Abstract

Two-photon fluorescence lifetime imaging (FLIM) of molecules can reveal important information on the local microenvironment. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic co-enzyme, has a lifetime that depends strongly on enzymatic binding. We present a custom image-processing algorithm for raw fluorescence lifetime and amplitude data that produces an image showing spatially distinct NADH fluorescence lifetimes in slices of rat and human brain. NADH FLIM images were collected in control and epileptic rat tissue. Differences in spatial patterns of NADH lifetimes support the hypothesis that NADH binding, and thus metabolic capacity, is significantly different between groups. This type of analysis can provide information on metabolic states in pathological material.

© 2008 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  24. W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
    [CrossRef] [PubMed]
  25. S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).
  26. S. Pelet, M. J. R. Previte, L. H. Laiho and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
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2007

L. Hertz, L. Peng, and G. A. Dienel, "Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis," J. Cereb. Blood Flow Metab. 27, 219-249 (2007).
[CrossRef]

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

2005

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

K. Suhling, P. M. French, and D. Phillips, "Time-resolved fluorescence microscopy," Photochem. Photobiol. Sci. 4, 13-22 (2005).
[CrossRef]

T. M. Melø, A. Nehlig, and U. Sonnewald, "Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy," J. Cereb. Blood Flow Metab. 25, 1254-1264 (2005).
[CrossRef] [PubMed]

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

2004

K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, and W. W. Webb, "Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis," Science 305, 99-103 (2004).
[CrossRef] [PubMed]

S. Pelet, M. J. R. Previte, L. H. Laiho and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

J. DeFelipe, "Cortical microanatomy and human brain disorders: epilepsy," Cortex 40, 232-233 (2004).
[CrossRef] [PubMed]

2003

H. Qu, H. Eloqayli, B. Müller, J. Aasly, and U. Sonnewald, "Glial-neuronal interactions following kainate injection in rats," Neurochem. Int. 42, 101-106 (2003).
[CrossRef]

O. Kann, S. Schuchmann, K. Buchheim, and U. Heinemann, "Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat," Neuroscience 119, 87-100 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

2002

L. D. Errante, A. Williamson, D. D. Spencer, and O. A. Petroff, "Gabapentin and vigabatrin increase GABA in the human neocortical slice," Epilepsy Res. 49, 203-210 (2002).
[CrossRef] [PubMed]

B. N. Smith and F. E. Dudek, "Network interactions mediated by new excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy," J. Neurophysiol. 87,1655-1658 (2002).
[PubMed]

2000

C. Dubé, S. Boyet, C. Marescaux, and A. Nehlig, "Progressive metabolic changes underlying the chronic reorganization of brain circuits during the silent phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat," Exp. Neurol. 162, 146-157 (2000).
[CrossRef] [PubMed]

1998

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

1996

C. L. Meier and F. E. Dudek, "Spontaneous and stimulation-induced synchronized burst afterdischarges in the isolated CA1 of kainate-treated rats," J. Neurophysiol. 76, 2231-2239 (1996).
[PubMed]

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

M. Tsacopoulos and P. J. Magistretti, "Metabolic coupling between glia and neurons," J. Neurosci. 16, 877-885 (1996).
[PubMed]

1992

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, "Fluorescence lifetime imaging of free and protein-bound NADH," Proc. Natl. Acad. Sci., U.S.A. 89, 1271-1275 (1992).
[CrossRef] [PubMed]

1989

D. A. McCormick and A. Williamson, "Convergence and divergence of neurotransmitter action in human cerebral cortex," Proc. Natl. Acad. Sci., U.S.A. 86, 8098-8102 (1989).
[CrossRef] [PubMed]

1986

T. J. Ashwood, B. Lancaster, and H. V. Wheal, "Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat," Exp. Brain Res. 62, 189-198 (1986).
[CrossRef] [PubMed]

1973

P. Lipton, "Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices," Biochem. J. 136, 999-1009 (1973).
[PubMed]

1962

S. J. Strickler and R. A. Berg, "Relationship between absorption intensity and fluorescence lifetime of molecules," J. Chem. Phys. 37, 814-822 (1962).
[CrossRef]

Aasly, J.

H. Qu, H. Eloqayli, B. Müller, J. Aasly, and U. Sonnewald, "Glial-neuronal interactions following kainate injection in rats," Neurochem. Int. 42, 101-106 (2003).
[CrossRef]

Alvestad, S.

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

Ashwood, T. J.

T. J. Ashwood, B. Lancaster, and H. V. Wheal, "Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat," Exp. Brain Res. 62, 189-198 (1986).
[CrossRef] [PubMed]

Behrens, C. J.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

Berg, R. A.

S. J. Strickler and R. A. Berg, "Relationship between absorption intensity and fluorescence lifetime of molecules," J. Chem. Phys. 37, 814-822 (1962).
[CrossRef]

Blahd, W. H.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

Boyet, S.

C. Dubé, S. Boyet, C. Marescaux, and A. Nehlig, "Progressive metabolic changes underlying the chronic reorganization of brain circuits during the silent phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat," Exp. Neurol. 162, 146-157 (2000).
[CrossRef] [PubMed]

Buchheim, K.

O. Kann, S. Schuchmann, K. Buchheim, and U. Heinemann, "Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat," Neuroscience 119, 87-100 (2003).
[CrossRef] [PubMed]

Chen, W. R.

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Chu, W. J.

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

Cornford, E. M.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

de Lanerolle, N. C.

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

DeFelipe, J.

J. DeFelipe, "Cortical microanatomy and human brain disorders: epilepsy," Cortex 40, 232-233 (2004).
[CrossRef] [PubMed]

Delgado-Escueta, A. V.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

Dienel, G. A.

L. Hertz, L. Peng, and G. A. Dienel, "Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis," J. Cereb. Blood Flow Metab. 27, 219-249 (2007).
[CrossRef]

Dubé, C.

C. Dubé, S. Boyet, C. Marescaux, and A. Nehlig, "Progressive metabolic changes underlying the chronic reorganization of brain circuits during the silent phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat," Exp. Neurol. 162, 146-157 (2000).
[CrossRef] [PubMed]

Dudek, F. E.

B. N. Smith and F. E. Dudek, "Network interactions mediated by new excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy," J. Neurophysiol. 87,1655-1658 (2002).
[PubMed]

C. L. Meier and F. E. Dudek, "Spontaneous and stimulation-induced synchronized burst afterdischarges in the isolated CA1 of kainate-treated rats," J. Neurophysiol. 76, 2231-2239 (1996).
[PubMed]

Elgavish, G. A.

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

Eloqayli, H.

H. Qu, H. Eloqayli, B. Müller, J. Aasly, and U. Sonnewald, "Glial-neuronal interactions following kainate injection in rats," Neurochem. Int. 42, 101-106 (2003).
[CrossRef]

Errante, L. D.

L. D. Errante, A. Williamson, D. D. Spencer, and O. A. Petroff, "Gabapentin and vigabatrin increase GABA in the human neocortical slice," Epilepsy Res. 49, 203-210 (2002).
[CrossRef] [PubMed]

Eyjolfsson, E.

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

Fisher, P. J.

K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, and W. W. Webb, "Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis," Science 305, 99-103 (2004).
[CrossRef] [PubMed]

French, P. M.

K. Suhling, P. M. French, and D. Phillips, "Time-resolved fluorescence microscopy," Photochem. Photobiol. Sci. 4, 13-22 (2005).
[CrossRef]

Gabriel, S.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

Gee, M. N.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

Gomes, W. A.

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

Hammer, J.

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

Heikal, A. A.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

Heinemann, U.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

O. Kann, S. Schuchmann, K. Buchheim, and U. Heinemann, "Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat," Neuroscience 119, 87-100 (2003).
[CrossRef] [PubMed]

Hertz, L.

L. Hertz, L. Peng, and G. A. Dienel, "Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis," J. Cereb. Blood Flow Metab. 27, 219-249 (2007).
[CrossRef]

Hetherington, H. P.

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Johnson, M. L.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, "Fluorescence lifetime imaging of free and protein-bound NADH," Proc. Natl. Acad. Sci., U.S.A. 89, 1271-1275 (1992).
[CrossRef] [PubMed]

Kann, O.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

O. Kann, S. Schuchmann, K. Buchheim, and U. Heinemann, "Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat," Neuroscience 119, 87-100 (2003).
[CrossRef] [PubMed]

Kasischke, K. A.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, and W. W. Webb, "Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis," Science 305, 99-103 (2004).
[CrossRef] [PubMed]

Kato, K.

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

Kovacs, R.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

Kuzniecky, R. I.

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

Lado, F. A.

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

Laiho, L. H.

S. Pelet, M. J. R. Previte, L. H. Laiho and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, "Fluorescence lifetime imaging of free and protein-bound NADH," Proc. Natl. Acad. Sci., U.S.A. 89, 1271-1275 (1992).
[CrossRef] [PubMed]

Lancaster, B.

T. J. Ashwood, B. Lancaster, and H. V. Wheal, "Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat," Exp. Brain Res. 62, 189-198 (1986).
[CrossRef] [PubMed]

Landaw, E. M.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

Lee, S.

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

Lehmann, T. N.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

Lipton, P.

P. Lipton, "Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices," Biochem. J. 136, 999-1009 (1973).
[PubMed]

Magistretti, P. J.

M. Tsacopoulos and P. J. Magistretti, "Metabolic coupling between glia and neurons," J. Neurosci. 16, 877-885 (1996).
[PubMed]

Mandelkern, M. A.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

Marescaux, C.

C. Dubé, S. Boyet, C. Marescaux, and A. Nehlig, "Progressive metabolic changes underlying the chronic reorganization of brain circuits during the silent phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat," Exp. Neurol. 162, 146-157 (2000).
[CrossRef] [PubMed]

Mason, G. F.

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

McCormick, D. A.

D. A. McCormick and A. Williamson, "Convergence and divergence of neurotransmitter action in human cerebral cortex," Proc. Natl. Acad. Sci., U.S.A. 86, 8098-8102 (1989).
[CrossRef] [PubMed]

Meier, C. L.

C. L. Meier and F. E. Dudek, "Spontaneous and stimulation-induced synchronized burst afterdischarges in the isolated CA1 of kainate-treated rats," J. Neurophysiol. 76, 2231-2239 (1996).
[PubMed]

Melø, T. M.

T. M. Melø, A. Nehlig, and U. Sonnewald, "Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy," J. Cereb. Blood Flow Metab. 25, 1254-1264 (2005).
[CrossRef] [PubMed]

Müller, B.

H. Qu, H. Eloqayli, B. Müller, J. Aasly, and U. Sonnewald, "Glial-neuronal interactions following kainate injection in rats," Neurochem. Int. 42, 101-106 (2003).
[CrossRef]

Nehlig, A.

T. M. Melø, A. Nehlig, and U. Sonnewald, "Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy," J. Cereb. Blood Flow Metab. 25, 1254-1264 (2005).
[CrossRef] [PubMed]

C. Dubé, S. Boyet, C. Marescaux, and A. Nehlig, "Progressive metabolic changes underlying the chronic reorganization of brain circuits during the silent phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat," Exp. Neurol. 162, 146-157 (2000).
[CrossRef] [PubMed]

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Njunting, M.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

Nowaczyk, K.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, "Fluorescence lifetime imaging of free and protein-bound NADH," Proc. Natl. Acad. Sci., U.S.A. 89, 1271-1275 (1992).
[CrossRef] [PubMed]

Otahal, J.

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

Ottersen, O. P.

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

Pan, C.

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

Pelet, S.

S. Pelet, M. J. R. Previte, L. H. Laiho and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Peng, L.

L. Hertz, L. Peng, and G. A. Dienel, "Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis," J. Cereb. Blood Flow Metab. 27, 219-249 (2007).
[CrossRef]

Petroff, O. A.

L. D. Errante, A. Williamson, D. D. Spencer, and O. A. Petroff, "Gabapentin and vigabatrin increase GABA in the human neocortical slice," Epilepsy Res. 49, 203-210 (2002).
[CrossRef] [PubMed]

Phillips, D.

K. Suhling, P. M. French, and D. Phillips, "Time-resolved fluorescence microscopy," Photochem. Photobiol. Sci. 4, 13-22 (2005).
[CrossRef]

Previte, M. J. R.

S. Pelet, M. J. R. Previte, L. H. Laiho and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Qu, H.

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

H. Qu, H. Eloqayli, B. Müller, J. Aasly, and U. Sonnewald, "Glial-neuronal interactions following kainate injection in rats," Neurochem. Int. 42, 101-106 (2003).
[CrossRef]

Schuchmann, S.

O. Kann, S. Schuchmann, K. Buchheim, and U. Heinemann, "Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat," Neuroscience 119, 87-100 (2003).
[CrossRef] [PubMed]

Shepherd, G. M.

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

Simor, T.

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

Smith, B. N.

B. N. Smith and F. E. Dudek, "Network interactions mediated by new excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy," J. Neurophysiol. 87,1655-1658 (2002).
[PubMed]

So, P. T. C.

S. Pelet, M. J. R. Previte, L. H. Laiho and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Sonnewald, U.

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

T. M. Melø, A. Nehlig, and U. Sonnewald, "Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy," J. Cereb. Blood Flow Metab. 25, 1254-1264 (2005).
[CrossRef] [PubMed]

H. Qu, H. Eloqayli, B. Müller, J. Aasly, and U. Sonnewald, "Glial-neuronal interactions following kainate injection in rats," Neurochem. Int. 42, 101-106 (2003).
[CrossRef]

Spencer, D. D.

L. D. Errante, A. Williamson, D. D. Spencer, and O. A. Petroff, "Gabapentin and vigabatrin increase GABA in the human neocortical slice," Epilepsy Res. 49, 203-210 (2002).
[CrossRef] [PubMed]

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

Strickler, S. J.

S. J. Strickler and R. A. Berg, "Relationship between absorption intensity and fluorescence lifetime of molecules," J. Chem. Phys. 37, 814-822 (1962).
[CrossRef]

Suhling, K.

K. Suhling, P. M. French, and D. Phillips, "Time-resolved fluorescence microscopy," Photochem. Photobiol. Sci. 4, 13-22 (2005).
[CrossRef]

Swartz, B. E.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

Szmacinski, H.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, "Fluorescence lifetime imaging of free and protein-bound NADH," Proc. Natl. Acad. Sci., U.S.A. 89, 1271-1275 (1992).
[CrossRef] [PubMed]

Takahashi, K.

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

Tsacopoulos, M.

M. Tsacopoulos and P. J. Magistretti, "Metabolic coupling between glia and neurons," J. Neurosci. 16, 877-885 (1996).
[PubMed]

Vishwasrao, H. D.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, and W. W. Webb, "Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis," Science 305, 99-103 (2004).
[CrossRef] [PubMed]

Webb, W. W.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, and W. W. Webb, "Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis," Science 305, 99-103 (2004).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Wheal, H. V.

T. J. Ashwood, B. Lancaster, and H. V. Wheal, "Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat," Exp. Brain Res. 62, 189-198 (1986).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Williamson, A.

L. D. Errante, A. Williamson, D. D. Spencer, and O. A. Petroff, "Gabapentin and vigabatrin increase GABA in the human neocortical slice," Epilepsy Res. 49, 203-210 (2002).
[CrossRef] [PubMed]

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

D. A. McCormick and A. Williamson, "Convergence and divergence of neurotransmitter action in human cerebral cortex," Proc. Natl. Acad. Sci., U.S.A. 86, 8098-8102 (1989).
[CrossRef] [PubMed]

Zipfel, W. R.

K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, and W. W. Webb, "Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis," Science 305, 99-103 (2004).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Ann. Neurol.

E. M. Cornford, M. N. Gee, B. E. Swartz, M. A. Mandelkern, W. H. Blahd, E. M. Landaw, and A. V. Delgado-Escueta, "Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx," Ann. Neurol. 43, 801-808 (1998).
[CrossRef] [PubMed]

Biochem. J.

P. Lipton, "Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices," Biochem. J. 136, 999-1009 (1973).
[PubMed]

Biophys. J.

S. Pelet, M. J. R. Previte, L. H. Laiho and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Brain

O. Kann, R. Kovacs, M. Njunting, C. J. Behrens, J. Otahal, T. N. Lehmann, S. Gabriel, and U. Heinemann, "Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans," Brain 128, 2396-2407 (2005).
[CrossRef] [PubMed]

Cortex

J. DeFelipe, "Cortical microanatomy and human brain disorders: epilepsy," Cortex 40, 232-233 (2004).
[CrossRef] [PubMed]

Epilepsy Res.

L. D. Errante, A. Williamson, D. D. Spencer, and O. A. Petroff, "Gabapentin and vigabatrin increase GABA in the human neocortical slice," Epilepsy Res. 49, 203-210 (2002).
[CrossRef] [PubMed]

Exp. Brain Res.

T. J. Ashwood, B. Lancaster, and H. V. Wheal, "Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat," Exp. Brain Res. 62, 189-198 (1986).
[CrossRef] [PubMed]

Exp. Neurol.

C. Dubé, S. Boyet, C. Marescaux, and A. Nehlig, "Progressive metabolic changes underlying the chronic reorganization of brain circuits during the silent phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat," Exp. Neurol. 162, 146-157 (2000).
[CrossRef] [PubMed]

J. Biol. Chem.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

J. Cereb. Blood Flow Metab.

L. Hertz, L. Peng, and G. A. Dienel, "Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis," J. Cereb. Blood Flow Metab. 27, 219-249 (2007).
[CrossRef]

T. M. Melø, A. Nehlig, and U. Sonnewald, "Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy," J. Cereb. Blood Flow Metab. 25, 1254-1264 (2005).
[CrossRef] [PubMed]

J. Chem. Phys.

S. J. Strickler and R. A. Berg, "Relationship between absorption intensity and fluorescence lifetime of molecules," J. Chem. Phys. 37, 814-822 (1962).
[CrossRef]

J. Neurophysiol.

C. L. Meier and F. E. Dudek, "Spontaneous and stimulation-induced synchronized burst afterdischarges in the isolated CA1 of kainate-treated rats," J. Neurophysiol. 76, 2231-2239 (1996).
[PubMed]

B. N. Smith and F. E. Dudek, "Network interactions mediated by new excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy," J. Neurophysiol. 87,1655-1658 (2002).
[PubMed]

J. Neurosci.

M. Tsacopoulos and P. J. Magistretti, "Metabolic coupling between glia and neurons," J. Neurosci. 16, 877-885 (1996).
[PubMed]

Magn. Reson. Med.

W. A. Gomes, F. A. Lado, N. C. de Lanerolle, K. Takahashi, C. Pan, and H. P. Hetherington, "Spectroscopic imaging of the pilocarpine model of human epilepsy suggests that early NAA reduction predicts epilepsy," Magn. Reson. Med. 58, 230-235 (2007).
[CrossRef] [PubMed]

Neurochem. Int.

H. Qu, H. Eloqayli, B. Müller, J. Aasly, and U. Sonnewald, "Glial-neuronal interactions following kainate injection in rats," Neurochem. Int. 42, 101-106 (2003).
[CrossRef]

Neurochem. Res.

S. Alvestad, J. Hammer, E. Eyjolfsson, H. Qu, O. P. Ottersen, and U. Sonnewald, "Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy," Neurochem. Res.33, 257-266 (2007).

Neurology

W. J. Chu, H. P. Hetherington, R. I. Kuzniecky, T. Simor, G. F. Mason, and G. A. Elgavish, "Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T," Neurology 51, 472-479 (1998).
[PubMed]

Neuroscience

O. Kann, S. Schuchmann, K. Buchheim, and U. Heinemann, "Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat," Neuroscience 119, 87-100 (2003).
[CrossRef] [PubMed]

Photochem. Photobiol. Sci.

K. Suhling, P. M. French, and D. Phillips, "Time-resolved fluorescence microscopy," Photochem. Photobiol. Sci. 4, 13-22 (2005).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A.

W. R. Chen, S. Lee, K. Kato, D. D. Spencer, G. M. Shepherd, and A. Williamson, "Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex," Proc. Natl. Acad. Sci. U.S.A. 93, 8011-8015 (1996).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci., U.S.A.

D. A. McCormick and A. Williamson, "Convergence and divergence of neurotransmitter action in human cerebral cortex," Proc. Natl. Acad. Sci., U.S.A. 86, 8098-8102 (1989).
[CrossRef] [PubMed]

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, "Fluorescence lifetime imaging of free and protein-bound NADH," Proc. Natl. Acad. Sci., U.S.A. 89, 1271-1275 (1992).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci., U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Science

K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, and W. W. Webb, "Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis," Science 305, 99-103 (2004).
[CrossRef] [PubMed]

Other

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, DOI:10,1117/12.590510 (2005).
[CrossRef]

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Figures (6)

Fig. 1.
Fig. 1.

The top row of the figure are sample NADH fluorescence lifetime decay fit curves (red line) for the same, individual pixel given a one- (a), two- (b), and three- (c) component fit. The calculated χ2 value for the pixel is provided, green line is the instrument response function, and blue dots are delayed photon counts arriving after the laser excitation pulse. The bottom row is the average χ2 value averaged from all the pixels in the image. The minimal χ2 value is achieved with a two-component fit (b). The χ2 value for a three-component fit increases due to increasing the number of fit parameters without significantly increasing the quality-of-fit.

Fig. 2.
Fig. 2.

(a). (c) Sample distribution of NADH lifetimes seen in the control and pilocarpine-treated rat hippocampus before bicuculline stimulation. Red line shows the NADH lifetime distribution obtained with the custom program. Three distinct NADH species are clearly identified: unbound NADH (red / S1) and two bound NADH species (green / S2 and blue / S3). Black line is the NADH distribution obtained from the Becker & Hickl FLIM analysis program where a single, amplitude-weighted average lifetime is obtained from each pixel. (b), (d) Sample NADH lifetime distributions obtained using the custom program and the Becker & Hickl program following bicuculline stimulation in control and pilocarpine-treated tissue, respectively.

Fig. 3.
Fig. 3.

(a). Concentration of NADH species varies across brain region and following bicuculline stimulation. There is a large difference in concentration between species #1 and #2 in all cases. (b) Concentration increases of each NADH species following bicuculline stimulation in the CA1 rat hippocampus. Notice the significant changes species #2 in the dendritic layer of the pilocarpine-treated rat.

Fig. 4.
Fig. 4.

Intensity (a) and Becker & Hickl FLIM (b) images generated using NADH autofluorescence in the pilocarpine-treated rat hippocampus (with bicuculline stimulation). Arrows in (a) point to astrocytes in the dendritic layer (D.L). Cell layer (C.L.) appears dark due to absence of NADH in the nuclei. The D.L. is bounded by a red, dotted line and the C.L. is bounded by a yellow, dotted line. NADH lifetimes in (b) correspond to the Becker & Hickl color histogram (c). Scale bar=20 µm.

Fig. 5.
Fig. 5.

Custom FLIM images generated from the histogram data used in Fig. 2 (pilocarpine-treated rat hippocampus). Custom FLIM image are separated into images that correspond to the color-coded lifetime shown in Fig. 2. Note how unbound NADH (a, red, ~300 ps) and heavily bound NADH (c, blue, ~4500 ps) appear with more cellular morphology, whereas lightly bound NADH (b, green, ~560 ps) is localized to the neuropil. Part (d) is a saturated, merged image of (a) and (b) to show the anti-localization of these two NADH species. Scale bar=20 µm.

Fig. 6.
Fig. 6.

Close-up intensity (a,c) and custom FLIM (b,d) images generated using NADH autofluorescence in the human cortex (a,b) and rat hippocampus (c,d), clearly showing neurons. The bright, punctate spots are NADH-rich mitochondria. Custom FLIM image reveals the intracellular composition of NADH: mitochondria contain primarily long lifetime NADH (blue, 3000 ps - 6000 ps) and unbound NADH (red, ~300 ps) is localized in the cytoplasm. Neuropil is composed largely of small-enzyme bound NADH (green, ~560 ps). Scale bar=20 µm.

Equations (3)

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F ( t ) i α i e t τ i
Fluorescence = ϕ σ I 2 · c i Q i = i α i 0 e t τ i d t = i α i τ i
α i = ϕ σ I 2 τ radiative · c i

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