Separating NADH and NADPH fluorescence in live cells and tissues using FLIM

Blacker, Thomas S.; Mann, Zoë F.; Gale, Jonathan E.; Ziegler, Mathias; Bain, Angus J.; Szabadkai, György; Duchen, Michael R.

doi:10.1038/ncomms4936

Cited by 474 publications

(484 citation statements)

References 57 publications

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“…In addition, initial superoxide signals produced by the NADPH oxidase can stimulate secondary mitochondrial superoxide and various ROS production. It could be also that proton channels play a role in UPE generation, as the NADPH-NADP + balance (Blacker et al, 2014) has a key role in redox and ROS-mediated processes that are the bases for photon emission processes. Kobayashi et al (1999b) reported an increase in UPE induced by the addition of 10 mm glutamate.…”

Section: Phosphene Generation In the Visual Cortex By Means Of Excessmentioning

confidence: 99%

Phosphene perception is due to the ultra-weak photon emission produced in various parts of the visual system: glutamate in the focus

Császár¹,

Scholkmann²,

Salari³

et al. 2016

Reviews in the Neurosciences

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AbstractPhosphenes are experienced sensations of light, when there is no light causing them. The physiological processes underlying this phenomenon are still not well understood. Previously, we proposed a novel biopsychophysical approach concerning the cause of phosphenes based on the assumption that cellular endogenous ultra-weak photon emission (UPE) is the biophysical cause leading to the sensation of phosphenes. Briefly summarized, the visual sensation of light (phosphenes) is likely to be due to the inherent perception of UPE of cells in the visual system. If the intensity of spontaneous or induced photon emission of cells in the visual system exceeds a distinct threshold, it is hypothesized that it can become a conscious light sensation. Discussing several new and previous experiments, we point out that the UPE theory of phosphenes should be really considered as a scientifically appropriate and provable mechanism to explain the physiological basis of phosphenes. In the present paper, we also present our idea that some experiments may support that the cortical phosphene lights are due to the glutamate-related excess UPE in the occipital cortex.

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Section: Phosphene Generation In the Visual Cortex By Means Of Excessmentioning

confidence: 99%

Phosphene perception is due to the ultra-weak photon emission produced in various parts of the visual system: glutamate in the focus

Császár¹,

Scholkmann²,

Salari³

et al. 2016

Reviews in the Neurosciences

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show abstract

“…The fluorescence lifetime, or the amount of time a fluorophore remains excited prior to decaying back to the ground state, provides complementary information to the redox ratio [16,19]. The fluorescence lifetime is sensitive to changes in cell microenvironment, such as pH, protein binding, and proximity to fluorescence quenchers such as oxygen [20][21][22]. Fluorescence lifetime imaging microscopy (FLIM) is an optical imaging technique capable of resolving these changes on a single-cell basis [23].…”

Section: Introductionmentioning

confidence: 99%

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

Cannon

Shah

Skala

2017

Biomed. Opt. Express

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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.

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“…Spatiotemporal partitioning of glycolytic and oxidative metabolism can be resolved by measuring cytoplasmic and mitochondrial NAD(P)H changes (Patterson et al, 2000). The different roles of NADH and NAD(P)H in ATP production and antioxidant defense can be separated by simultaneously measuring two-photon NAD(P)H and one-photon lipoamide dehydrogenase autofluorescence (Rocheleau et al, 2004) or by fluorescence lifetime imaging (Blacker et al, 2014). The dynamics of lipid partitioning and fatty acid oxidation can be modeled by monitoring electron transfer flavoprotein autofluorescence (Lam et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Multifunctional in vivo imaging of pancreatic islets during diabetes development

Ward

et al. 2016

Journal of Cell Science

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Pancreatic islet dysfunction leading to insufficient glucose-stimulated insulin secretion triggers the clinical onset of diabetes. How islet dysfunction develops is not well understood at the cellular level, partly owing to the lack of approaches to study single islets longitudinally in vivo. Here, we present a noninvasive, high-resolution system to quantitatively image real-time glucose metabolism from single islets in vivo, currently not available with any other method. In addition, this multifunctional system simultaneously reports islet function, proliferation, vasculature and macrophage infiltration in vivo from the same set of images. Applying our method to a longitudinal high-fat diet study revealed changes in islet function as well as alternations in islet microenvironment. More importantly, this label-free system enabled us to image real-time glucose metabolism directly from single human islets in vivo for the first time, opening the door to noninvasive longitudinal in vivo studies of healthy and diabetic human islets.

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Separating NADH and NADPH fluorescence in live cells and tissues using FLIM

Cited by 474 publications

References 57 publications

Phosphene perception is due to the ultra-weak photon emission produced in various parts of the visual system: glutamate in the focus

Phosphene perception is due to the ultra-weak photon emission produced in various parts of the visual system: glutamate in the focus

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

Multifunctional in vivo imaging of pancreatic islets during diabetes development

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