In this study, we explore the use of red blood cell and hemoglobin autofluorescence ad potential long-term biomarkers for diabetes. We found that under 370 nm excitation, both red blood cells (RBC) and Hb fluorescence in the 420-600 nm region. At early time points following diabetic induction in rats, autofluorescence increases in lysed Hb is more dramatic compared to that of RBC. Moreover, we found significance variance of Hb autofluorescence despite relatively constant HbA1c levels. The results of our study suggest that with additional development, RBC and hemoglobin autofluorescence from may be used as long-term glycemic markers for monitoring diabetic complications in the clinical setting.
In nonlinear optical imaging of biological samples, epi-illumination collection of fluorescent signals results in half of the signal loss. We enhanced the collected luminescent signal by using a multilayer coating of tantalum pentoxide (Ta2O5) and cerium oxide (SiO2). Our coatings are biocompatible, allowing visual inspection of samples and optimizing the collection of luminescent signals. This method was confirmed on a number of samples, including sulforhodamine solutions, fluorescent microspheres and labeled 3T3 cells. In all cases, the coated coverslips were used to enhance the signal by approximately 2 times. Image analysis also shows that signal enhancement does not result in additional photobleaching. Our results indicate that the designed coated coverslip enhances multi-photon microscopy detection signals.
Hypericin (Hyp) has received attention due to its high phototoxicity against viruses and anti-tumor photoactivity. Using
two-photon imaging, we demonstrated that Hyp induced photosensitized modification of collagen fibers in native tissues.
Dynamics of photo-processes was monitored by time-lapse multiphoton imaging. We showed that Hyp–mediated
processes in collagen tissues may be used for the selective modification of collagen fibers.
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.
Fractional photothermolysis (FP) induces discrete columns of photothermal damage in skin dermis, thereby promoting collagen regeneration. This technique has been widely used for treating wrinkles, sun damage, and scar. In this study, we evaluate the potential of multiphoton microscopy as a noninvasive imaging modality for the monitoring of skin rejuvenation following FP treatment. The dorsal skin of a nude mouse underwent FP treatment in order to induce microthermal zones (MTZs). We evaluated the effect of FP on skin remodeling at 7 and 14 days after treatment. Corresponding histology was performed for comparison. After 14 days of FP treatment at 10 mJ, the second harmonic generation signal recovered faster than the skin treated with 30 mJ, indicating a more rapid regeneration of dermal collagen at 10 mJ. Our results indicate that nonlinear optical microscopy is effective in detecting the damaged areas of MTZ and monitoring collagen regeneration following FP treatment.
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 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.
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