NADH Lifetime Research Articles

The Fluorescence Lifetime of Bound NADH: Clues from the Phasor Plots

The Fluorescence Lifetime of Bound NADH: Clues from the Phasor Plots

Characterization of the Metabolic State and Molecular Crowding in Breast Cancer Spheroids

Spheroids are 3D cultures of cells specifically grown in order to create a single spherical cell mass, which can be used as a model for studying the behavior of tumors in a controlled environment. Despite being a simplistic model, it allows for the rapid creation of the samples and displays a more complex physiology when compared to 2D cultures [1]. The spheroids grown for this study are derived from two different breast adenocarcinoma cell lines, MCF7 and MDA-MB231, which display less and more invasive behavior, respectively. From 20,000 cells, our protocol provides spheres with a diameter of approximately 400 μm. The spheres were then transferred to a collagen Type-1 gel matrix in order to mimic the extracellular matrix. Once obtained and transferred in the collagen, the spheres were observed at different time points over 24 hours. We measured their single cell metabolic signature by analyzing the NADH lifetime with the Phasor-FLIM approach [2] and assessed their molecular crowding using the ACDAN probe [3] and analyzing its emission spectrum. The main advantage of these techniques is the minimal interaction with the system, since NADH Phasor-FLIM is a label-free approach and ACDAN does not directly interact with the cellular machinery. Furthermore, both techniques can be implemented in a 2-photon microscope, which allows for deep sample penetration and reduced phototoxicity. Our preliminary results show a significant difference in the above-mentioned properties between the cells located at the center and at the periphery of the sphere. Furthermore, we could observe the early stage of metastatic behavior of our tumor model and analyze the characteristics of the cells invading the collagen matrix. [1] Costa et al Biotechnol. Adv. (2016) [2] Stringari et al Sci. Rep. (2012) [3] Thoke et al Sci. Rep. (2017)

Metabolic Profiling of Live Cancer Tissues Using NAD(P)H Fluorescence Lifetime Imaging.

Altered metabolism is a hallmark of cancer, both resulting from and driving oncogenesis. The NAD and NADP redox couples play a key role in a large number of the metabolic pathways involved. In their reduced forms, NADH and NADPH, these molecules are intrinsically fluorescent. As the average time for fluorescence to be emitted following excitation by a laser pulse, the fluorescence lifetime, is exquisitely sensitive to changes in the local environment of the fluorophore, imaging the fluorescence lifetime of NADH and NADPH offers the potential for label-free monitoring of metabolic changes inside living tumors. Here, we describe the biological, photophysical, and methodological considerations required to establish fluorescence lifetime imaging (FLIM) of NAD(P)H as a routine method for profiling the metabolism of living cancer cells and tissues.

Effect of Fixation and Mounting on Fluorescence Lifetime of Cellular Autofluorescence

Fluorescence lifetime measurements are often performed on live as well as fixed cells and tissues. Fixation and mounting processes are routinely used in cellular research or clinical diagnosis. In this paper, the effects of fixation and mounting on the fluorescence lifetime of cellular autofluorescence were studied by fluorescence lifetime imaging microscopy over time. Two endogenous fluorescent fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), showed different results between live cells and fixed cells. The average lifetime of NADH in live HeLa cells was about 1.02 ns, while maintained about 1.57 ns during the fixation periods of 14 days. The average lifetimes of FAD in live and fixed HeLa cells within 11 days were similar around 1.75 ns but increased to 2.10 ns after 12 days. The free and bound states of the two kinds of fluorophores were further analyzed. It was found that the bound-FAD had two different groups, which was related to the cell division cycle. The effect of mounting medium on fluorescence lifetimes was also studied, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.

Collagen density modulates triple-negative breast cancer cell metabolism through adhesion-mediated contractility

Extracellular matrix (ECM) mechanical properties upregulate cancer invasion, cell contractility, and focal adhesion formation. Alteration in energy metabolism is a known characteristic of cancer cells (i.e., Warburg effect) and modulates cell invasion. There is little evidence to show if collagen density can alter cancer cell metabolism. We investigated changes in energy metabolism due to collagen density in five breast cell lines by measuring the fluorescence lifetime of NADH. We found that only triple-negative breast cancer cells, MDA-MB231 and MDA-MB468 cells, had an increased population of bound NADH, indicating an oxidative phosphorylation (OXPHOS) signature, as collagen density decreased. When inhibiting ROCK and cell contractility, MDA-MB231 cells on glass shifted from glycolysis (GLY) to OXPHOS, confirming the intricate relationship between mechanosensing and metabolism. MCF10A cells showed less significant changes in metabolism, shifting towards GLY as collagen density decreased. The MCF-7 and T-47D, less invasive breast cancer cells, compared to the MDA-MB231 and MDA-MB468 cells, showed no changes regardless of substrate. In addition, OXPHOS or GLY inhibitors in MDA-MB231 cells showed dramatic shifts from OXPHOS to GLY or vice versa. These results provide an important link between cellular metabolism, contractility, and collagen density in human breast cancer.

Abstract 3472: Dual PI3K/mTOR inhibition in PIK3CA mutant pancreatic cancers

Abstract Background: Pancreatic cancer is one of the most deadly cancer diagnoses with limited long-term survival for patients, even with early-stage disease. 3-5% of these cancers have mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway. There is growing interest in targeting cancers with this mutation using specific pathway inhibitors. We have previously proven that dual PI3K/mTOR inhibition is sufficient to decrease tumor growth in mice with a PI3K p110* transgene. Here we aim to determine if this treatment can induce a response in pancreatic cancers with the human PIK3CA H1047R hotspot mutation. Methods: Transgenic mice were developed using a cre lox-p system (Pdx1-cre) to develop PIK3CA H1047R (PCPK mice) pancreatic adenocarcinomas. Tumors were harvested and grown in spheroid culture. These were treated with increasing concentrations of BEZ235, a dual PI3K/mTOR inhibitor. Treatment groups were compared using spheroid diameter changes and metabolic activity via NADH and FAD+ quantitation. Additionally, immunofluorescence staining was performed and quantified on treated spheroid cultures. PET/CT diagnostics were performed and analyzed pre- and post- treatment with BEZ-235 on a cohort of these experimental mice. Treated tissues were resected for immunohistochemistry and Western blot analyses. Results: Organotypic PCPK spheroid cultures were treated with BEZ235 in the culture media for 48 hours. Pre- and post-brightfield imaging demonstrated a modest reduction in the percent change in spheroid diameter compared to control (control: 41.8%, BEZ235 100nM: 9%, BEZ235 200nM: -7.7%, BEZ235 400nM: -14.3%; p<0.001). This stability of the spheroid size correlated with minimal differences between the control and treated spheroids using the optical metabolic imaging, including nonsignificant changes in NADH lifetime, FAD+ lifetime and the optical redox ratio (p=NS). PCPK mice (n=10) aged until they developed cancers and then were treated with BEZ235 for 14 days. Pre- and post-positron emission tomography/computed tomography (PET/CT) were performed. Nonsignificant changes in tumor volume and PET avidity were seen between the treatment groups. Both in vitro and in vivo treatments with BEZ235 proved to have no significant change in tumor avidity in this model. Immunoblotting demonstrated persistent activation of the PI3K pathway despite BEZ235 treatment in these spheres and mice as measured by persistent phosphorylation of RPS6 and 4EBP1. Conclusions: Despite significant responses being previously detected in mice with pancreatic tumors initiated by the PI3K p110* transgene, BEZ235 did not result in a similar treatment effect in PCPK mice possessing the human PIK3CA H1047R hotspot mutation. Further studies will examine the potential resistance mechanisms of these tumors to this therapy and potential ways in which to overcome this resistance. Citation Format: Susan Payne, Mitchell Depke, Alex Yueh, Joseph T. Sharick, Peter F. Favreau, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Melissa Skala, Dustin A. Deming. Dual PI3K/mTOR inhibition in PIK3CA mutant pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3472.

Fluorescence Lifetime of NADH Reveals PARP-Dependent Increase of Oxidative Phosphorylation Critical for Cell Survival

Fluorescence Lifetime of NADH Reveals PARP-Dependent Increase of Oxidative Phosphorylation Critical for Cell Survival

Effects of Nanosecond Pulsed Electric Field on Intracellular NADH Autofluorescence: A Comparison between Normal and Cancer Cells.

Intracellular fluorescence lifetime and intensity images of the endogenous fluorophore of nicotinamide adenine dinucleotide (NADH) have been observed before and after application of nanosecond pulsed electric field (nsPEF) in normal and cancer cells, that is, in Wistar-King-Aptekman rat fetus fibroblast (WFB) cells and W31 cells, which are the malignant transformed cells from WFB. The application of nsPEF induces a change both in intensity and lifetime of NADH, indicating that the intracellular function is affected by application of nsPEF in both normal and cancer cells. The application of nsPEF induces an increase in the fluorescence lifetime of NADH and a morphological change, which is attributed to the induction of apoptosis by nsPEF. The field effect on the intensity and lifetime clearly depends on the pulse width, and magnitude of the field-induced increase in the fluorescence lifetime of NADH has a tendency to increase with a decreasing pulse width. It is also found that apoptosis can be induced only in cancer cells using a suitable nsPEF, showing a possibility that ultrashort pulsed electric field is applicable for drug-free cancer therapy.

Autofluorescence imaging captures heterogeneous drug response differences between 2D and 3D breast cancer cultures.

Two-photon microscopy of cellular autofluorescence intensity and lifetime (optical metabolic imaging, or OMI) is a promising tool for preclinical drug development. OMI, which exploits the endogenous fluorescence from the metabolic coenzymes NAD(P)H and FAD, is sensitive to changes in cell metabolism produced by drug treatment. Previous studies have shown that drug response, genetic expression, cell-cell communication, and cell signaling in 3D culture match those of the original in vivo tumor, but not those of 2D culture. The goal of this study is to use OMI to quantify dynamic cell-level metabolic differences in drug response in 2D cell lines vs. 3D organoids generated from xenograft tumors of the same cell origin. BT474 cells and Herceptin-resistant BT474 (HR6) cells were tested. Cells were treated with vehicle control, Herceptin, XL147 (PI3K inhibitor), and the combination. The OMI index was used to quantify response, and is a linear combination of the redox ratio (intensity of NAD(P)H divided by FAD), mean NADH lifetime, and mean FAD lifetime. The results confirm that the OMI index resolves significant differences (p<0.05) in drug response for 2D vs. 3D cultures, specifically for BT474 cells 24 hours after Herceptin treatment, for HR6 cells 24 and 72 hours after combination treatment, and for HR6 cells 72 hours after XL147 treatment. Cell-level analysis of the OMI index also reveals differences in the number of cell sub-populations in 2D vs. 3D culture at 24, 48, and 72 hours post-treatment in control and treated groups. Finally, significant increases (p<0.05) in the mean lifetime of NADH and FAD were measured in 2D vs. 3D for both cell lines at 72 hours post-treatment in control and all treatment groups. These whole-population differences in the mean NADH and FAD lifetimes are supported by differences in the number of cell sub-populations in 2D vs. 3D. Overall, these studies confirm that OMI is sensitive to differences in drug response in 2D vs. 3D, and provides further information on dynamic changes in the relative abundance of metabolic cell sub-populations that contribute to this difference.

Abstract B36: Interrogating cellular metabolism to improve therapy for EGFR mutant NSCLC

Abstract Tyrosine kinase inhibitors (TKIs) that target the EGFR are the standard of care for patients with EGFR mutant non-small lung cancer (NSCLC). However, TKIs are not curative; most patients with EGFR-mutant NSCLC treated with EGFR TKIs develop resistance within 9 to 14 months. The tumor microenvironment is thought to influence the fitness of cancer cells and drive the evolution of a tumor, but the effect of altered environmental conditions on oncogenic signaling and therapeutic response is not well understood. In the present study, we used the Operetta® High Content Imaging System to track cell dynamics over time and found that microenvironmental stress, specifically nutrient starvation, had a differential effect on growth rates of TKIs sensitive and resistant cells. Furthermore, we discovered that TKIs sensitive and resistant cells have differential metabolic signaling in response to this microenvironmental stress. To further interrogate cellular metabolism in TKIs sensitive and resistant cells, we utilized fluorescence lifetime imaging microscopy (FLIM), a state-of-the-art live-cell imaging technique to measure the autofluorescence lifetime of metabolic coenzyme NADH, the principle electron acceptor in glycolysis and electron donor in oxidative phosphorylation. Taken together, our data suggest that TKIs sensitive and resistant cells have adopted different metabolic strategies to cope with stressful environmental conditions, and interrogating tumor metabolic adaptations would inform the design of new therapeutic strategies to delay or prevent the outgrowth of resistant clones during treatment of EGFR mutant NSCLC. Citation Format: Chun-Te Chiang, Chi-Li Chiu, Cosimo Arnesano, Scott E. Fraser, David B. Agus, Dan L. Ruderman, Shannon M. Mumenthaler. Interrogating cellular metabolism to improve therapy for EGFR mutant NSCLC. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B36.

SU‐G‐TeP3‐10: Radiation Induces Prompt Live‐Cell Metabolic Fluxes

Purpose:To compare metabolic dynamics and HIF‐1α expression following radiation between a cancerous cell line (UM‐SCC‐22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF‐1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF‐1 might give a better understanding of the mechanisms responsible for radiosensitivity.Methods:Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH. Expression of HIF‐1α was measured by immunofluorescence for both cell lines with and without irradiation. Radiation response is also monitored with additional treatment of a HIF‐1α inhibitor (chrysin) as well as a radical scavenger (glutathione). Changes in oxygen consumption and respiratory capacity are also monitored using the Seahorse XF analyzer.Results:An increase in HIF‐1α was found to be in response to radiation for the cancer cell line, but not the normal cell line. Radiation was found to shift metabolism toward glycolytic pathways in cancer cells as measured by oxygen consumption and respiratory capacity. Radiation response was found to be muted by addition of glutathione to cell media. HIF‐1α inhibition similarly muted radiation response in cancer.Conclusion:The HIF‐1 protein complex is a key regulator cellular metabolism through the regulation of glycolysis and glucose transport enzymes. Moreover, HIF‐1 has shown radio‐protective effects in tumor vascular endothelia, and has been implicated in metastatic aggression. Monitoring interplay between metabolism and the HIF‐1 protein complex can give a more fundamental understanding of radiotherapy response.

Label-free in vivo cellular-level detection and imaging of apoptosis.

Cell death plays a critical role in health and homeostasis as well as in the pathogenesis and treatment of a broad spectrum of diseases and can be broadly divided into two main categories: apoptosis, or programmed cell death, and necrosis, or acute cell death. While these processes have been characterized extensively in vitro, label-free detection of apoptosis and necrosis at the cellular level in vivo has yet to be shown. In this study, for the first time, fluorescence lifetime imaging microscopy (FLIM) of intracellular reduced nicotinamide adenine dinucleotide (NADH) was utilized to assess the metabolic response of in vivo mouse epidermal keratinocytes following induction of apoptosis and necrosis. Results show significantly elevated levels of both the mean lifetime of NADH and the intracellular ratio of protein bound-to-free NADH in the apoptotic compared to the necrotic tissue. In addition, the longitudinal profiles of these two cell death processes show remarkable differences. By identifying and extracting these temporal metabolic signatures, apoptosis in single cells can be studied in native tissue environments within the living organism.

Longitudinal in vivo tracking of adverse effects following topical steroid treatment.

Topical steroids are known for their anti-inflammatory properties and are commonly prescribed to treat many adverse skin conditions such as eczema and psoriasis. While these treatments are known to be effective, adverse effects including skin atrophy are common. In this study, the progression of these effects is investigated in an in vivo mouse model using multimodal optical microscopy. Utilizing a system capable of performing two-photon excitation fluorescence microscopy (TPEF) of reduced nicotinamide adenine dinucleotide (NADH) to visualize the epidermal cell layers and second harmonic generation (SHG) microscopy to identify collagen in the dermis, these processes can be studied at the cellular level. Fluorescence lifetime imaging microscopy (FLIM) is also utilized to image intracellular NADH levels to obtain molecular information regarding metabolic activity following steroid treatment. In this study, fluticasone propionate (FP)-treated, mometasone furoate (MF)-treated and untreated animals were imaged longitudinally using a custom-built multimodal optical microscope. Prolonged steroid treatment over the course of 21 days is shown to result in a significant increase in mean fluorescence lifetime of NADH, suggesting a faster rate of maturation of epidermal keratinocytes. Alterations to collagen organization and the structural microenvironment are also observed. These results give insight into the structural and biochemical processes of skin atrophy associated with prolonged steroid treatment.

Ototoxic Aminoglycosides Inhibit NADH Metabolism and Increase Reactive Oxygen Species Formation in Cochlear Cells

Despite causing permanent hearing loss by damaging inner ear sensory cells, aminoglycosides (AGs) remain one of the most widely used classes of antibiotics in the world. Although the mechanisms of cochlear sensory cell damage are not fully known, reactive oxygen species (ROS) may be involved. Mitochondrial-specific ROS formation was evaluated in acutely-cultured murine cochlear explants exposed to gentamicin (GM), a representative ototoxic AG antibiotic. Superoxide (O2.-) and hydrogen peroxide (H2O2) ROS were measured using MitoSox Red and Dihydrorhodamine 123, respectively. Acute GM did not significantly increase O2.-formation but did increase H2O2 formation in all cell types within one hour of exposure. At the same point, GM significantly increased manganese superoxide dismutase (mnSOD) levels while significantly decreasing copper/zinc superoxide dismutase (cuznSOD) in cochlear sensory cells. This suggests 1) a rapid conversion of highly reactive O2.-to H2O2 during the acute stage of ototoxic antibiotic exposure and 2) that the endogenous antioxidant system is significantly altered by ototoxic antibiotics. Fluorescence intensity-based measurements of nicotinamide adenine dinucleotide (NADH) were used to determine if increased ROS production was correlated with acute GM-induced changes in mitochondrial metabolism. Acute exposure also triggered a redistribution of NADH in the seven distinct NADH lifetime pools identified in cochlear cells. This project provides a base for understanding the mechanisms of mitochondrial ROS production in cochlear cells exposed to ototoxic antibiotics. Understanding the nature of ototoxic antibiotic-induced changes in mitochondrial metabolism are critical for developing hearing loss treatment and prevention strategies. Supported by the National Institute on Deafness and Other Communication Disorders (NIDCD,RO3DC012109), and COBRe (8P20GM103471-09) to HJS and a Ferlic Undergraduate Research Scholarship to DD.

Quantification of the Metabolic State in Cell-Model of Parkinson's Disease by Fluorescence Lifetime Imaging Microscopy.

Intracellular endogenous fluorescent co-enzymes, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), play a pivotal role in cellular metabolism; quantitative assessment of their presence in living cells can be exploited to monitor cellular energetics in Parkinson’s disease (PD), a neurodegenerative disorder. Here, we applied two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to noninvasively measure the fluorescence lifetime components of NADH and FAD, and their relative contributions in MPP+ (1-methyl-4-phenylpyridinium) treated neuronal cells, derived from PC12 cells treated with nerve growth factor (NGF), to mimic PD conditions. A systematic FLIM data analysis showed a statistically significant (p < 0.001) decrease in the fluorescence lifetime of both free and protein-bound NADH, as well as free and protein-bound FAD in MPP+ treated cells. On the relative contributions of the free and protein-bound NADH and FAD to the life time, however, both the free NADH contribution and the corresponding protein-bound FAD contribution increase significantly (p < 0.001) in MPP+ treated cells, compared to control cells. These results, which indicate a shift in energy production in the MPP+ treated cells from oxidative phosphorylation towards anaerobic glycolysis, can potentially be used as cellular metabolic metrics to assess the condition of PD at the cellular level.

SU‐C‐303‐02: Correlating Metabolic Response to Radiation Therapy with HIF‐1alpha Expression

Purpose:To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient‐specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM‐SCC‐22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF‐1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient‐specific adaptive radiotherapy.Methods:Measurements of metabolism and HIF‐1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x‐ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF‐1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package.Results:FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF‐1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles.Conclusion:Radiation induces significant changes in metabolic activity and HIF‐1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

Quantitative optical imaging of primary tumor organoid metabolism predicts drug response in breast cancer.

There is a need for technologies to predict the efficacy of cancer treatment in individual patients. Here, we show that optical metabolic imaging of organoids derived from primary tumors can predict the therapeutic response of xenografts and measure antitumor drug responses in human tumor-derived organoids. Optical metabolic imaging quantifies the fluorescence intensity and lifetime of NADH and FAD, coenzymes of metabolism. As early as 24 hours after treatment with clinically relevant anticancer drugs, the optical metabolic imaging index of responsive organoids decreased (P < 0.001) and was further reduced when effective therapies were combined (P < 5 × 10(-6)), with no change in drug-resistant organoids. Drug response in xenograft-derived organoids was validated with tumor growth measurements in vivo and staining for proliferation and apoptosis. Heterogeneous cellular responses to drug treatment were also resolved in organoids. Optical metabolic imaging shows potential as a high-throughput screen to test the efficacy of a panel of drugs to select optimal drug combinations. Cancer Res; 74(18); 5184-94. ©2014 AACR.

Time-resolved fluorescence lifetime for cutaneous melanoma detection.

Melanoma is the most aggressive skin cancer type. It is characterized by pigmented lesions with high tissue invasion and metastatic potential. The early detection of melanoma is extremely important to improve patient prognosis and survival rate, since it can progress to the deadly metastatic stage. Presently, the melanoma diagnosis is based on the clinical analysis of the macroscopic lesion characteristics such as shape, color, borders following the ABCD rules. The aim of this study is to evaluate the time-resolved fluorescence lifetime of NADH and FAD molecules to detect cutaneous melanoma in an experimental in vivo model. Forty-two lesions were analyzed and the data was classified using linear discriminant analysis, a sensitivity of 99.4%, specificity of 97.4% and accuracy of 98.4% were achieved. These results show the potential of this fluorescence spectroscopy for melanoma detection.

WE‐E‐BRE‐12: Tumor Microenvironment Dynamics Following Radiation

Purpose:This work aims to understand the radiation‐induced interplay between tumor oxygenation and metabolic activity. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient‐specific adaptive radiotherapy.Methods:Using patient‐derived xenografts of head and neck cancer we assessed tumor oxygenation via fiber‐optic probe monitored hemoglobin saturation and Blood Oxygen Level Dependent (BOLD) MRI. Measurements were taken before and after a 10 Gy dose of radiation. Changes in metabolic activity were measured via Fluorescence Lifetime IMaging (FLIM) with the appropriate controls following a 10 Gy dose of radiation. FLIM can non‐invasively monitor changes in fluorescence in response to the microenvironment including being able to detect free and bound states of the intrinsically fluorescent metabolite NADH (Nicotinamide Adenine Dinucleotide). With this information FLIM can accurately quantify the metabolic state of cells that have been radiated. To model the observed changes, a two‐compartment, source‐sink simulation relating hemoglobin saturation and metabolic activity was performed using MATLAB.Results:Hemoglobin saturation as measured by interstitial probe and BOLD‐MRI decreased by 30% within 15 minutes following radiation. FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways. Simulation of radiation‐induced alterations in tumor oxygenation demonstrated that these changes can be the result of changes in either vasculature or metabolic activity.Conclusion:Radiation induces significant changes in hemoglobin saturation and metabolic activity. These alterations occur on time scales approximately the duration of common radiation treatments. Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

Studies on external electric field effects on absorption and fluorescence spectra of NADH

Electric field effects on absorption and fluorescence spectra have been investigated for NADH that is a representative autofluorescent chromophore in cells. The change in electric dipole moment following absorption is significant in the electroabsorption spectrum, indicating charge transfer character in the excited state. The fluorescence intensity decreases in the presence of an electric field, which arises from the field-induced increase in the rate of the non-radiative process. The blue shift of the fluorescence spectrum and the increase in the fluorescence lifetime of NADH are measured in yeast cells, which is discussed in terms of a local electric field around NADH.