NADH Imaging Research Articles

Multimodal Phasor Approach to study breast cancer cells invasion in 3D spheroid model.

We implemented a multimodal set of functional imaging techniques optimized for deep-tissue imaging to investigate how cancer cells invade surrounding tissues and how their physiological properties change in the process. As a model for cancer invasion of the extracellular matrix, we created 3D spheroids from triple-negative breast cancer cells (MDA-MB-231) and non-tumorigenic breast epithelial cells (MCF-10A). We analyzed multiple hallmarks of cancer within the same spheroid by combining a number of imaging techniques, such as metabolic imaging of NADH by Fluorescence Lifetime Imaging Microscopy (NADH-FLIM), hyperspectral imaging of a solvatochromic lipophilic dye (Nile Red) and extracellular matrix imaging by Second Harmonic Generation (SHG). We included phasor-based bioimage analysis of spheroids at three different time points, tracking both morphological and biological properties, including cellular metabolism, fatty acids storage, and collagen organization. Employing this multimodal deep-imaging framework, we observed and quantified cancer cell plasticity in response to changes in the environment composition.

The use of NADH anisotropy to investigate mitochondrial cristae alignment

Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340–360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative—especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.

Abstract A037: Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer

Abstract Metastatic breast cancer is currently the most deadly and incurable stage of breast cancer. A recent metabolic screen has identified sialic acid as a key player in progression of breast cancer metastasis. In the present study, we metabolically target the hypersialylation of metastatic breast cancer cells using appropriately designed nutritionary interventions. Our rationales are the following: (a) The aberrantly sialylated, hyperbranched glycocalyx promotes metastasis by facilitating cellular migration and immune evasion. (b) As hypersialylation relies heavily on enhanced sialic acid biosynthesis from the oncogenic glycolytic surge and preferential dietary uptake of glucose by cancer cells, it stands to reason that dietary changes can impact the extent of sialylation of the glycocalyx. Considering these points, we hypothesized that it was possible to control the degree of glycocalyx sialylation in breast cancer cells using an appropriate nutritionary regimen, which will in turn modulate the progression to invasive phenotype. The goal is to harness the metabolism of cancer cells themselves to curb their metastatic potential. Using a specific ketone-body forming metabolite called 3-hydroxybutyrate (HB) and lowering of glucose concentration, the cell culture medium was modified to mimic ketogenic diet. The response of metastatic breast cancer cells and normal breast epithelial cells was then monitored. The two main measures of cellular response were: cell surface sialylation via fluorescence microscopy and fluorescence lifetime imaging (FLIM) of intracellular NADH as a marker of metabolic index. We are currently investigating the consequences of nutritionary interventions on metastatic potential of breast cancer cells by measuring their impact on cellular proliferation, clonogenicity and migratory abilities. We have observed that the main metabolite of ketogenic diet, HB, tends to protect metastatic breast cancer cells from DOX treatment when supplemented in a low-glucose (LG) medium. However, the extent of increase in cell surface sialylation is very modest in comparison to that in normal breast epithelial cells. This differential increase in sialylation implies that a ketogenic diet might significantly promote the proliferation of healthy cells over the proliferation of metastatic breast cancer cells. FLIM measurements indicate that the chemotherapeutic, Doxorubicin tends to induce an initial preference for glycolysis in invasive breast cancer cells. While an HB rich LG media by itself promotes glycolysis in these cells, it suppresses any further Doxorubicin induced onset of glycolysis pointing to a protective mechanism against the chemotherapeutic. Through this ongoing study, we aim to decipher the right set of nutrients that would “deshield” the breast cancer cells and lower their metastatic disposition. The ultimate goal of our research is to determine a paradigm of drugs and diet that metastatic breast cancer cells are most responsive to, and that would translate into a viable remedy for metastatic breast cancer with minimal side-effects. Citation Format: Mohini Kamra, Yuan-I Chen, Paula C Delgado, Tim Yeh, Amy Brock, Sapun H Parekh. Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A037.

Abstract 2461: Integrated lifetime and spectral phasor imaging of biomarker expression and metabolism in hormone receptor positive breast cancer models

Abstract In HR+ (hormone receptor positive) breast cancer, endocrine therapies that directly antagonize estrogen receptor or reduce synthesis of its ligand can be highly effective, but endocrine-resistance occurs frequently. Metabolic dysregulation is a mechanistic driver of endocrine-resistant HR+ breast cancer, but most existing methods cannot be multiplexed with assays for biomarker expression. Spectral phasor imaging of multiplex immunofluorescence (mIF) staining for expression of hormone receptors and other biomarkers combined with phasor lifetime analysis of NADH to quantify metabolic state will provide integrated spatial maps of metabolism and biomarker expression in endocrine-resistant HR breast cancer. We used the HCI-013 (013) and HCI-013EI (013EI) patient-derived xenograft (PDX) system, derived from a HR+, invasive lobular metastatic breast cancer which harbors the Y537S ESR1 mutation. Tumors were stained with a multiplex immunofluorescence (mIF) panel for pan-cytokeratin, ER, PR, HER2, and Ki67, detected by spectrally separate OPAL dyes. Hyperspectral phasor fluorescence spectra were obtained using successive single photon excitation at 488 nm, 561 nm and 640 nm and using multiphoton excitation at 1024 nm. Single photon excitation excites specific dyes, and multiphoton excites the entire panel of dyes simultaneously. The emission was collected using a Zeiss 880 laser scanning microscope equipped with a 32 channel spectral detector. Phasor coordinates of each component were resolved into fractional intensities of the five genes. In the same tissues and sections, phasor fluorescence lifetime imaging (FLIM) of NADH was used to quantify metabolic heterogeneity. NADH is present in two forms: free and protein-bound NADH. They have different fluorescence lifetimes and the ratio of these two species are correlated with NAD+/NADH ratio. Free NADH is associated with increased glycolysis. We observed greater metabolic heterogeneity in 013EI PDX samples, with similar levels of free NADH (glycolysis) at the tumor border, but significantly increased bound NADH (oxidative phosphorylation) in the tumor core. Spectral phasor data show differential expression of PR and PanCK (for 640 nm excitation), and HER2 and ERalpha (for 561 nm excitation) in the edge and center of cancer in 013 and 013EI variants. Our results show how a combination of spectral phasor and phasor-FLIM can be used to define metabolic changes in endocrine-resistant, HR+ PDX models and correlate them with biomarker expression. Ongoing work is quantitatively mapping these measurements at single-pixel resolution, and applying this imaging workflow to RNAscope in situ hybridization assays for gene expression. Combining multiphoton spectral (RNAscope) and FLIM (NADH) will allow pixelwise correlation of the gene expression and metabolic changes. Citation Format: Belen Torrado, Alexander Vallmitjana, Ayodeji Olukoya, Shaymaa Bahnassy, Hillary Stires, Aaron Rozeboom, Rebecca B. Riggins, Suman Ranjit. Integrated lifetime and spectral phasor imaging of biomarker expression and metabolism in hormone receptor positive breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2461.

Luminescent Turn-On Upconversion Nanoprobes for Monitoring NADH In Vivo

Mitochondrial damage is implicated with a variety of pathological processes and is featured by the dramatically raised NADH level. To date, the detection and in situ imaging of NADH in living animals is still a challenge. Herein, we designed an upconversion luminescent nanoprobe for the in vivo imaging of NADH. The Nile blue (NB) with a strong absorption band at 500–700 nm can quench the red luminescence of the upconversion nanoparticle at 654 nm by the fluorescence resonance energy transfer effect. Under the presence of NADH, the NB was bleached, so that the fluorescence of the probe was “turned on”. The nanoprobe achieved highly selective detection of NADH, and the in vivo monitoring of NADH was also demonstrated on living mice.

A coarse-grained NADH redox model enables inference of subcellular metabolic fluxes from fluorescence lifetime imaging.

Mitochondrial metabolism is of central importance to diverse aspects of cell and developmental biology. Defects in mitochondria are associated with many diseases, including cancer, neuropathology, and infertility. Our understanding of mitochondrial metabolism in situ and dysfunction in diseases are limited by the lack of techniques to measure mitochondrial metabolic fluxes with sufficient spatiotemporal resolution. Herein, we developed a new method to infer mitochondrial metabolic fluxes in living cells with subcellular resolution from fluorescence lifetime imaging of NADH. This result is based on the use of a generic coarse-grained NADH redox model. We tested the model in mouse oocytes and human tissue culture cells subject to a wide variety of perturbations by comparing predicted fluxes through the electron transport chain (ETC) to direct measurements of oxygen consumption rate. Interpreting the fluorescence lifetime imaging microscopy measurements of NADH using this model, we discovered a homeostasis of ETC flux in mouse oocytes: perturbations of nutrient supply and energy demand of the cell do not change ETC flux despite significantly impacting NADH metabolic state. Furthermore, we observed a subcellular spatial gradient of ETC flux in mouse oocytes and found that this gradient is primarily a result of a spatially heterogeneous mitochondrial proton leak. We concluded from these observations that ETC flux in mouse oocytes is not controlled by energy demand or supply, but by the intrinsic rates of mitochondrial respiration.

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots

AbstractReduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in living cells due to its role as an electron carrier in redox reactions, and its concentration is an important indicator of cell metabolic state. Abnormal NADH levels are associated with age‐related metabolic diseases and neurodegenerative disorders, creating a demand for a simple, rapid analytical method for point‐of‐care NADH sensing. Here we develop a series of NADH‐sensitive semiconducting polymer dots (Pdots) as nanoprobes for NADH measurement, and test their performance in vitro and in vivo. NADH sensing is based on electron transfer from semiconducting polymer chains in the Pdot to NADH upon UV excitation, quenching Pdot fluorescence emission. In polyfluorene‐based Pdots, this mechanism resulted in an on‐off NADH sensor; in DPA‐CNPPV Pdots, UV excitation resulted in NADH‐sensitive emission at two wavelengths, enabling ratiometric detection. Ratiometric NADH detection using DPA‐CNPPV Pdots exhibits high sensitivity (3.1 μM limit of detection), excellent selectivity versus other analytes, reversibility, and a fast response (less than 5 s). We demonstrate applications of the ratiometric NADH‐sensing Pdots including smartphone‐based NADH imaging for point‐of‐care use.

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots.

Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in living cells due to its role as an electron carrier in redox reactions, and its concentration is an important indicator of cell metabolic state. Abnormal NADH levels are associated with age-related metabolic diseases and neurodegenerative disorders, creating a demand for a simple, rapid analytical method for point-of-care NADH sensing. Here we develop a series of NADH-sensitive semiconducting polymer dots (Pdots) as nanoprobes for NADH measurement, and test their performance in vitro and in vivo. NADH sensing is based on electron transfer from semiconducting polymer chains in the Pdot to NADH upon UV excitation, quenching Pdot fluorescence emission. In polyfluorene-based Pdots, this mechanism resulted in an on-off NADH sensor; in DPA-CNPPV Pdots, UV excitation resulted in NADH-sensitive emission at two wavelengths, enabling ratiometric detection. Ratiometric NADH detection using DPA-CNPPV Pdots exhibits high sensitivity (3.1 μM limit of detection), excellent selectivity versus other analytes, reversibility, and a fast response (less than 5 s). We demonstrate applications of the ratiometric NADH-sensing Pdots including smartphone-based NADH imaging for point-of-care use.

Simultaneous Two-Photon Fluorescence Microscopy of NADH and FAD Using Pixel-to-Pixel Wavelength-Switching

Two-photon fluorescence (TPF) microscopy of intrinsic fluorophores provides physiological and pathological information from biological tissues. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are two endogenous fluorescent coenzymes existing on the intracellular scale. Autofluorescence images of NADH and FAD have been applied to noninvasively record changes during metabolism, according to their distributions and concentrations. However, the widely used sequential (non-simultaneous) excitation scheme results in artifacts caused by sample motion or laser power fluctuation. The single-wavelength illumination scheme suffers from low excitation efficiency and spectral bleed-through. In this paper, we demonstrate a new imaging system simultaneously capturing autofluorescence images from NADH and FAD, with high excitation efficiency and negligible spectral bleed-through. Two temporally multiplexed and spatially overlapped excitation beams were achieved with fast-switching light paths based on an electro-optic modulator. The switching beams were centered at 750 and 860 nm, enabling independent excitations of NADH and FAD. Autofluorescence images of NADH and FAD were acquired at the wavelength ranges of 415–455 nm and 500–550 nm, respectively. The electro-optic modulator was synchronized with the pixel clock from the microscope, achieving pixel-to-pixel wavelength-switching. The capability of the system was demonstrated by performing TPF imaging of freshly excised mouse colon tissues. The microenvironment of the colon wall was depicted by the distributions of colonocytes, goblet cells, and crypts of Lieberkühn, and the relative concentrations of NADH and FAD were estimated. The experimental results show that the system can effectively perform simultaneous imaging of NADH and FAD, and is considered a promising tool for investigations into metabolism-associated processes and diseases.

Macroscopic fluorescence-lifetime imaging of NADH and protoporphyrin IX improves the detection and grading of 5-aminolevulinic acid-stained brain tumors

Maximal safe tumor resection remains the key prognostic factor for improved prognosis in brain tumor patients. Despite 5-aminolevulinic acid-based fluorescence guidance the neurosurgeon is, however, not able to visualize most low-grade gliomas (LGG) and infiltration zone of high-grade gliomas (HGG). To overcome the need for a more sensitive visualization, we investigated the potential of macroscopic, wide-field fluorescence lifetime imaging of nicotinamide adenine dinucleotide (NADH) and protoporphyrin IX (PPIX) in selected human brain tumors. For future intraoperative use, the imaging system offered a square field of view of 11 mm at 250 mm free working distance. We performed imaging of tumor tissue ex vivo, including LGG and HGG as well as brain metastases obtained from 21 patients undergoing fluorescence-guided surgery. Half of all samples showed visible fluorescence during surgery, which was associated with significant increase in PPIX fluorescence lifetime. While the PPIX lifetime was significantly different between specific tumor tissue types, the NADH lifetimes did not differ significantly among them. However, mainly necrotic areas exhibited significantly lower NADH lifetimes compared to compact tumor in HGG. Our pilot study indicates that combined fluorescence lifetime imaging of NADH/PPIX represents a sensitive tool to visualize brain tumor tissue not detectable with conventional 5-ALA fluorescence.

Abstract 5727: Metabolic status and adaptability of breast cancer stem cells

Abstract Breast cancer stem cells (BCSCs) represent the subpopulation of malignant cells that cause tumor initiation, metastasis, and recurrence. BCSCs resist therapy with radiation and standard drugs, emphasizing the need to identify new vulnerabilities as therapeutic targets. Here, we investigated the metabolism of BCSCs at single-cell resolution using molecular imaging and scRNA sequencing techniques. We imaged retention of the fluorescent dye PKH26 or expression of a CRISPR/Cas9-engineered ALDH1A3-mCherry promoter-reporter to identify BCSCs and metabolic state of cells by two-photon microscopy with fluorescence lifetime imaging (FLIM) of endogenous NADH. We previously reported that sorted BCSCs exhibited enhanced metabolic plasticity relative to bulk tumor cells in 2D culture and rapidly metabolically adapted to the glycolysis inhibitor 2-deoxyglucose (2DG). Since cells rewire signaling and metabolism in 3D environments, we utilized two-photon microscopy to quantify metabolism in secondary mammospheres and living animals. In both intact spheres and orthotopic tumor xenografts, BCSCs marked by either fluorescent reporter utilized glycolysis to a greater extent than bulk cancer cells. We validated our findings by correlating cellular entropy as a marker for cancer stem cells with metabolic pathway preferences on a single-cell basis. When treated with 2DG, mammospheres showed increased OXPHOS and a significant decrease in the percentage of ALDH1A3-mCherry+ cells. These data 1) highlight capabilities of FLIM to measure the metabolism of single cancer cells in physiologic environments; 2) correlate molecular phenotypes with real-time molecular imaging profiles of metabolism and stem-ness; and 3) reveal that BCSCs rely on glycolysis, suggesting a potential target for metabolic therapy. Citation Format: Johanna M. Buschhaus, Ayse J. Muniz, Ming Luo, Joseph P. Burnett, Nathan A. Truchan, Michael D. Brooks, Brock A. Humphries, Kathy E. Luker, Joerg Lahann, Duxin Sun, Max S. Wicha, Gary D. Luker. Metabolic status and adaptability of breast cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5727.

Abstract 3874: Monitoring immune-tumor cell interactions in 3D microfluidic models with optical metabolic imaging and molecular profiling

Abstract The tumor microenvironment is defined by nutrient starvation, waste product accumulation, hypoxia, and acidosis, which collectively contribute towards immune exhaustion and suppression. Models that mimic the tumor microenvironment will be instrumental to develop new therapies that improve immune response. Microfluidic models can mimic the tumor metabolic microenvironment to elucidate mechanisms that lead to weakened tumor immunity and to new tumor immunotherapies. We developed microfluidic models where cancer cells were cultured as a dense mass embedded in a 3D matrix. The microfluidic platform has lateral channels that are lined with endothelial cells to mimic the tumor vasculature. These vessels can be perfused with immune cells or drugs that extravasate into the tumor mass. Immune cells were isolated from the tumor mass within these microfluidic models and gene expression was analyzed to quantify changes in immune cell function under tumor metabolic microenvironment conditions. Optical metabolic imaging of NADH and FAD autofluorescence with 2-photon microscopy monitored metabolic dynamics during tumor-immune cell interactions. Immunotherapy drugs were also perfused through the microfluidic devices to measure drug efficacy. Changes in immune cell function after drug exposure were analyzed by optical metabolic imaging to examine metabolic dynamics, alongside fluorescence microscopy to visualize cytotoxicity changes. Natural killer cells exhibited directional migration towards the tumor, which indicates that natural killer cells can detect the presence of the tumor several hundreds of microns away. Real-time microscopy revealed that natural killer cells destroyed tumor cells at the tumor periphery and notably at the innermost tumor core. Gene expression analysis of immune cells and tumor cells cultured in the microfluidic model reveals that the tumor cells have created an environment consistent with immunosuppression, immune exhaustion, and nutrient starvation. Metabolic imaging reveals that immune cells exposed to the tumor microenvironment have an altered metabolic profile, specifically decreased redox ratio that persists even under normal culture conditions. The microfluidic model provides a system to examine immune cytotoxicity, metabolic dynamics during tumor-immune interactions, and the impact of immunotherapies in enhancing immune cytotoxicity. Optical metabolic imaging and microfluidic models provide novel insight into metabolic dynamics with tumor cell and immune cell interactions within the tumor metabolic microenvironment. Future studies will expand this technology towards studying immune exhaustion and dysfunction in other immune therapies. Citation Format: Shujah H. Rehman, Jose M. Ayuso, Maria Maria Virumbrales-Munoz, Patrick H. McMinn, David J. Beebe, Melissa C. Skala. Monitoring immune-tumor cell interactions in 3D microfluidic models with optical metabolic imaging and molecular profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3874.

Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia.

This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages. Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5-0% over the course of ~1.5 h, then 5% O2 was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration. Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen. Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength.

.Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

Cystamine-cobalt complex based fluorescent sensor for detection of NADH and cancer cell imaging

The pronounced demand of sensing of biological analytes which play a significant role in progress of cancer cells encouraged us to develop a novel chemosensor having high selectivity and sensitivity for NADH. Therefore, imine linked dipodal receptor (L1) was synthesized and its organic nanoparticles (L1-ONP) were prepared using reprecipitation method to enhance their water solubility. These ONPs form a metal complex with Co(II) which was characterized by UV–vis absorption and Fluorescence Spectroscopy. The ONPs show highly sensitive affinity having a low detection limit (LOD) of 16.95 nM for the cobalt ions. The prepared ONPs metal complex exhibits high selectivity and sensitivity to sense the NADH with a very low LOD of 83.89 nM without any interference from other potentially interfering biological molecules. Fluorescence response from NADH was also not affected by pH and UV irradiation. The sensor was also employed for detection of NADH in MCF-7 cells.

Two-Photon Imaging of NADH in SKH1 Mice Reveals Changes in Keratinocyte Metabolism with Chronic UVA Exposure

Cancer development is characterized by altered cell metabolism. Alterations of growth-factor signaling pathways adjust the metabolic phenotype to better support tumorigenesis and disease progression, which affects the concentration and binding characteristics of endogenous coenzymes such as nicotinamide adenine dinucleotide (NADH) and flavoproteins. To characterize metabolic changes induced by chronic UVA exposure in vivo, we designed and constructed a multiphoton laser scanning imaging platform specifically for in situ metabolic imaging of NADH within the skin of anesthetized mice. Age matched SKH1 hairless mice were divided equally into sham and UV treatment groups, with the treatment group receiving daily illumination of 15 kJ/m2 UVA. NADH phasor FLIM was used to characterize metabolism of keratinocytes as a function of cell layer within the epidermis in both groups over a period of months. Early changes in the enzyme-bound-to-free ratio of NADH were clearly evident prior to the onset of disease, with UVA- treated mice showing significantly greater proportion of enzyme-bound NADH. Basal keratinocytes generally had a lower bound-to-free ratio than keratinocytes from stratum spinosum or granulosa layers. Keratinocytes in vivo had a relatively greater fraction of bound NADH compared to squamous cell carcinoma cells in vitro. Changes in skin metabolism with normal aging was also clearly evident. This study demonstrates the feasibility and challenges of phasor FLIM metabolic imaging for long term longitudinal studies. This study was made possible by grants from the National Institute for General Medical Science (NIGMS) (5P20GM103427), a component of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Two-photon fluorescence lifetime imaging of intrinsic NADH in three-dimensional tumor models.

Most studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like three-dimensional (3D) models. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior. © 2018 International Society for Advancement of Cytometry.

NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research.

More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker-free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research. © 2018 International Society for Advancement of Cytometry.

Dual probing of redox species, NAD(P)H and HOCl, with a benzo[a]phenoxazine based far red-emitting dye

We report a benzo[a]phenoxazine-based far red-emitting probe which selectively senses reduced nicotinamide adenine dinucleotide (NADH) and its phosphate ester NADPH, the most indispensable coenzymes found in all living cells, with “turn-off” fluorescence response at 650 nm. The original probe can be regenerated quantitatively through oxidation using hypochlorous acid, a reactive oxygen species, which was accompanied by restored fluorescence at 650 nm. This probe was also applied for fluorescence imaging of NADH in HeLa cells.

Multicolor Two-Photon Fluorescence Lifetimes Microscopy by Wavelenght Mixing for Efficient and Simultaneus NADH and FAD Imaging Reveals Metabolic Shifts Associated to Cellular Differentiation and Oxidative Stress in Living Tissues

Multicolor Two-Photon Fluorescence Lifetimes Microscopy by Wavelenght Mixing for Efficient and Simultaneus NADH and FAD Imaging Reveals Metabolic Shifts Associated to Cellular Differentiation and Oxidative Stress in Living Tissues