Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was approximately 1.3 ns. The NADH average fluorescence lifetime increased to approximately 3.5 ns within 15 min after 1 microM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.

Abstract. The metabolic changes of human mesenchymal stem cells ͑hMSCs͒ during osteogenic differentiation were accessed by reduced nicotinamide adenine dinucleotide ͑NADH͒ fluorescence lifetime. An increase in mean fluorescence lifetime and decrease in the ratio between free NADH and protein-bound NADH correlated with our previously reported increase in the adenosine triphosphate ͑ATP͒ level of hMSCs during differentiation. These findings suggest that NADH fluorescence lifetime may serve as a new optical biomarker for noninvasive selection of stem cells from differentiated progenies. Keywords: microscopy; fluorescence lifetime; stem cell.Paper 08176L received Jun. 5, 2008; accepted for publication Aug. 14, 2008; published online Oct. 9, 2008. Stem cells give rise to tissue progenitor cells, which can differentiate into specific progenies and have potential use in regenerative medicine, disease treatment, and developmental biology. Efforts have been made to search for reliable biomarkers to identify stem cells ex vivo 1 and in vivo 2 so as to gain a better insight into the biology and physiology of stem cells, as well as to increase the selection efficiency from a given cell pool. However, many of the markers are invasive even in in vivo imaging approaches because stem cells were preloaded ex vivo by radionuclide, ferromagnetic, or reporter labeling, 2 which decreases the clinical usefulness of these methods. Recently, a noninvasive biomarker using proton nuclear magnetic resonance spectroscopy ͑ 1 H-MRS͒ has been identified for detection of neural stem and progenitor cells in the human brain in vivo.3 Although the identity of this 1 H-MRS-detected biomarker is not known, it is suggestive of a metabolic profile of fatty acids. In fact, one generally accepted property of stem cells that differs from their differentiated progenies is a lower metabolic rate accompanied by a lower adenosine triphosphate ͑ATP͒ content. 4 The shift from anaerobic glycolysis to the more efficient mitochondrial oxidative metabolism has been demonstrated in the differentiation of cardiomyocytes 5 and human mesenchymal stem cells ͑hMSCs͒. 6 The preference of stem cells to produce energy by glycolysis instead of oxidative phosphorylation is similar to that of cancer cells, which has been termed the Warburg effect.Optical detection/imaging techniques have been employed to study cell metabolism in a noninvasive manner by monitoring the intrinsic fluorescence signal of reduced nicotinamide adenine dinucleotide ͑NADH͒, a key coenzyme in glycolysis and oxidative metabolism. Two measurement schemes are possible: fluorescence lifetime 7 and fluorescence intensity. 8 In the fluorescence lifetime measurement scheme, a fluorescence decay curve is typically fitted to a twocomponent exponential decay function F͑t͒ = a 1 exp͑−t / 1 ͒ + a 2 exp͑−t / 2 ͒, where 1 and 2 correspond to the short and long fluorescence lifetimes of NADH and were reported to be ϳ400 to 500 ps and ϳ2000 to 2500 ps for free and bound NADH, respectively.7 a 1 and a 2 are the c...