Reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime has been broadly used as a metabolic indicator for stem cell imaging. However, the direct relationship between NADH fluorescence lifetime and metabolic pathway and activity remains to be clarified. In this study, we measured the NADH fluorescence lifetime of human mesenchymal stem cells (hMSCs) as well as the metabolic indictors, such as adenosine triphosphate (ATP) level, oxygen consumption, and lactate release, up to 4 weeks under normal osteogenic differentiation and oxidative phosphorylation-attenuated/inhibited differentiation by oligomycin A (OA) treatment. NADH fluorescence lifetime was positively correlated with oxygen consumption and ATP level during energy transformation from glycolysis to oxidative phosphorylation. Under OA treatment, oxidative phosphorylation was attenuated/inhibited (i.e., oxygen consumption remained the same as controls or lower), cells showed attenuated differentiation under glycolysis, and NADH fluorescence lifetime change was not detected. Increased expression of the overall complex proteins was observed in addition to Complex I. We suggested special caution needs to be exercised while interpreting NADH fluorescence lifetime signal in terms of stem cell differentiation.
Noninvasive detection of cell death has the potential for definitive diagnosis and monitoring treatment outcomes in real time. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence intensity has long been used as a noninvasive optical probe of metabolic states. NADH fluorescence lifetime has recently been studied for its potential as an alternative optical probe of cellular metabolic states and cell death. In this study, we investigated the potential using NADH fluorescence intensity and/or lifetime to detect poly(adenosine-5′-diphosphate-ribose) polymerase-1 (PARP-1)-mediated cell death in HeLa cells. We also examined if NADH signals respond to treatment by pyruvate. The mechanism of PARP-1-mediated cell death has been well studied that extensive PARP-1 activation leads to cytosolic nicotinamide adenine dinucleotide depletion resulting in glycolytic inhibition, mitochondrial failure, and death. Pyruvate could restore electron transport chain to prevent energy failure and death. Our results show that NADH fluorescence lifetime, not intensity, responded to PARP-1-mediated cell death and the rescue effect of pyruvate. This lifetime change of NADH fluorescence happened before the collapse of mitochondrial membrane potential and mitochondrial uncoupling. Together with our previous findings in staurosporine-induced cell death, we suggest that NADH fluorescence lifetime increase during cell death is mainly due to increased protein-protein interactions but not the intracellular NADH content.
In vivo noninvasive detection of apoptosis represents a new tool that may yield a more definite diagnosis, a more accurate prognosis, and help improve therapies for human diseases. The intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) may be a potential optical biomarker for the apoptosis detection because NADH is involved in the respiration for the mitochondrial membrane potential (ΔΨ) formation and adenosine-5′-triphosphate (ATP) synthesis, and the depletion of ΔΨ and ATP level is the hallmark of apoptosis. We have previously observed the NADH fluorescence lifetime change is associated with staurosporine (STS)-induced mitochondria-mediated apoptosis. However, its relationship with mitochondrial functions such as ΔΨ, ATP, and oxygen consumption rate is not clear. In this study, we investigated this relationship. Our results indicate that the NADH fluorescence lifetime increased when ΔΨ and ATP levels were equal to or higher than their values of controls and decreased before the depletion of ΔΨ and ATP, and the oxygen consumption rate did not change. These findings suggest that the increased NADH fluorescence lifetime in STS-induced cell death occurred before the depletion of ΔΨ and ATP and activation of caspase 3, and was not simply caused by cellular metabolic change. Furthermore, the NADH fluorescence lifetime change is associated with the pace of apoptosis.
Fluorescence lifetime of NADH had been used as an optical marker for monitoring cellular metabolism. In our pervious
studies, we have demonstrated that NADH lifetime of hMSCs increase gradually with time of osteogenic differentiation.
In this study, we measured NADH lifetime of hMSCs from a different donor as well as the corresponding metabolic
indices such as ATP level, oxygen consumption and lactate release. We also measure the quantity of Complex I, III, IV
and V. The results show that during differentiation more oxygen consumed, higher ATP level expressed and less lactate
released, and the increase of NADH lifetime was associated with ATP level. Higher expression of the total Complex
protein was observed at 3 and 4 weeks after differentiation than controls. However, Complex I expression did not show
significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change
of NADH lifetime was associated with the metabolic change during osteogenic differentiation of hMSCs. The increase
of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria after differentiation.
The photon-scattering imaging data of Liposyn II intravenous emulsion solution samples of different
concentrations and different thicknesses is reported and analyzed. The scattering Mueller matrix
element m11 data shows that the maximum number of multi-photon scatterings is an increasing
function of concentration and sample thickness.
Tissue is optically anisotropic and highly photon-scattering medium. It has long been treated as optically
diffusive medium in bio-medical applications. The diffusion equation of isotropic photon-density wave (PDW)
was widely applied to interpret the data of reflectance spectroscopy and biomedical imaging experiments. In
our recent transmission Stokes imaging experiment of the rat liver samples, the Mueller matrix elements were
measured and analyzed theoretically. The measured data of depolarization constant has shown that the optical
property is not perfectly diffusive. Based upon our recently developed theoretical model of anisotropic and
highly photon-scattering medium, the simulated results of anisotropy, photon-scattering and depolarization
property for the reflectance/backscattering experiment are reported.
The metabolic changes of human mesenchymal stem cells (hMSCs) during osteogenic differentiation were accessed by reduced nicotinamide adenine dinucleotide (NADH) 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 (ATP) 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.
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 ~1.3 ns. The NADH average fluorescence lifetime increased to ~3.5 ns within 15 min after 1 µM 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.
(PDT). This technique has been extensively validated in tissue phantoms; however, validation in patients has been limited. This pilot study compares blood oxygenation and photosensitizer tissue uptake measured by multiwavelength DRS with ex vivo assays of the hypoxia marker, 2-(2-nitroimida-zol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5), and the photosensitizer (motexafin lutetium, MLu) from tissues at the same tumor site of three tumors in two patients with intra-abdominal cancers. Similar in vivo and ex vivo measurements of MLu concentration are carried out in murine radiation-induced fibrosarcoma (RIF) tumors (n=9). The selection of optimal DRS wavelength range and source-detector separations is discussed and implemented, and the association between in vivo and ex vivo measurements is examined. The results demonstrate a negative correlation between blood oxygen saturation (StO2) and EF5 binding, consistent with published relationships between EF5 binding and electrode measured pO2, and between electrode measured pO2 and StO2. A tight correspondence is observed between in vivo DRS and ex vivo measured MLu concentration in the RIF tumors; similar data are positively correlated in the human intraperitoneal tumors. These results further demonstrate the potential of in vivo DRS measurements in clinical PDT.
We evaluate Photofrin-mediated photodynamic therapy (PDT) in a phase 2 clinical trial as an adjuvant to surgery to treat peritoneal carcinomatosis. We extract tissue optical [reduced scattering (µ), absorption (µa), and attenuation coefficients (µeff)] and physiological [blood oxygen saturation (%StO2), total hemoglobin concentration (THC), and photosensitizer concentration (cPhotofrin)] properties in 12 patients using a diffuse reflectance instrument and algorithms based on the diffusion equation. Before PDT, in normal intraperitoneal tissues %StO2 and THC ranged between 32 to 100% and 19 to 263 µM, respectively; corresponding data from tumor tissues ranged between 11 to 44% and 61 to 224 µM. Tumor %StO2 is significantly lower than oxygenation of normal intraperitoneal tissues in the same patients. The mean (±standard error of mean) penetration depth () in millimeters at 630 nm is 4.8(±0.6) for small bowel, 5.2 (±0.67) for large bowel, 3.39(±0.29) for peritoneum, 5.19(±1.4) for skin, 1.0(±0.1) for liver, and 3.02(±0.66) for tumor. cPhotofrin in micromolars is 4.9(±2.3) for small bowel, 4.8(±2.3) for large bowel, 3.0 (±1.0) for peritoneum, 2.5(±0.9) for skin, and 7.4(±2.8) for tumor. In all tissues examined, mean cPhotofrin tends to decrease after PDT, perhaps due to photobleaching. These results provide benchmark in-vivo tissue optical property data, and demonstrate the feasibility of in-situ measurements during clinical PDT treatments.
Changes in blood flow and oxygenation during and after PDT provide information about tumor vessel and cellular damage. The characterization of these changes may improve our understanding of PDT mechanisms and help predict treatment efficacy. We have designed a hybrid system that can non-invasively measure in vivo hemodynamic changes and provide independent information about tumor oxygenation and blood flow. Diffuse correlation spectroscopy (DCS) monitors blood flow by measuring the optical phase shifts caused by moving blood cells, while diffuse photon density wave (DPDW) spectroscopy measures tissue absorption and scattering. When mounted on a camera, our unique probe allows non-contact measurements that avoid compressing the tumor and altering blood flow. An optical filter mounted in front of the camera lens cut off light below 650nm, which allowed monitoring of blood flow during PDT. The utility of the hybrid system was demonstrated by monitoring the hemodynamic changes during and after PDT in mice bearing the experimental radiation-induced fibrosarcoma (RIF). For the first time, we non-invasively and continually monitored the in vivo flow changes during PDT. Relative oxygen consumption was calculated using flow values measured by DCS and oxygenation measured by a broadband absorption spectrometer. During PDT an initial rapid increase in blood flow was found, followed by a decrease and slow recovery. After PDT, substantial and continued reductions in blood saturation, blood flow and oxygen consumption were found after 3 hours, suggesting that permanent damage to tumor cells and blood vessels had occurred. The comparison of flow values after PDT as measured by DCS and by Power Doppler ultrasound (CWFA) demonstrated that both techniques non-invasively detected similar global changes in tumor blood flow or perfusion after PDT.
Photodynamic therapy (PDT) employs a combination of photosensitizing chemical, light, and oxygen Knowledge of tissue optical properties, including absorption (μa) and reduce scattering coefficients (μs’), makes possible to derive blood oxygen saturation, light penetration depth, and drug concentration, which are important to ensure PDT treatment efficacy at the specific wavelengths. We have developed an absorption spectroscopy system to measure μa and μs’ in the spectral range 600-800nm using a contact linear probe with a source fiber and multiple source-detector separation distances less than 1 cm. The μa and μs’ were recovered based on diffusion approximations of the photon transport equation. We measured tissue optical properties among various organs of patients with intraperitoneal malignancies for an on-going Phase II PDT protocol. The results from 12 patients showed various effective penetration depth from site to site and from organ to organ. The percentage oxygen saturation (%StO2) are similar before and after PDT. Before PDT, meff (mean (standard deviation) (number of patients)) in cm-1 at 630nm are 2.4 (0.2) (12) in small bowel, 2.2(0.4) (9) in large bowel, 4.2(2.7) (7) in tumor, 3.3 (0.3) (10) in peritoneum, 2.7 (0.3) (11) in skin, and 10.1 (0.6) (10) in liver. %StO2 is 60-80% for most organs but 30-40% for tumor.
We have developed a full field optical coherence microscopy (OCM) system operating at several frames per second. Depth ranging capability is compared between OCM and scanning confocal microscopy by imaging a test chart imbedded in 10 percent intralipid and imaging an onion. For sufficiently dense scattering, OCM was able to resolve test chart features and onion structures which were not detectable using confocal imaging alone.
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