Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells

Abstract

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.

An automated multiwell plate reading flim microscope for live cell autofluorescence lifetime assays

“…3.5 are consistent with changes in NADH°uorescence lifetime parameters following a shift in cell metabolism toward glycolysis reported by Skala et al 39 We note, however, that apoptosis induced by staurosporine has previously been linked to an increase in mean NADH°uorescence lifetime. 30,40 The di®erence between this observation and our results may be due to the speci¯c modes of action of cisplatin and staurosporine and/or the rate at which apoptosis occurs with these two compounds but further work would be required to elucidate this.…”

“…28,29 There is potentially an additional source of cellular auto°uorescence detected in our experiments arising from NADPH, which is spectrally indistinguishable from NADH, although it is normally considered that the relative abundance and quantum yield of NADPH is su±-ciently low that it may be ignored. 30 Thus, a complete description of NADH°uorescence would require a multiexponential model with four or more components. With the limited number of auto-°u orescence photons typically detected, however, it is generally not possible to¯t FLIM data to such complex models.…”

Intravital multiphoton microscopy can model uptake and excretion of fluorescein in hepatic ischemia-reperfusion injury

“…As a result, NAD(P)H may be recruited by similar proteins related to oxidative repair, causing the lifetime change we observe in both solar-exposed skin and the liver after I/R injury. 33 Interestingly, these lifetime changes are in sharp contrast to ischemic necrosis and apoptosis, which are associated with a significant increase in the fluorescence lifetime of NAD(P)H. 11,34,35 While these processes also involve oxidative damage, the outcome is ultimately cell death and a breakdown of internal compartmentalization within the cell, 36 which may alter protein recruitment of NAD(P)H and thus result in the lifetime changes we observe. Further studies are required to identify the protein-binding associated changes for NAD(P)H in response to I/R injury and cell death.…”

Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging

“…For MPM-FLIM, images obtained in emission channel 350-450 nm under 740 nm excitation mainly reflect the fluorescence lifetime of NADH, which is sensitive to the changes in the cellular energy metabolism and microvascular oxygen supply [25]. Free NADH is mainly located in the cytoplasm, and involved in adenosine triphosphate (ATP) synthesis without oxygen in glycolysis, while protein-bound NADH is located in mitochondrial membrane, producing ATP in aerobic conditions [9,33]. The free and protein-bound forms of NADH have similar excitation and emission wavelengths, but can be separated by their distinct fluorescence lifetimes [34].…”