondrial dysfunction due to laser damage by 2-photon FLIM microscopy.

Mitochondria are the central organelles in cellular bio-energetics with key roles to play in energy metabolism and cell fate decisions. Fluorescence Lifetime Imaging microscopy (FLIM) is used to track metabolic changes by following the intrinsic co-enzymes NAD(P)H and FAD, present in metabolic pathways. FLIM records-lifetimes and the relative fractions of free (unbound) and bound states of NAD(P)H and FAD are achieved by multiphoton excitation of a pulsed femto-second infra-red laser. Optimization of multiphoton laser power levels is critical to achieve sufficient photon counts for correct lifetime fitting while avoiding phototoxic effects. We have characterized two photon (2p) laser induced changes at the intra-cellular level, specifically in the mitochondria, where damage was assessed at rising 2p laser average power excitation. Our results show that NAD(P)H-a2%-the lifetime-based enzyme bound fraction, an indicator of mitochondrial OXPHOS activity is increased by rising average power, while inducing changes in the mitochondria at higher power levels, quantified by different probes. Treatment response tracked by means of NAD(P)H-a2% can be confounded by laser-induced damage producing the same effect. Our study demonstrates that 2p-laser power optimization is critical by characterizing changes in the mitochondria at increasing laser average power.

Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay.

Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment. We have used Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify mitochondrial metabolic response in PCa cells by tracking auto-fluorescent NAD(P)H, FAD and tryptophan (Trp) lifetimes and their enzyme-bound fractions as markers, before and after treatment with anti-cancer drug doxorubicin. A 3-channel FLIM assay and quantitative analysis of these markers for cellular metabolism show in response to doxorubicin, NAD(P)H mean fluorescence lifetime (τm) and enzyme-bound (a2%) fraction increased, FAD enzyme-bound (a1%) fraction was decreased, NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio and ROS increased, followed by induction of apoptosis. For the first time, a FRET assay in PCa cells shows Trp-quenching due to Trp-NAD(P)H interactions, correlating energy transfer efficiencies (E%) vs NAD(P)H-a2%/FAD-a1% as sensitive parameters in predicting drug response. Applying this FLIM assay as early predictor of drug response would meet one of the important goals in cancer treatment.