Flavin Adenine Dinucleotide Fluorescence Research Articles

High levels of FAD autofluorescence indicate pathology preceding cell death

Flavin adenine dinucleotide (FAD) autofluorescence from cells reports on the enzymatic activity which involves FAD as a cofactor. Most of the cellular FAD fluorescence comes from complex II of the electron transport chain in mitochondria and can be assessed with inhibitor analysis. The intensity of FAD autofluorescence is not homogeneous and vary between cells in tissue and in cell culture types. Using primary co-culture of neurons and astrocytes, and human skin fibroblasts we have found that very high FAD autofluorescence is a result of an overactivation of the mitochondrial complex II from ETC and from the activity of monoamine oxidases. Cells with high FAD autofluorescence were mostly intact and were not co-labelled with indicators for necrosis or apoptosis. However, cells with high FAD fluorescence showed activation of apoptosis and necrosis within 24 h after initial measurements. Thus, high level of FAD autofluorescence is an indicator of cell pathology and reveals an upcoming apoptosis and necrosis.

Breast tissue analysis using a clinically compatible combined time-resolved fluorescence and diffuse reflectance (TRF-DR) system.

This work aims to develop a clinically compatible system that can perform breast tissue analysis in a more time efficient process than conventional histopathological assessment. The potential for such a system to be used in vivo in the operating room or surgical suite to improve patient outcome is investigated. In this work, 80 matched pairs of invasive ductal carcinoma and adjacent normal breast tissue were measured in a combined time-resolved fluorescence and diffuse reflectance (DA) system. Following measurement, the fluorescence intensity of collagen and flavin adenine dinucleotide (FAD); the fluorescence lifetime of collagen, nicotinamide adenine dinucleotide (NADH), and FAD; the DA; absorption coefficient; and reduced scattering coefficient were extracted. Samples then underwent histological processing and H&E staining to classify composition as tumor, fibroglandular, and/or adipose tissue. Statistically significant differences in the collagen and FAD fluorescence intensity, collagen and FAD fluorescence lifetime, DA, and scattering coefficient were found between each tissue group. The NADH fluorescence lifetime and absorption coefficient were statistically different between the tumor and fibroglandular groups, and the tumor and adipose groups. While many breast tissue analysis studies label fibroglandular and adipose together as "normal" breast tissue, this work indicates that some differences between tumor and fibroglandular tissue are not the same as differences between tumor and adipose tissue. Observations of the reduced scatter coefficient may also indicate further classification to include fibro-adipose may be necessary. Future work would benefit from the additional tissue classification. With observable differences in optical parameters between the three tissue types, this system shows promise as a breast analysis tool in a clinical setting. With further work involving samples of mixed composition, this combined system could potentially be used intraoperatively for rapid margin assessment.

Spectroscopic Study of Time-Varying Optical Redox Ratio in NADH/FAD Solution.

Autofluorescence imaging has been widely applied as advanced noninvasive diagnostics for in vivo and ex vivo tissues. The optical redox ratio (ORR), which is defined as the fluorescence intensity ratio between reduced nicotine adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), has been used as a diagnostic parameter strongly, because NADH and FAD play an important role in energetic and respiratory metabolism as coenzymes. The ORR method has provided successful assessment in cancer diagnosis including breast, cervical, and oral cancer; few studies have been reported about optical and chemical interference between two molecules resulting in a change in ORR values. In this study, we investigated the variations in ORR values of NADH/FAD mixtures dissolved in tris(hydroxymethyl)aminomethane, phosphate buffer, and deionized water environments. In vitro solutions were prepared in various concentration ratios and the experimental and theoretical ORR values were obtained from fluorescence and absorption spectra in time series. Based on the spectroscopic analysis, we concluded that the inner filter effect causes an instant decrease in FAD fluorescence just after dissolution and that the oxidation-reduction coupled with oxygenation reaction results in time-varying decreases in NADH fluorescence and FAD emission.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA\u2013induced metabolic stress in reconstructed human skin

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.

Influence of nanoenvironment in reverse micelles on the fluorescence of flavins

Riboflavin (RF) and flavin adenine dinucleotide (FAD) are two chromophores in ubiquitous blue-light photoreceptors where molecular motions are partially constrained. However, most previous studies on their fluorescence were performed in solutions. In this work, we studied their fluorescence in Aerosol-OT (AOT) and cetyltrimethylammonium bromide (CTAB) reverse micelles (RMs) by using steady-state UV–visible spectroscopy and femtosecond transient absorption spectroscopy. Compared with in aqueous solution, fluorescence of RF in AOT RMs remains nearly constant, but it is quenched in CTAB RMs induced by intersystem crossing promoted by bromide anion. In addition, fluorescence of FAD in AOT RMs is strengthened because the stacked conformation is less favorable in AOT RMs than in aqueous solution. Moreover, the fluorescence quenching of FAD in CTAB RMs by intersystem crossing mainly affects the unstacked FAD because the intramolecular charge transfer in stacked FAD is faster than intersystem crossing by more than 40 folds. These results determine the dependence of fluorescence of flavins on the ions, polarity and steric constraints in nanoenvironment.

Intravital Metabolic Autofluorescence Imaging Captures Macrophage Heterogeneity Across Normal and Cancerous Tissue.

Macrophages are dynamic immune cells that govern both normal tissue function and disease progression. However, standard methods to measure heterogeneity in macrophage function within tissues require tissue excision and fixation, which limits our understanding of diverse macrophage function in vivo. Two-photon microscopy of the endogenous metabolic co-enzymes NAD(P)H and flavin adenine dinucleotide (FAD) (metabolic autofluorescence imaging) enables dynamic imaging of mouse models in vivo. Here, we demonstrate metabolic autofluorescence imaging to assess cell-level macrophage heterogeneity in response to normal and cancerous tissue microenvironments in vivo. NAD(P)H and FAD fluorescence intensities and lifetimes were measured for both tissue-resident macrophages in mouse ear dermis and tumor-associated macrophages in pancreatic flank tumors. Metabolic and spatial organization of macrophages were determined by performing metabolic autofluorescence imaging and single macrophage segmentation in mice engineered for macrophage-specific fluorescent protein expression. Tumor-associated macrophages exhibited decreased optical redox ratio [NAD(P)H divided by FAD intensity] compared to dermal macrophages, indicating that tumor-associated macrophages are more oxidized than dermal macrophages. The mean fluorescence lifetimes of NAD(P)H and FAD were longer in dermal macrophages than in tumor-associated macrophages, which reflects changes in NAD(P)H and FAD protein-binding activities. Dermal macrophages had greater heterogeneity in optical redox ratio, NAD(P)H mean lifetime, and FAD mean lifetime compared to tumor-associated macrophages. Similarly, standard markers of macrophage phenotype (CD206 and CD86) assessed by immunofluorescence revealed greater heterogeneity in dermal macrophages compared to tumor-associated macrophages. Ultimately, metabolic autofluorescence imaging provides a novel tool to assess tissue-specific macrophage behavior and cell-level heterogeneity in vivo in animal models.

The forensic implications of the relationship between the proteolytic enzymes activity and the changes in NADH and FAD fluorescence intensity in skeletal muscle when determining the time of death (experimental study)

Evaluation of the relationship between the activity of proteolytic enzymes (cathepsin D and calpains) and the dynamics of the fluorescence intensity of the coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the rats' skeletal muscles in relation to the time of death. The proteolytic activity of enzymes in rat skeletal muscle was determined at the postmortem time points corresponding to the most significant changes in the dynamics of coenzymes NADH and FAD fluorescence intensity. The proteolytic enzymes activity was found to be low during the period of increasing intensity of NADH fluorescence observed within 3 hours after death. An increase in the activity of proteolytic enzymes was registered in 4.5 hours after death which corresponds to the initial point of decrease in NADH fluorescence intensity. In 24 hours post-mortem, corresponding to increased FAD fluorescence intensity a significant decrease in the activity of calpains was found. The results of the study suggest that the nature of the postmortem dynamics of the fluorescence intensity of coenzymes is largely due to the peculiarities of intracellular proteolysis. The study results suggest that the pattern of post mortem changes in coenzyme fluorescence intensity is largely attributable to the specifics of intracellular proteolysis. The relationship between coenzyme fluorescence and molecular mechanisms of cell death confirms the viability and feasibility of laser-induced spectroscopy for post-mortem changes assessment when determining the time of death.

Mechanobiology of Cartilage Impact Via Real-Time Metabolic Imaging.

Cartilage loading is important in both structural and biological contexts, with overloading known to cause osteoarthritis (OA). Cellular metabolism, which can be evaluated through the relative measures of glycolysis and oxidative phosphorylation, is important in disease processes across tissues. Details of structural damage coupled with cellular metabolism in cartilage have not been evaluated. Therefore, the aim of this study was to characterize the time- and location-dependent metabolic response to traumatic impact loading in articular cartilage. Cartilage samples from porcine femoral condyles underwent a single traumatic injury that created cracks in most samples. Before and up to 30 min after loading, samples underwent optical metabolic imaging. Optical metabolic imaging measures the fluorescent intensity of byproducts of the two metabolic pathways, flavin adenine dinucleotide for oxidative phosphorylation and nicotinamide adenine dinucleotide ± phosphatefor glycolysis, as well as the redox ratio between them. Images were taken at varied distances from the center of the impact. Shortly after impact, fluorescence intensity in both channels decreased, while redox ratio was unchanged. The most dramatic metabolic response was measured closest to the impact center, with suppressed fluorescence in both channels relative to baseline. Redox ratio varied nonlinearly as a function of distance from the impact. Finally, both lower and higher magnitude loading reduced flavin adenine dinucleotide fluorescence, whereas reduced nicotinamide adenine dinucleotide ± phosphatefluorescence was associated only with low strain loads and high contact pressure loads, respectively. In conclusion, this study performed novel analysis of metabolic activity following induction of cartilage damage and demonstrated time-, distance-, and load-dependent response to traumatic impact loading.

The early effects of uninephrectomy on rat kidney metabolic state using optical imaging.

Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney, although it is uncertain if this alters the mitochondrial redox state and how soon such changes may occur. A custom-designed fluorescence cryo-imaging technique was used to quantitatively assess the effect of UNX by measuring the levels of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the remaining kidney. Kidneys were snap-frozen 3 days following UNX, and the intrinsic fluorescence of NADH and FAD were optically acquired. The 3D images were created to characterize the NADH/FAD redox ratios (RR) of the right kidneys, which underwent UNX and the remaining kidneys 3 days following UNX. Both the NADPH-oxidases (Nox2 and Nox4) and the mitochondria are the main sources of reactive oxygen species (ROS) production in tubular epithelial cells. Responses to the UNX were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt-sensitive (SS) rats and SS rats in which NADPH-oxidase isoform 4 (Nox4) was knocked out (SSNox4-/- ). The results found that each of the strains exhibited similar increase in kidney weights averaging 17% after 3 days of UNX. SD and SSNox4-/- rats both exhibited global reductions of the RR (P < .05) with a similar tendency observed in SS rats (P < .08), indicating increased ROS production. The unexpected reduction of the RR in the remnant kidneys of SSNox4-/- rats indicates that mechanisms independent of H2 O2 produced from Nox4 may be responsible for this global increase of ROS. We propose that the reduced RR was largely a consequence of enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney. The data indicate that mitochondria become the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.

Flavin Adenine Dinucleotide Fluorescence as an Early Marker of Mitochondrial Impairment During Brain Hypoxia.

Multimodal continuous bedside monitoring is increasingly recognized as a promising option for early treatment stratification in patients at risk for ischemia during neurocritical care. Modalities used at present are, for example, oxygen availability and subdural electrocorticography. The assessment of mitochondrial function could be an interesting complement to these modalities. For instance, flavin adenine dinucleotide (FAD) fluorescence permits direct insight into the mitochondrial redox state. Therefore, we explored the possibility of using FAD fluorometry to monitor consequences of hypoxia in brain tissue in vitro and in vivo. By combining experimental results with computational modeling, we identified the potential source responsible for the fluorescence signal and gained insight into the hypoxia-associated metabolic changes in neuronal energy metabolism. In vitro, hypoxia was characterized by a reductive shift of FAD, impairment of synaptic transmission and increasing interstitial potassium [K+]o. Computer simulations predicted FAD changes to originate from the citric acid cycle enzyme α-ketoglutarate dehydrogenase and pyruvate dehydrogenase. In vivo, the FAD signal during early hypoxia displayed a reductive shift followed by a short oxidation associated with terminal spreading depolarization. In silico, initial tissue hypoxia followed by a transient re-oxygenation phase due to glucose depletion might explain FAD dynamics in vivo. Our work suggests that FAD fluorescence could be readily used to monitor mitochondrial function during hypoxia and represents a potential diagnostic tool to differentiate underlying metabolic processes for complementation of multimodal brain monitoring.

Decay Times and Anisotropy in Polarized Fluorescence of Flavin Adenine Dinucleotide Determined with Subnanosecond Resolution

Optical properties of biological coenzyme FAD in an aqueous solution have been studied. Two fluorescence lifetimes, a rotational diffusion time, and an anisotropy parameter have been determined from experiments by recording the decay of polarized fluorescence excited by picosecond laser pulses. The results obtained are discussed and compared with the results of other authors.

Fluorescence Quenching-Based Evaluation of Glucose Oxidase Composite with Conducting Polymer, Polypyrrole

Glucose oxidase (GOx) composites with conducting polymers (e.g., polypyrrole (Ppy)) are excellent nanobiomaterials suitable for the design of bioelectronic devices such as biosensors and biofuel cells. Here, we address the spectroscopic properties of GOx, flavin adenine dinucleotide (FAD), and composites of these compounds with polypyrrole (Ppy). The exploration of native GOx and FAD solutions confirmed that about 5% of FAD dissociated from GOx during the period of solution preparation, and this fraction remained constant for 1 month. It has been found that the Ppy, which formed composites with FAD and GOx, facilitated the removal of FAD molecules from GOx and twice reduced the fluorescence decay rate. Differences in the FAD and Ppy average fluorescence relaxation times showed that the FAD composite with Ppy and Ppy effectively quenched the FAD fluorescence and FAD could not freely unfold. The intramolecular electron transfer took place between adenine and isoalloxazine moieties over the first 5 ps after ...

Intrinsic fluorescence for cervical precancer detection using polarized light based in-house fabricated portable device

An in-house fabricated portable device has been tested to detect cervical precancer through the intrinsic fluorescence from human cervix of the whole uterus in a clinical setting. A previously validated technique based on simultaneously acquired polarized fluorescence and polarized elastic scattering spectra from a turbid medium is used to extract the intrinsic fluorescence. Using a diode laser at 405nm, intrinsic fluorescence of flavin adenine dinucleotide, which is the dominant fluorophore and other contributing fluorophores in the epithelium of cervical tissue, has been extracted. Different grades of cervical precancer (cervical intraepithelial neoplasia; CIN) have been discriminated using principal component analysis-based Mahalanobis distance and linear discriminant analysis. Normal, CIN I and CIN II samples have been discriminated from one another with high sensitivity and specificity at 95% confidence level. This ex vivo study with cervix of whole uterus samples immediately after hysterectomy in a clinical environment indicates that the in-house fabricated portable device has the potential to be used as a screening tool for in vivo precancer detection using intrinsic fluorescence.

Quantitative, Label-Free Evaluation of Tissue-Engineered Skeletal Muscle Through Multiphoton Microscopy.

The lack of tools for assessing engineered tissue viability and function in a noninvasive manner is a major regulatory and translational challenge facing tissue engineers. Label-free, nonlinear optical molecular imaging (OMI) has utilized endogenous nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence to indicate metabolic activity. Similarly, second harmonic generation (SHG) signals from myosin and collagen can measure overall muscle structural integrity and function. The purpose of this study was to demonstrate these OMI techniques for the first time in engineered skeletal muscle and to develop a novel method for evaluating our engineered skeletal muscle units (SMUs) before implantation. Three experimental groups were studied: Control, Steroid Supplemented, and Metabolically Stressed SMUs. After imaging and analysis in ImageJ, a redox ratio (RR) metric was calculated to indicate metabolic activity, and a structure ratio metric was calculated to reflect structural composition. In addition, function was evaluated as tetanic force production in response to electrical stimulation. In living tissues, the RRs successfully distinguished control and metabolically stressed SMUs in both monolayer and 3D form. OMI of myosin and collagen SHG similarly differentiated control SMUs from the steroid supplemented group. With respect to function, steroid supplementation significantly increased active force generation. When comparing functional and OMI measures, a significant correlation was present between overall myosin density and active force generation. This work demonstrates the potential for using label-free OMI to evaluate tissue-engineered skeletal muscle constructs. The positive correlation between structural OMI measures and force production suggests that OMI could potentially serve as an accurate predictor of functional behaviors, such as integration and tissue regeneration, after implantation. This noninvasive OMI methodology, demonstrated for the first time in engineered skeletal muscle, could prove invaluable for assessing our tissue engineering technology and confirming release criteria for validation.

On chip two-photon metabolic imaging for drug toxicity testing.

We have developed a microfluidic system suitable to be incorporated with a metabolic imaging method to monitor the drug response of cells cultured on a chip. The cells were perfusion-cultured to mimic the blood flow in vivo. Label-free optical measurements and imaging of nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence intensity and morphological changes were evaluated non-invasively. Drug responses calculated using redox ratio imaging were compared with the drug toxicity testing results obtained with a traditional well-plate system. We found that our method can accurately monitor the cell viability and drug response and that the IC50 value obtained from imaging analysis was sensitive and comparable with a commonly used cell viability assay: MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) assay. Our method could serve as a fast, non-invasive, and reliable way for drug screening and toxicity testing as well as enabling real-time monitoring of in vitro cultured cells.

A neuronal lactate uptake inhibitor slows recovery of extracellular ion concentration changes in the hippocampal CA3 region by affecting energy metabolism.

Astrocyte-derived lactate supports pathologically enhanced neuronal metabolism, but its role under physiological conditions is still a matter of debate. Here, we determined the contribution of astrocytic neuronal lactate shuttle for maintenance of ion homeostasis and energy metabolism. We tested for the effects of α-cyano-4-hydroxycinnamic acid (4-CIN), which could interfere with energy metabolism by blocking monocarboxylate-transporter 2 (MCT2)-mediated neuronal lactate uptake, on evoked potentials, stimulus-induced changes in K+, Na+, Ca2+, and oxygen concentrations as well as on changes in flavin adenine dinucleotide (FAD) autofluorescence in the hippocampal area CA3. MCT2 blockade by 4-CIN reduced synaptically evoked but not antidromic population spikes. This effect was dependent on the activation of KATP channels indicating reduced neuronal ATP synthesis. By contrast, lactate receptor activation by 3,5-dihydroxybenzoic acid (3,5-DHBA) resulted in increased antidromic and orthodromic population spikes suggesting that 4-CIN effects are not mediated by lactate accumulation and subsequent activation of lactate receptors. Recovery kinetics of all ion transients were prolonged and baseline K+ concentration became elevated by blockade of lactate uptake. Lactate contributed to oxidative metabolism as both baseline respiration and stimulus-induced changes in Po2 were decreased, while FAD fluorescence increased likely due to a reduced conversion of FAD into FADH2 These data suggest that lactate shuttle contributes to regulation of ion homeostatsis and synaptic signaling even in the presence of ample glucose.

Abstract 1673: Optical metabolic imaging of response to radiation in radiation-sensitive and resistant lung cancer cells

Abstract Tumor resistance to radiation therapy is a significant obstacle that patients and doctors face while treating tumors. Meaningful changes in therapy are contingent upon the ability to identify an unfavorable response very early (first week) in the course of treatment. Optical imaging can perform noninvasive, frequent and quantitative measurements of tumor biology at the point of care. The goal of this study was to identify optically measurable metabolic endpoints in a matched model of radiation resistance before and after treatment. A human lung cancer cell line was induced to develop radiation resistance through repeated exposure to a clinically relevant dose of X-ray radiation (2 Gy). We used a fluorescent glucose analog called 2-NBDG to quantify glucose uptake. We measured the endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), metabolic coenzymes found primarily in the cytoplasm and mitochondria, respectively. We determined the redox ratio [FAD/(NADH + FAD)] in both cell lines. We evaluated the behavior of the cellular antioxidant system by comparing the levels of reactive oxygen species (ROS) and ROS scavengers, such as glutathione (GSH) for each cell group. Finally, we analyzed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a metabolic flux analyzer. Radiation-resistant A549 lung cancer cells were induced by exposure to 25 fractions of X-ray radiation (2.2 Gy/fraction). Clonogenic assays were performed to confirm radiation resistance. All fluorescence images were obtained using a multiphoton microscope. 2-NBDG, ROS, GSH, NADH, and FAD fluorescence were excited at 960, 930, 780, 755 and 860 nanometers, respectively. The cells were exposed to 2 Gy 60 minutes prior to the Seahorse assay. All experiments were performed in both the parental A549 cell line (A549C) and the radiation-resistant A549 cell line (A549R) at baseline (prior to radiation) and after radiation. At baseline, the A549R cells revealed a significantly lower level of oxidative stress and a higher level of GSH. Interestingly, glucose uptake, quantified using 2-NBDG fluorescence, was significantly lower in the A549R cells compared to the A549C cells at baseline. The redox ratio was significantly higher in the A549R cells prior to radiation, indicating a preference for mitochondrial respiration. However, after radiation, the OCR of the A549R cells was significantly lower than the parental cell line, indicating a lower use of mitochondrial respiration. There were no significant differences in ECAR levels between both cell lines after radiation. Our results indicate that the radiation-resistant A549 lung cancer cells present very distinguishable metabolic properties at baseline and after exposure to radiation relative to the parental cell line that could potentially be exploited in vivo. Citation Format: Kinan Alhallak, Ruud P.M. Dings, Narasimhan Rajaram. Optical metabolic imaging of response to radiation in radiation-sensitive and resistant lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1673.

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO2 , field potentials and changes in extracellular potassium concentration ([K+ ]o ). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K+ ]o and pO2 changes as compared to continuous illumination.

Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

Optimization of sample cooling temperature for redox cryo-imaging.

Cryo-imaging techniques have been widely used to measure the metabolic state of tissues by capturing reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. However, NADH and FAD fluorescence is sensitive to changes in temperature, which may result in unreliable redox ratio calculations. Here, the relationship between the measured redox ratio and sample surface temperature was analyzed using a standard phantom solution and biological tissues. The results indicated that a temperature < - 100°C was a suitable cryo-imaging temperature window in which redox ratio measuring was immune to temperature fluctuations. These results may serve as a reference for designing and optimizing redox cryo-imaging experiments for quantitatively mapping the metabolic state of biological samples.