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.

Two-photon absorption coefficients of GaN for below band gap ultraviolet wavelength and midgap infrared wavelength were measured by using femtosecond pulsewidth autocorrelation and Z-scan techniques. Large two-photon absorption coefficients were obtained. Taking advantage of the large two-photon absorption, we have demonstrated two-photon confocal imaging of a GaN thin film. Direct correlation was found between the yellow luminescence and suppression of bandedge luminescence.

Formulation of oxidative phosphorylation and its first observation by means of fluorescence spectroscopy in the 1960s led to the acceptance of bioenergetics as a new field of studies. The new discipline grew fast with the increasing number of papers, related to the energy generation in mitochondria, advancement of the instrumentation, and improvement of observation techniques. As such, fluorescence lifetime imaging microscopy (FLIM) has gained popularity as a sensitive technique to monitor the functional/conformational states of nicotinamide adenine dinucleotide reduced (NADH)one of the main compounds of oxidative phosphorylation. We hereby review the development and current application of cellular metabolism observation via NADH FLIM, illustrating it with the examples of both physiological (cell density, apoptosis, necrosis) and pathological states (inhibition of the electron transfer chain).

Nano-composites of quantum dots (QDs) and gold nanorods (GNRs) or silica-coated GNRs (GNRs_SiO2) were synthesized. The attached GNRs modify the excitation intensity and spontaneous emission of QDs through the surface plasmonic effects. The fluorescence from QDs is enhanced and can be optimized by modifying the thickness of silica coated on GNRs, under both one- and two-photon excitations. The measurements of fluorescence intensity and lifetime demonstrate that the enhancement may be attributed to the matching of the localized surface plasmon resonance of GNR to the excitation wavelength. In addition to enhancing QD-fluorescence in QD-GNR@SiO2, GNRs also present as an effective contrast agent for bio-imaging, through light scattering and or two-photon emission, as well as for photo-thermal therapy. The composite's multifunctional characteristics are highly valuable and to be exploited in bio-applications.

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