Abstract

Autofluorescence of endogenous molecules can provide valuable insights in both basic research and clinical applications. One such technique is fluorescence lifetime imaging (FLIM) of NAD(P)H, which serves as a correlate of glycolysis and electron transport chain rates in metabolically active tissue. A powerful advantage of NAD(P)H-FLIM is the ability to measure cell-specific metabolism within heterogeneous tissues. Cell-type specific identification is most commonly achieved with directed green fluorescent protein (GFP) expression. However, we demonstrate that NAD(P)H-FLIM should not be analyzed in GFP-expressing cells, as GFP molecules themselves emit photons in the blue spectrum with short fluorescence lifetimes when imaged using two-photon excitation at 750 nm. This is substantially different from the reported GFP emission wavelength and lifetime after two-photon excitation at 910 nm. These blue GFP photons are indistinguishable from free NAD(P)H by both emission spectra and fluorescence lifetime. Therefore, NAD(P)H-FLIM in GFP-expressing cells will lead to incorrect interpretations of metabolic rates, and thus, these techniques should not be combined.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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2018 (2)

P. Büttner, R. Galli, D. Husser, and A. Bollmann, “Label-free imaging of myocardial remodeling in atrial fibrillation using nonlinear optical microscopy: A feasibility study,” J. Atr. Fibrillation 10(5), 1644 (2018).
[Crossref] [PubMed]

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

2017 (1)

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

2016 (2)

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

2015 (2)

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

M. Lombardo, D. Merino, P. Loza-Alvarez, and G. Lombardo, “Translational label-free nonlinear imaging biomarkers to classify the human corneal microstructure,” Biomed. Opt. Express 6(8), 2803–2818 (2015).
[Crossref] [PubMed]

2014 (1)

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

2013 (5)

M. A. Yaseen, S. Sakadžić, W. Wu, W. Becker, K. A. Kasischke, and D. A. Boas, “In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH,” Biomed. Opt. Express 4(2), 307–321 (2013).
[Crossref] [PubMed]

J. Vergen, C. Hecht, L. V Zholudeva, M. M. Marquardt, and M. G. Nichols, “Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging,” Microsc Microanal. 18(4), 761 (2013).

J. Mandl, T. Mészáros, G. Bánhegyi, and M. Csala, “Minireview: Endoplasmic Reticulum Stress: Control in Protein, Lipid, and Signal Homeostasis,” Mol. Endocrinol. 27(3), 384–393 (2013).
[Crossref] [PubMed]

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
[Crossref] [PubMed]

2012 (2)

B. Schwendele, B. Brawek, M. Hermes, and O. Garaschuk, “High-resolution in vivo imaging of microglia using a versatile nongenetically encoded marker,” Eur. J. Immunol. 42(8), 2193–2196 (2012).
[Crossref] [PubMed]

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential,” PLoS One 7(11), e48014 (2012).
[Crossref] [PubMed]

2011 (1)

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

2008 (1)

T. Nakabayashi, H.-P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci. 7(6), 668–670 (2008).
[Crossref] [PubMed]

2007 (1)

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

2005 (2)

G. Jung, J. Wiehler, and A. Zumbusch, “The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222,” Biophys. J. 88(3), 1932–1947 (2005).
[Crossref] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

2004 (1)

H. Schneckenburger, M. Wagner, P. Weber, W. S. L. Strauss, and R. Sailer, “Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode,” J. Fluoresc. 14(5), 649–654 (2004).
[Crossref] [PubMed]

2003 (1)

S. T. Hess, E. D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal, “Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells,” Biophys. J. 85(4), 2566–2580 (2003).
[Crossref] [PubMed]

2000 (1)

A. Volkmer, V. Subramaniam, D. J. Birch, and T. M. Jovin, “One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins,” Biophys. J. 78(3), 1589–1598 (2000).
[Crossref] [PubMed]

1996 (2)

M. Chattoraj, B. A. King, G. U. Bublitz, and S. G. Boxer, “Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer,” Proc. Natl. Acad. Sci. U.S.A. 93(16), 8362–8367 (1996).
[Crossref] [PubMed]

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

1994 (1)

M. Chalfie, Y. Tu, G. Euskirchen, and W. W. Ward, “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263(5148), 802–805 (1994).

1992 (1)

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(4), 1271–1275 (1992).
[Crossref] [PubMed]

1990 (1)

W. J. Streit, “An Improved Staining Method for Rat Microglial Cells Using the Lectin from Griffonia simplicifolia,” J. Histochem. Cytochem. 38(11), 1683–1686 (1990).
[Crossref] [PubMed]

1989 (1)

O. Marcillat, Y. Zhang, and K. J. Davies, “Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin,” Biochem. J. 259(1), 181–189 (1989).
[Crossref] [PubMed]

1969 (1)

O. Shimomura and F. H. Johnson, “Properties of the Bioluminescent Protein Aequorin,” Biochemistry 8(10), 3991–3997 (1969).
[Crossref] [PubMed]

Aguirre-Ghiso, J.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

Alfonso-García, A.

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

Alonzo, C. A.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

Andersen, B.

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

Annunziato, L.

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
[Crossref] [PubMed]

Bain, A. J.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Balu, M.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

Bánhegyi, G.

J. Mandl, T. Mészáros, G. Bánhegyi, and M. Csala, “Minireview: Endoplasmic Reticulum Stress: Control in Protein, Lipid, and Signal Homeostasis,” Mol. Endocrinol. 27(3), 384–393 (2013).
[Crossref] [PubMed]

Becker, W.

Bieser, G.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Birch, D. J.

A. Volkmer, V. Subramaniam, D. J. Birch, and T. M. Jovin, “One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins,” Biophys. J. 78(3), 1589–1598 (2000).
[Crossref] [PubMed]

Bird, D. K.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Blacker, T. S.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Boas, D. A.

Bollmann, A.

P. Büttner, R. Galli, D. Husser, and A. Bollmann, “Label-free imaging of myocardial remodeling in atrial fibrillation using nonlinear optical microscopy: A feasibility study,” J. Atr. Fibrillation 10(5), 1644 (2018).
[Crossref] [PubMed]

Boscia, F.

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
[Crossref] [PubMed]

Boxer, S. G.

M. Chattoraj, B. A. King, G. U. Bublitz, and S. G. Boxer, “Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer,” Proc. Natl. Acad. Sci. U.S.A. 93(16), 8362–8367 (1996).
[Crossref] [PubMed]

Brawek, B.

B. Schwendele, B. Brawek, M. Hermes, and O. Garaschuk, “High-resolution in vivo imaging of microglia using a versatile nongenetically encoded marker,” Eur. J. Immunol. 42(8), 2193–2196 (2012).
[Crossref] [PubMed]

Bublitz, G. U.

M. Chattoraj, B. A. King, G. U. Bublitz, and S. G. Boxer, “Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer,” Proc. Natl. Acad. Sci. U.S.A. 93(16), 8362–8367 (1996).
[Crossref] [PubMed]

Büttner, P.

P. Büttner, R. Galli, D. Husser, and A. Bollmann, “Label-free imaging of myocardial remodeling in atrial fibrillation using nonlinear optical microscopy: A feasibility study,” J. Atr. Fibrillation 10(5), 1644 (2018).
[Crossref] [PubMed]

Casamassa, A.

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
[Crossref] [PubMed]

Castracane, J.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

Cerchia, L.

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
[Crossref] [PubMed]

Chalfie, M.

M. Chalfie, Y. Tu, G. Euskirchen, and W. W. Ward, “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263(5148), 802–805 (1994).

Chattoraj, M.

M. Chattoraj, B. A. King, G. U. Bublitz, and S. G. Boxer, “Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer,” Proc. Natl. Acad. Sci. U.S.A. 93(16), 8362–8367 (1996).
[Crossref] [PubMed]

Cinquin, A.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Cinquin, O.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
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Condeelis, J.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

Crosignani, V.

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

Csala, M.

J. Mandl, T. Mészáros, G. Bánhegyi, and M. Csala, “Minireview: Endoplasmic Reticulum Stress: Control in Protein, Lipid, and Signal Homeostasis,” Mol. Endocrinol. 27(3), 384–393 (2013).
[Crossref] [PubMed]

Datta, R.

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
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Davies, K. J.

O. Marcillat, Y. Zhang, and K. J. Davies, “Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin,” Biochem. J. 259(1), 181–189 (1989).
[Crossref] [PubMed]

de Franciscis, V.

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
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C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
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C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
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T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
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Eickhoff, J.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

Eliceiri, K. W.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Entenberg, D.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

Esposito, C. L.

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
[Crossref] [PubMed]

Euskirchen, G.

M. Chalfie, Y. Tu, G. Euskirchen, and W. W. Ward, “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263(5148), 802–805 (1994).

Fei, Y.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
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Flanagan, L. A.

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential,” PLoS One 7(11), e48014 (2012).
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Gale, J. E.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Galli, R.

P. Büttner, R. Galli, D. Husser, and A. Bollmann, “Label-free imaging of myocardial remodeling in atrial fibrillation using nonlinear optical microscopy: A feasibility study,” J. Atr. Fibrillation 10(5), 1644 (2018).
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Ganini, D.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
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Garaschuk, O.

B. Schwendele, B. Brawek, M. Hermes, and O. Garaschuk, “High-resolution in vivo imaging of microglia using a versatile nongenetically encoded marker,” Eur. J. Immunol. 42(8), 2193–2196 (2012).
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Gendron-fitzpatrick, A.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

Geyfman, M.

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

Gratton, E.

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential,” PLoS One 7(11), e48014 (2012).
[Crossref] [PubMed]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Harris, R. M.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

Hecht, C.

J. Vergen, C. Hecht, L. V Zholudeva, M. M. Marquardt, and M. G. Nichols, “Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging,” Microsc Microanal. 18(4), 761 (2013).

Heikal, A. A.

S. T. Hess, E. D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal, “Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells,” Biophys. J. 85(4), 2566–2580 (2003).
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Heinecke, R.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Hermes, M.

B. Schwendele, B. Brawek, M. Hermes, and O. Garaschuk, “High-resolution in vivo imaging of microglia using a versatile nongenetically encoded marker,” Eur. J. Immunol. 42(8), 2193–2196 (2012).
[Crossref] [PubMed]

Hess, S. T.

S. T. Hess, E. D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal, “Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells,” Biophys. J. 85(4), 2566–2580 (2003).
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W. Li, K. D. Houston, and J. P. Houston, “Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry,” Sci. Rep.7, 1–11 (2017).
[Crossref]

Houston, K. D.

W. Li, K. D. Houston, and J. P. Houston, “Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry,” Sci. Rep.7, 1–11 (2017).
[Crossref]

Hu, C. W.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Husser, D.

P. Büttner, R. Galli, D. Husser, and A. Bollmann, “Label-free imaging of myocardial remodeling in atrial fibrillation using nonlinear optical microscopy: A feasibility study,” J. Atr. Fibrillation 10(5), 1644 (2018).
[Crossref] [PubMed]

Inman, D. R.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

Jiang, J.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

Jing, Y.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
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Johnson, F. H.

O. Shimomura and F. H. Johnson, “Properties of the Bioluminescent Protein Aequorin,” Biochemistry 8(10), 3991–3997 (1969).
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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(4), 1271–1275 (1992).
[Crossref] [PubMed]

Jonsson, T.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Jovin, T. M.

A. Volkmer, V. Subramaniam, D. J. Birch, and T. M. Jovin, “One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins,” Biophys. J. 78(3), 1589–1598 (2000).
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Jung, G.

G. Jung, J. Wiehler, and A. Zumbusch, “The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222,” Biophys. J. 88(3), 1932–1947 (2005).
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Kao, F. J.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Kasischke, K. A.

Keely, P. J.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Kelly, K. M.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

King, B. A.

M. Chattoraj, B. A. King, G. U. Bublitz, and S. G. Boxer, “Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer,” Proc. Natl. Acad. Sci. U.S.A. 93(16), 8362–8367 (1996).
[Crossref] [PubMed]

Kinjo, M.

T. Nakabayashi, H.-P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci. 7(6), 668–670 (2008).
[Crossref] [PubMed]

Kompa, F. P. C.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Kumar, A.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

Kumar, V.

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

Kummer, H. L. A.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

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(4), 1271–1275 (1992).
[Crossref] [PubMed]

Leinisch, F.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

Li, W.

W. Li, K. D. Houston, and J. P. Houston, “Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry,” Sci. Rep.7, 1–11 (2017).
[Crossref]

Liu, W. F.

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

Liu, Z.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

Lombardo, G.

Lombardo, M.

Loza-Alvarez, P.

Luu, T. U.

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

Lyn, R. K.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Ma, J.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

Malone, C. C.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

Mandl, J.

J. Mandl, T. Mészáros, G. Bánhegyi, and M. Csala, “Minireview: Endoplasmic Reticulum Stress: Control in Protein, Lipid, and Signal Homeostasis,” Mol. Endocrinol. 27(3), 384–393 (2013).
[Crossref] [PubMed]

Mann, Z. F.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Marcillat, O.

O. Marcillat, Y. Zhang, and K. J. Davies, “Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin,” Biochem. J. 259(1), 181–189 (1989).
[Crossref] [PubMed]

Marquardt, M. M.

J. Vergen, C. Hecht, L. V Zholudeva, M. M. Marquardt, and M. G. Nichols, “Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging,” Microsc Microanal. 18(4), 761 (2013).

Mason, R. P.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

Mazumder, N.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

McLauchlan, J.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Merino, D.

Mészáros, T.

J. Mandl, T. Mészáros, G. Bánhegyi, and M. Csala, “Minireview: Endoplasmic Reticulum Stress: Control in Protein, Lipid, and Signal Homeostasis,” Mol. Endocrinol. 27(3), 384–393 (2013).
[Crossref] [PubMed]

Mi, L.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

Michel-beyerle, M. E.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Moffatt, D. J.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Nakabayashi, T.

T. Nakabayashi, H.-P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci. 7(6), 668–670 (2008).
[Crossref] [PubMed]

Nichols, M. G.

J. Vergen, C. Hecht, L. V Zholudeva, M. M. Marquardt, and M. G. Nichols, “Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging,” Microsc Microanal. 18(4), 761 (2013).

Nourse, J. L.

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential,” PLoS One 7(11), e48014 (2012).
[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(4), 1271–1275 (1992).
[Crossref] [PubMed]

Ohta, N.

T. Nakabayashi, H.-P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci. 7(6), 668–670 (2008).
[Crossref] [PubMed]

Petrovich, R. M.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

Pezacki, J. P.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Ponik, S. M.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

Potma, E. O.

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

Pouli, D.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

Quinn, K. P.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

Ramanujam, N.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Riching, K. M.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

Ridsdale, A.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Sailer, R.

H. Schneckenburger, M. Wagner, P. Weber, W. S. L. Strauss, and R. Sailer, “Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode,” J. Fluoresc. 14(5), 649–654 (2004).
[Crossref] [PubMed]

Sakadžic, S.

Schneckenburger, H.

H. Schneckenburger, M. Wagner, P. Weber, W. S. L. Strauss, and R. Sailer, “Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode,” J. Fluoresc. 14(5), 649–654 (2004).
[Crossref] [PubMed]

Schwendele, B.

B. Schwendele, B. Brawek, M. Hermes, and O. Garaschuk, “High-resolution in vivo imaging of microglia using a versatile nongenetically encoded marker,” Eur. J. Immunol. 42(8), 2193–2196 (2012).
[Crossref] [PubMed]

Sheets, E. D.

S. T. Hess, E. D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal, “Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells,” Biophys. J. 85(4), 2566–2580 (2003).
[Crossref] [PubMed]

Shimomura, O.

O. Shimomura and F. H. Johnson, “Properties of the Bioluminescent Protein Aequorin,” Biochemistry 8(10), 3991–3997 (1969).
[Crossref] [PubMed]

Silva, C. M.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Singaravelu, R.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Skala, M. C.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

Smith, T. D.

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

Song, C.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

Stolow, A.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Strauss, W. S. L.

H. Schneckenburger, M. Wagner, P. Weber, W. S. L. Strauss, and R. Sailer, “Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode,” J. Fluoresc. 14(5), 649–654 (2004).
[Crossref] [PubMed]

Streit, W. J.

W. J. Streit, “An Improved Staining Method for Rat Microglial Cells Using the Lectin from Griffonia simplicifolia,” J. Histochem. Cytochem. 38(11), 1683–1686 (1990).
[Crossref] [PubMed]

Stringari, C.

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential,” PLoS One 7(11), e48014 (2012).
[Crossref] [PubMed]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Subramaniam, V.

A. Volkmer, V. Subramaniam, D. J. Birch, and T. M. Jovin, “One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins,” Biophys. J. 78(3), 1589–1598 (2000).
[Crossref] [PubMed]

Szabadkai, G.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[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(4), 1271–1275 (1992).
[Crossref] [PubMed]

Szulczewski, J. M.

J. M. Szulczewski, D. R. Inman, D. Entenberg, S. M. Ponik, J. Aguirre-Ghiso, J. Castracane, J. Condeelis, K. W. Eliceiri, and P. J. Keely, “In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging,” Sci. Rep. 6(1), 25086 (2016).
[Crossref] [PubMed]

Takahashi, J. S.

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

Tokar, E. J.

D. Ganini, F. Leinisch, A. Kumar, J. Jiang, E. J. Tokar, C. C. Malone, R. M. Petrovich, and R. P. Mason, “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells,” Redox Biol. 12(March), 462–468 (2017).
[Crossref] [PubMed]

Tromberg, B. J.

D. Pouli, M. Balu, C. A. Alonzo, Z. Liu, K. P. Quinn, R. M. Harris, K. M. Kelly, and B. J. Tromberg, “Imaging mitochondrial dynamics in human skin reveals depth- dependent hypoxia and malignant potential for diagnosis,” Sci. Transl. Med.8, 367ra169 (2017).

Tsai, H. R.

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Tu, Y.

M. Chalfie, Y. Tu, G. Euskirchen, and W. W. Ward, “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263(5148), 802–805 (1994).

Vaughan, E. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Vergen, J.

J. Vergen, C. Hecht, L. V Zholudeva, M. M. Marquardt, and M. G. Nichols, “Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging,” Microsc Microanal. 18(4), 761 (2013).

Volkmer, A.

A. Volkmer, V. Subramaniam, D. J. Birch, and T. M. Jovin, “One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins,” Biophys. J. 78(3), 1589–1598 (2000).
[Crossref] [PubMed]

Vrotsos, K. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Wagenknecht-Wiesner, A.

S. T. Hess, E. D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal, “Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells,” Biophys. J. 85(4), 2566–2580 (2003).
[Crossref] [PubMed]

Wagner, M.

H. Schneckenburger, M. Wagner, P. Weber, W. S. L. Strauss, and R. Sailer, “Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode,” J. Fluoresc. 14(5), 649–654 (2004).
[Crossref] [PubMed]

Wang, H.

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

Wang, H.-P.

T. Nakabayashi, H.-P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci. 7(6), 668–670 (2008).
[Crossref] [PubMed]

Wang, X.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

Wang, Y.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

Ward, W. W.

M. Chalfie, Y. Tu, G. Euskirchen, and W. W. Ward, “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263(5148), 802–805 (1994).

Weber, P.

H. Schneckenburger, M. Wagner, P. Weber, W. S. L. Strauss, and R. Sailer, “Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode,” J. Fluoresc. 14(5), 649–654 (2004).
[Crossref] [PubMed]

White, J. G.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Wiehler, J.

G. Jung, J. Wiehler, and A. Zumbusch, “The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222,” Biophys. J. 88(3), 1932–1947 (2005).
[Crossref] [PubMed]

Wu, W.

Xie, Y.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

Yan, L.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Yang, M. M.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Yaseen, M. A.

Youvan, D. C.

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Zhang, Q.

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

Zhang, Y.

O. Marcillat, Y. Zhang, and K. J. Davies, “Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin,” Biochem. J. 259(1), 181–189 (1989).
[Crossref] [PubMed]

Zholudeva, L. V

J. Vergen, C. Hecht, L. V Zholudeva, M. M. Marquardt, and M. G. Nichols, “Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging,” Microsc Microanal. 18(4), 761 (2013).

Ziegler, M.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Zumbusch, A.

G. Jung, J. Wiehler, and A. Zumbusch, “The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222,” Biophys. J. 88(3), 1932–1947 (2005).
[Crossref] [PubMed]

Biochem. J. (1)

O. Marcillat, Y. Zhang, and K. J. Davies, “Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin,” Biochem. J. 259(1), 181–189 (1989).
[Crossref] [PubMed]

Biochemistry (1)

O. Shimomura and F. H. Johnson, “Properties of the Bioluminescent Protein Aequorin,” Biochemistry 8(10), 3991–3997 (1969).
[Crossref] [PubMed]

Biomed. Opt. Express (2)

Biophys. J. (3)

S. T. Hess, E. D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal, “Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells,” Biophys. J. 85(4), 2566–2580 (2003).
[Crossref] [PubMed]

A. Volkmer, V. Subramaniam, D. J. Birch, and T. M. Jovin, “One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins,” Biophys. J. 78(3), 1589–1598 (2000).
[Crossref] [PubMed]

G. Jung, J. Wiehler, and A. Zumbusch, “The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222,” Biophys. J. 88(3), 1932–1947 (2005).
[Crossref] [PubMed]

Cancer Res. (1)

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

Cell Reports (1)

C. Stringari, H. Wang, M. Geyfman, V. Crosignani, V. Kumar, J. S. Takahashi, B. Andersen, and E. Gratton, “In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells,” Cell Reports 10(1), 1–7 (2015).
[Crossref] [PubMed]

Chem. Phys. (1)

H. L. A. Kummer, R. Heinecke, F. P. C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, and M. E. Michel-beyerle, “Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions,” Chem. Phys. 213, 1 (1996).

Eur. J. Immunol. (1)

B. Schwendele, B. Brawek, M. Hermes, and O. Garaschuk, “High-resolution in vivo imaging of microglia using a versatile nongenetically encoded marker,” Eur. J. Immunol. 42(8), 2193–2196 (2012).
[Crossref] [PubMed]

J. Atr. Fibrillation (1)

P. Büttner, R. Galli, D. Husser, and A. Bollmann, “Label-free imaging of myocardial remodeling in atrial fibrillation using nonlinear optical microscopy: A feasibility study,” J. Atr. Fibrillation 10(5), 1644 (2018).
[Crossref] [PubMed]

J. Biomed. Opt. (2)

A. Alfonso-García, T. D. Smith, R. Datta, T. U. Luu, E. Gratton, E. O. Potma, and W. F. Liu, “Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 21(4), 046005 (2016).
[Crossref] [PubMed]

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo Multiphoton Fluorescence Lifetime Imaging of Protein-bound and Free NADH in Normal and Pre-cancerous Epithelia,” J. Biomed. Opt. 12(2), 1–19 (2007).
[Crossref] [PubMed]

J. Biophotonics (1)

Y. Jing, Y. Wang, X. Wang, C. Song, J. Ma, Y. Xie, Y. Fei, Q. Zhang, and L. Mi, “Label-free imaging and spectroscopy for early detection of cervical cancer,” J. Biophotonics 11(5), e201700245 (2018).
[Crossref] [PubMed]

J. Fluoresc. (1)

H. Schneckenburger, M. Wagner, P. Weber, W. S. L. Strauss, and R. Sailer, “Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode,” J. Fluoresc. 14(5), 649–654 (2004).
[Crossref] [PubMed]

J. Histochem. Cytochem. (1)

W. J. Streit, “An Improved Staining Method for Rat Microglial Cells Using the Lectin from Griffonia simplicifolia,” J. Histochem. Cytochem. 38(11), 1683–1686 (1990).
[Crossref] [PubMed]

J. Neurochem. (1)

F. Boscia, C. L. Esposito, A. Casamassa, V. de Franciscis, L. Annunziato, and L. Cerchia, “The isolectin IB4 binds RET receptor tyrosine kinase in microglia,” J. Neurochem. 126(4), 428–436 (2013).
[Crossref] [PubMed]

Microsc Microanal (1)

J. Vergen, C. Hecht, L. V Zholudeva, M. M. Marquardt, and M. G. Nichols, “Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging,” Microsc Microanal. 18(4), 761 (2013).

Mol. Endocrinol. (1)

J. Mandl, T. Mészáros, G. Bánhegyi, and M. Csala, “Minireview: Endoplasmic Reticulum Stress: Control in Protein, Lipid, and Signal Homeostasis,” Mol. Endocrinol. 27(3), 384–393 (2013).
[Crossref] [PubMed]

Nat. Commun. (1)

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Photochem. Photobiol. Sci. (1)

T. Nakabayashi, H.-P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci. 7(6), 668–670 (2008).
[Crossref] [PubMed]

PLoS One (2)

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential,” PLoS One 7(11), e48014 (2012).
[Crossref] [PubMed]

N. Mazumder, R. K. Lyn, R. Singaravelu, A. Ridsdale, D. J. Moffatt, C. W. Hu, H. R. Tsai, J. McLauchlan, A. Stolow, F. J. Kao, and J. P. Pezacki, “Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein,” PLoS One 8(6), e66738 (2013).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (3)

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

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[Crossref]

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Figures (7)

Fig. 1
Fig. 1 NAD(P)H-FLIM. (a) Experiments were performed in the CA1 region of acute hippocampal slices. (b) A two-photon laser excites electrons of fluorescent molecules. Upon electron decay to ground state, a photon is released and detected by the hybrid PMT. The time of photon arrival is compared to the time of initial laser pulse, and the lifetime of the excited electron is calculated. (c) On a two-photon microscope, emitted light is passed through a series of long-pass dichroic mirrors and filter sets for NAD(P)H (460/50 nm), GFP (535/50 nm), or tomato lectin 594-DyLight (630/75 nm). (d) Endogenous NADH is autofluorescent and has a short lifetime of 400 ps when free, which increases to 2000 ps when enzymatically bound. (e) Based on photon arrival times, a biexponential curve (solid blue line) is fit to the data to separate photons into free or bound NADH components (dotted lines).
Fig. 2
Fig. 2 Recombinant GFP lifetime across excitation and emission settings. Recombinant GFP protein in a sealed pipette was imaged with 910 nm or 750 nm excitation wavelengths. Either a green (535/50 nm) or blue (460/50 nm) emission filter was used before collecting light with the FLIM hybrid detector. Mean GFP lifetimes are over 2500 ps using 910 nm excitation with either the green (a), or the blue emission filter (b). Mean lifetime is also over 2500 ps using 750 nm excitation and the green emission filter (c). Using 750 nm excitation and the blue emission filter, the mean lifetime of GFP is 459 ps (d). Distribution of lifetimes are plotted as histograms showing the normalized number of pixels across mean lifetimes.
Fig. 3
Fig. 3 Recombinant GFP emission lifetime and intensity spectra. Recombinant GFP protein in a sealed pipette was imaged from 700 nm to 950 nm excitation wavelengths at intervals of 25 nm. Either a blue (460/50 nm) or green (535/50 nm) emission filter was used before collecting light with the FLIM hybrid detector. Mean GFP lifetimes (τm; bold line) and the corresponding short (τ1) and long (τ2) lifetimes are shown across excitation lifetimes for blue (a) and green (c) emission. τ1 and τ2 lifetimes were fixed based on the average unfixed values from 700 – 825 nm. The relative contribution of the short (α1) and long (α2) lifetimes are represented as percentage of the total photons (dashed line). Intensities of blue (b) and green (d) GFP emission were measured across excitation wavelengths as the number of photons collected and normalized to the peak emission intensity.
Fig. 4
Fig. 4 NAD(P)H measurements of Thy1-EGFP neurons in situ. (a) Non-EGFP neurons are identified by transmitted light. (b) NAD(P)H mean lifetime is 1183 ps, representing a mix of bound and free NAD(P)H species. Thy1-EGFP neurons (c) were imaged with either 750 nm (d) or 910 nm (f) excitation, using the NAD(P)H (460/50 nm) emission filter. Distribution of lifetimes are plotted as histograms showing the normalized number of pixels across mean lifetimes. Only pixels within the neuron mask (dotted line in b, d, and f) are considered. (e) Decay curves within traced mask generated with 750 nm (top) or 910 nm (bottom) excitation.
Fig. 5
Fig. 5 EGFP-expressing microglia have shorter NAD(P)H mean lifetimes. Wild type microglia identified by tomato lectin 594-DyLight (a), and corresponding NAD(P)H lifetime measurement (b). EGFP-expressing microglia imaged at 750 nm excitation with a 535/50 nm filter (c), or 460/50 nm filter to collect NAD(P)H emission (d). Distribution of lifetimes are plotted as histograms showing the normalized number of pixels across mean lifetimes. Only pixels within microglia cell masks (dotted line in b and d) are considered. (e) Comparison of mean NAD(P)H lifetime and coefficients of variation (%CV) from microglia with or without EGFP expression. Microglia were identified by transmitted light and imaged before (f,g,h), and after (i,j,k) tomato lectin application. **** p< 0.0001 by Student’s t-test; error bars show standard deviation of the mean.
Fig. 6
Fig. 6 ROS scavengers do not increase NAD(P)H lifetimes in EGFP expressing microglia. Acute hippocampal slices with EGFP-positive microglia were incubated for 1 hour in 5 mM N-acetyl cysteine and 2 mM ascorbic acid before imaging (a). Microglial NAD(P)H lifetime in the presence of ROS scavengers was ~583 ps. Distribution of lifetimes are plotted as a histogram showing the normalized number of pixels across mean lifetimes. Only pixels within the microglia cell mask (dotted line) are considered (b). (c) ROS scavengers did not significantly increase the mean lifetimes of EGFP-positive microglia, which were still significantly shorter than microglia from wild type slices identified by lectin (same lectin data set as presented in Fig. 5(e)). **** p< 0.0001 by one-way ANOVA; error bars show standard deviation of the mean.
Fig. 7
Fig. 7 Exogenous protein expression does not cause short NAD(P)H lifetime measurements. Microglia were identified by tomato lectin staining in acute hippocampal slices from mice expressing a Cre protein under the CX3CR1 promoter (a). The NAD(P)H lifetime in these cells was 998 ps (b). Distribution of lifetimes are plotted as a histogram showing the normalized number of pixels across mean lifetimes. Only pixels within the microglia cell mask (dotted line) are considered. (c) NAD(P)H lifetimes in Cre-expressing microglia are significantly higher and with a larger coefficient of variation than from EGFP-positive cells. **** p< 0.0001 by Student’s t-test; error bars show standard deviation of the mean.

Equations (2)

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F( t )= α 1 e t/ τ 1 + α 2 e t/ τ 2
τ m = α 1 τ 1 + α 2 τ 2

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