Ammasi Periasamy

Two‐photon (2P) fluorescence lifetime imaging microscopy (FLIM) was used to track cellular metabolism with drug treatment of auto‐fluorescent coenzymes NAD(P)H and FAD in living cancer cells. Simultaneous excitation at 800 nm of both coenzymes was compared with traditional sequential 740/890 nm plus another alternative of 740/800 nm, before and after adding doxorubicin in an imaging time course. Changes of redox states at single cell resolution were compared by three analysis methods: our recently introduced fluorescence lifetime redox ratio (FLIRR: NAD(P)H‐a ₂%/FAD‐a ₁%), machine‐learning (ML) algorithms using principal component (PCA) and non‐linear multi‐Feature autoencoder (AE) analysis. While all three led to similar biological conclusions (early drug response), the ML models provided statistically the most robust significant results. The advantage of the single 800 nm excitation of both coenzymes for metabolic imaging in above mentioned analysis is demonstrated.

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.

Significance: Here, we present unequivocal evidence of physical association of AMPK holoenzymes with mitochondrial reticulum (mitoAMPK) across multiple mouse tissues with evidence of conservation in human skeletal muscle and heart. We demonstrate that mitoAMPK is activated heterogeneously across the mitochondrial reticulum by mitochondrial energetic stress. Finally, we present evidence that suggests activation of mitoAMPK in skeletal muscle is required for mitophagy. We propose that mitoAMPK responds to mitochondrial microenvironment cues to maintain energetic homeostasis through mitochondrial quality control.

Increasingly, the auto-fluorescent coenzymes NAD(P)H and FAD are being tracked by multi-photon fluorescence lifetime microscopy (FLIM) and used as versatile markers for changes in mammalian metabolism. The cellular redox state of different cell model systems, organoids and tissue sections is investigated in a range of pathologies where the metabolism is disrupted or reprogrammed; the latter is particularly relevant in cancer biology. Yet, the actual optimized process of acquiring images by FLIM, execute a correct lifetime fitting procedure and subsequent processing and analysis can be challenging for new users. Questions remain of how to optimize FLIM experiments, whether any potential photo-bleaching affects FLIM results and whether fixed specimens can be used in experiments. We have broken down the multi-step sequence into best-practice application of FLIM for NAD(P)H and FAD imaging, with images generated by a time-correlated-single-photon-counting (TCSPC) system, fitted with Becker & Hickl software and further processed with open-source ImageJ/Fiji and Python software.

Fluorescence lifetime imaging microscopy (FLIM) is applied in broad areas of the life sciences and industrial fields for its ability to capture information from a smaller focal volume, inter alia being independent of fluorophore concentration, but sensitive to environmental changes such as pH and temperature and other advantages, which can all be exploited in scientific investigations.^{1 – 9} Fluorescence lifetime is of particular interest for quantitative studies in scattering and absorbing samples, such as tissue sections, where intensity-based methods are problematic.^{10 – 14} When FLIM is combined with multiphoton (MP) excitation, greater focal depth is achieved, important for thicker tissue specimens, and out-of-focus fluorescence is avoided with a smaller focal volume, without the need for a confocal pinhole. Two FLIM methods are available: frequency-domain FLIM and time-domain FLIM.^{15 – 20} This paper uses the latter, also called time-correlated single-photon counting (TCSPC).²¹ MP excitation illuminates molecules by infrared ranges that would otherwise require single-photon excitation in the UV region, generally undesirable to live cells, because of phototoxicity at longer exposure.²²

Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)‐based redox ratio NAD(P)H‐a2%/FAD‐a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H‐a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto‐fluorescent NAD(P)H, FAD lifetime is acquired by 2‐ photon excitation and Tryptophan by 3‐photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields‐of‐view (FoV) at each interval allows single‐cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H‐a2%, as does the NADPH/NADH ratio, highlighting a multi‐parametric assay to track metabolic changes in live specimens. © 2019 International Society for Advancement of Cytometry

Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses.

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 (τ_m) 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.