Abstract

An optical method is presented that allows the measurement of the triplet lifetime of a fluorescent molecule. This is a characteristic specific to each fluorophore. Based on differences in triplet lifetimes of two fluorescent species (autofluorescence versus label), this novel approach measures relative quantities of a transmembrane receptor and associated fluorescently labeled ligand during its recycling in living cells. Similarly to fluorescence-lifetime based methods, our approach is almost insensitive to photobleaching. A simple theory for unmixing two known triplet lifetimes is presented along with validation of the method by measurements of transferrin recycling in a model system based on chinese hamster ovarian cells (CHO). Transferrin is the delivery carrier for Fe3+ to the cell.

© 2012 OSA

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  1. M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
    [CrossRef] [PubMed]
  2. T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
    [CrossRef] [PubMed]
  3. W. Rumsey, J. Vanderkooi, and D. Wilson, “Imaging of phosphorescence: A novel method for measuring oxygen distribution in perfused tissue,” Science241, 1649–1651 (1988).
    [CrossRef] [PubMed]
  4. J. Vanderkooi, G. Maniara, T. Green, and D. Wilson, “An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence,” J. Biol. Chem.262, 5476–5482 (1987).
    [PubMed]
  5. G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
    [CrossRef] [PubMed]
  6. H. Li and Z. M. Qian, “Transferrin/transferrin receptor-mediated drug delivery,” Med. Res. Rev.22, 225–250 (2002).
    [CrossRef] [PubMed]
  7. B. Alberts, Molecular Biology of the Cell (Garland Science - Taylor&Francis group, 2008).
  8. D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
    [CrossRef] [PubMed]
  9. Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
    [CrossRef] [PubMed]
  10. P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
    [CrossRef] [PubMed]
  11. E. Daro, P. Van Der Sluijs, T. Galli, and I. Mellman, “Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling,” Proc. Natl. Acad. Sci. USA93, 9559–9564 (1996).
    [CrossRef] [PubMed]
  12. J. Gruenberg and F. Maxfield, “Membrane transport in the endocytic pathway,” Curr. Opin. Cell Biol.7, 552–563 (1995).
    [CrossRef] [PubMed]
  13. R. Ghosh, D. Gelman, and F. Maxfield, “Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy,” J. Cell Sci.107, 2177–2189 (1994).
    [PubMed]
  14. D. M. Sipe and R. F. Murphy, “High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling,” Proc. Natl. Acad. Sci. USA84, 7119–7123 (1987).
    [CrossRef] [PubMed]
  15. T. McGraw and F. Maxfield, “Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain,” Cell Regul.1, 369–377 (1990).
    [PubMed]
  16. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).
    [CrossRef]
  17. F. Maxfield and T. McGraw, “Endocytic recycling,” Nat. Rev. Mol. Cell Biol.5, 121–132 (2004).
    [CrossRef] [PubMed]
  18. B. Grant and J. Donaldson, “Pathways and mechanisms of endocytic recycling,” Nat. Rev. Mol. Cell Biol.10, 597–608 (2009).
    [CrossRef] [PubMed]
  19. A. Durrbach, D. Louvard, and E. Coudrier, “Actin filaments facilitate two steps of endocytosis,” J. Cell Sci.109, 457–465 (1996).
    [PubMed]
  20. N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
    [CrossRef]

2011 (1)

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

2010 (1)

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

2009 (1)

B. Grant and J. Donaldson, “Pathways and mechanisms of endocytic recycling,” Nat. Rev. Mol. Cell Biol.10, 597–608 (2009).
[CrossRef] [PubMed]

2007 (1)

T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
[CrossRef] [PubMed]

2005 (2)

G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
[CrossRef]

2004 (1)

F. Maxfield and T. McGraw, “Endocytic recycling,” Nat. Rev. Mol. Cell Biol.5, 121–132 (2004).
[CrossRef] [PubMed]

2002 (2)

H. Li and Z. M. Qian, “Transferrin/transferrin receptor-mediated drug delivery,” Med. Res. Rev.22, 225–250 (2002).
[CrossRef] [PubMed]

D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
[CrossRef] [PubMed]

1996 (2)

A. Durrbach, D. Louvard, and E. Coudrier, “Actin filaments facilitate two steps of endocytosis,” J. Cell Sci.109, 457–465 (1996).
[PubMed]

E. Daro, P. Van Der Sluijs, T. Galli, and I. Mellman, “Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling,” Proc. Natl. Acad. Sci. USA93, 9559–9564 (1996).
[CrossRef] [PubMed]

1995 (1)

J. Gruenberg and F. Maxfield, “Membrane transport in the endocytic pathway,” Curr. Opin. Cell Biol.7, 552–563 (1995).
[CrossRef] [PubMed]

1994 (1)

R. Ghosh, D. Gelman, and F. Maxfield, “Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy,” J. Cell Sci.107, 2177–2189 (1994).
[PubMed]

1991 (1)

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

1990 (1)

T. McGraw and F. Maxfield, “Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain,” Cell Regul.1, 369–377 (1990).
[PubMed]

1988 (1)

W. Rumsey, J. Vanderkooi, and D. Wilson, “Imaging of phosphorescence: A novel method for measuring oxygen distribution in perfused tissue,” Science241, 1649–1651 (1988).
[CrossRef] [PubMed]

1987 (2)

J. Vanderkooi, G. Maniara, T. Green, and D. Wilson, “An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence,” J. Biol. Chem.262, 5476–5482 (1987).
[PubMed]

D. M. Sipe and R. F. Murphy, “High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling,” Proc. Natl. Acad. Sci. USA84, 7119–7123 (1987).
[CrossRef] [PubMed]

Alberts, B.

B. Alberts, Molecular Biology of the Cell (Garland Science - Taylor&Francis group, 2008).

Atwal, J.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Baravalle, G.

G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

Bayer, N.

G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

Blom, H.

T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
[CrossRef] [PubMed]

Coudrier, E.

A. Durrbach, D. Louvard, and E. Coudrier, “Actin filaments facilitate two steps of endocytosis,” J. Cell Sci.109, 457–465 (1996).
[PubMed]

Daro, E.

E. Daro, P. Van Der Sluijs, T. Galli, and I. Mellman, “Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling,” Proc. Natl. Acad. Sci. USA93, 9559–9564 (1996).
[CrossRef] [PubMed]

Dennis, M. S.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Donaldson, J.

B. Grant and J. Donaldson, “Pathways and mechanisms of endocytic recycling,” Nat. Rev. Mol. Cell Biol.10, 597–608 (2009).
[CrossRef] [PubMed]

Durrbach, A.

A. Durrbach, D. Louvard, and E. Coudrier, “Actin filaments facilitate two steps of endocytosis,” J. Cell Sci.109, 457–465 (1996).
[PubMed]

Elliott, J. M.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Formey, A.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Friden, P.

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

Fuchs, R.

G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

Galli, T.

E. Daro, P. Van Der Sluijs, T. Galli, and I. Mellman, “Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling,” Proc. Natl. Acad. Sci. USA93, 9559–9564 (1996).
[CrossRef] [PubMed]

Geissbuehler, M.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Gelman, D.

R. Ghosh, D. Gelman, and F. Maxfield, “Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy,” J. Cell Sci.107, 2177–2189 (1994).
[PubMed]

Ghosh, R.

R. Ghosh, D. Gelman, and F. Maxfield, “Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy,” J. Cell Sci.107, 2177–2189 (1994).
[PubMed]

Girard, P.

N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
[CrossRef]

Grant, B.

B. Grant and J. Donaldson, “Pathways and mechanisms of endocytic recycling,” Nat. Rev. Mol. Cell Biol.10, 597–608 (2009).
[CrossRef] [PubMed]

Green, T.

J. Vanderkooi, G. Maniara, T. Green, and D. Wilson, “An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence,” J. Biol. Chem.262, 5476–5482 (1987).
[PubMed]

Gruenberg, J.

J. Gruenberg and F. Maxfield, “Membrane transport in the endocytic pathway,” Curr. Opin. Cell Biol.7, 552–563 (1995).
[CrossRef] [PubMed]

Hacker, D.

N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
[CrossRef]

Hinz, B.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Hoyte, K.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Huber, M.

G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

Johnsson, K.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Jordan, M.

N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
[CrossRef]

Kenrick, M.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).
[CrossRef]

Lasser, T.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Leutenegger, M.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Li, H.

H. Li and Z. M. Qian, “Transferrin/transferrin receptor-mediated drug delivery,” Med. Res. Rev.22, 225–250 (2002).
[CrossRef] [PubMed]

Louvard, D.

A. Durrbach, D. Louvard, and E. Coudrier, “Actin filaments facilitate two steps of endocytosis,” J. Cell Sci.109, 457–465 (1996).
[PubMed]

Lu, Y.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Luk, W.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Malfroy, B.

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

Maniara, G.

J. Vanderkooi, G. Maniara, T. Green, and D. Wilson, “An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence,” J. Biol. Chem.262, 5476–5482 (1987).
[PubMed]

Märki, I.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Maxfield, F.

F. Maxfield and T. McGraw, “Endocytic recycling,” Nat. Rev. Mol. Cell Biol.5, 121–132 (2004).
[CrossRef] [PubMed]

J. Gruenberg and F. Maxfield, “Membrane transport in the endocytic pathway,” Curr. Opin. Cell Biol.7, 552–563 (1995).
[CrossRef] [PubMed]

R. Ghosh, D. Gelman, and F. Maxfield, “Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy,” J. Cell Sci.107, 2177–2189 (1994).
[PubMed]

T. McGraw and F. Maxfield, “Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain,” Cell Regul.1, 369–377 (1990).
[PubMed]

McGraw, T.

F. Maxfield and T. McGraw, “Endocytic recycling,” Nat. Rev. Mol. Cell Biol.5, 121–132 (2004).
[CrossRef] [PubMed]

T. McGraw and F. Maxfield, “Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain,” Cell Regul.1, 369–377 (1990).
[PubMed]

Mellman, I.

D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
[CrossRef] [PubMed]

E. Daro, P. Van Der Sluijs, T. Galli, and I. Mellman, “Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling,” Proc. Natl. Acad. Sci. USA93, 9559–9564 (1996).
[CrossRef] [PubMed]

Muller, N.

N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
[CrossRef]

Murphy, R.

G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

Murphy, R. F.

D. M. Sipe and R. F. Murphy, “High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling,” Proc. Natl. Acad. Sci. USA84, 7119–7123 (1987).
[CrossRef] [PubMed]

Musso, G.

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

O’Connell, C.

D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
[CrossRef] [PubMed]

Pelletier, L.

D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
[CrossRef] [PubMed]

Persson, G.

T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
[CrossRef] [PubMed]

Prabhu, S.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Qian, Z. M.

H. Li and Z. M. Qian, “Transferrin/transferrin receptor-mediated drug delivery,” Med. Res. Rev.22, 225–250 (2002).
[CrossRef] [PubMed]

Rumsey, W.

W. Rumsey, J. Vanderkooi, and D. Wilson, “Imaging of phosphorescence: A novel method for measuring oxygen distribution in perfused tissue,” Science241, 1649–1651 (1988).
[CrossRef] [PubMed]

Sandén, T.

T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
[CrossRef] [PubMed]

Schober, D.

G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

Sheff, D.

D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
[CrossRef] [PubMed]

Sipe, D. M.

D. M. Sipe and R. F. Murphy, “High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling,” Proc. Natl. Acad. Sci. USA84, 7119–7123 (1987).
[CrossRef] [PubMed]

Spielmann, T.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Starzyk, R.

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

Taylor, M.

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

Thyberg, P.

T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
[CrossRef] [PubMed]

Van De Ville, D.

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Van Der Sluijs, P.

E. Daro, P. Van Der Sluijs, T. Galli, and I. Mellman, “Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling,” Proc. Natl. Acad. Sci. USA93, 9559–9564 (1996).
[CrossRef] [PubMed]

Vanderkooi, J.

W. Rumsey, J. Vanderkooi, and D. Wilson, “Imaging of phosphorescence: A novel method for measuring oxygen distribution in perfused tissue,” Science241, 1649–1651 (1988).
[CrossRef] [PubMed]

J. Vanderkooi, G. Maniara, T. Green, and D. Wilson, “An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence,” J. Biol. Chem.262, 5476–5482 (1987).
[PubMed]

Walus, L.

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

Warren, G.

D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
[CrossRef] [PubMed]

Watts, R. J.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Widengren, J.

T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
[CrossRef] [PubMed]

Wilson, D.

W. Rumsey, J. Vanderkooi, and D. Wilson, “Imaging of phosphorescence: A novel method for measuring oxygen distribution in perfused tissue,” Science241, 1649–1651 (1988).
[CrossRef] [PubMed]

J. Vanderkooi, G. Maniara, T. Green, and D. Wilson, “An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence,” J. Biol. Chem.262, 5476–5482 (1987).
[PubMed]

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N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
[CrossRef]

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Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Zhang, Y.

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Anal. Chem. (1)

T. Sandén, G. Persson, P. Thyberg, H. Blom, and J. Widengren, “Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording,” Anal. Chem.79, 3330–3341 (2007).
[CrossRef] [PubMed]

Biophys. J. (1)

M. Geissbuehler, T. Spielmann, A. Formey, I. Märki, M. Leutenegger, B. Hinz, K. Johnsson, D. Van De Ville, and T. Lasser, “Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5),” Biophys. J.98, 339–349 (2010).
[CrossRef] [PubMed]

Biotechnol. Bioeng. (1)

N. Muller, P. Girard, D. Hacker, M. Jordan, and F. Wurm, “Orbital shaker technology for the cultivation of mammalian cells in suspension,” Biotechnol. Bioeng.89, 400–406 (2005).
[CrossRef]

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G. Baravalle, D. Schober, M. Huber, N. Bayer, R. Murphy, and R. Fuchs, “Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature,” Cell. Tissue Res.320, 99–113 (2005).
[CrossRef] [PubMed]

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J. Gruenberg and F. Maxfield, “Membrane transport in the endocytic pathway,” Curr. Opin. Cell Biol.7, 552–563 (1995).
[CrossRef] [PubMed]

J. Biol. Chem. (1)

J. Vanderkooi, G. Maniara, T. Green, and D. Wilson, “An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence,” J. Biol. Chem.262, 5476–5482 (1987).
[PubMed]

J. Cell Biol. (1)

D. Sheff, L. Pelletier, C. O’Connell, G. Warren, and I. Mellman, “Transferrin receptor recycling in the absence of perinuclear recycling endosomes,” J. Cell Biol.156, 797–804 (2002).
[CrossRef] [PubMed]

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H. Li and Z. M. Qian, “Transferrin/transferrin receptor-mediated drug delivery,” Med. Res. Rev.22, 225–250 (2002).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol. (2)

F. Maxfield and T. McGraw, “Endocytic recycling,” Nat. Rev. Mol. Cell Biol.5, 121–132 (2004).
[CrossRef] [PubMed]

B. Grant and J. Donaldson, “Pathways and mechanisms of endocytic recycling,” Nat. Rev. Mol. Cell Biol.10, 597–608 (2009).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. USA (3)

D. M. Sipe and R. F. Murphy, “High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling,” Proc. Natl. Acad. Sci. USA84, 7119–7123 (1987).
[CrossRef] [PubMed]

P. Friden, L. Walus, G. Musso, M. Taylor, B. Malfroy, and R. Starzyk, “Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier,” Proc. Natl. Acad. Sci. USA88, 4771–4775 (1991).
[CrossRef] [PubMed]

E. Daro, P. Van Der Sluijs, T. Galli, and I. Mellman, “Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling,” Proc. Natl. Acad. Sci. USA93, 9559–9564 (1996).
[CrossRef] [PubMed]

Sci. Transl. Med. (1)

Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target,” Sci. Transl. Med.3, 84ra44 (2011).
[CrossRef] [PubMed]

Science (1)

W. Rumsey, J. Vanderkooi, and D. Wilson, “Imaging of phosphorescence: A novel method for measuring oxygen distribution in perfused tissue,” Science241, 1649–1651 (1988).
[CrossRef] [PubMed]

Other (2)

B. Alberts, Molecular Biology of the Cell (Garland Science - Taylor&Francis group, 2008).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).
[CrossRef]

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Figures (6)

Fig. 1
Fig. 1

Transferrin uptake and recycling pathways as described in various publications [5, 8, 12, 13, 1719]. The pH and typical half-lifetimes are indicative values adopted from [17].

Fig. 2
Fig. 2

(a) Jablonski diagram of a fluorescent molecule with three states. (b) Simulation of fluorescence emission when using a pulsed excitation with an iso-dosis of light illumination. Three different cases of τT and ΦT are shown. For all curves τs = 2.3 ns, and τex = 2.1 ns (corresponding to an illumination intensity of 10 mW/μm2 for TMR) and pulse widths vary from 1 to 50 μs with a repetition rate of 50 μs. (c) Schematic drawing of the triplet lifetime imaging setup. Beamshaping ensured a spot radius of ≈175 μm at the sample plane, resulting in a maximum intensity of ≈0.6 mW/μm2. The cell-incubator maintained optimal temperature (37°C), humidity and carbon dioxide content of the atmosphere in the observation chamber (CO2 at 5%).

Fig. 3
Fig. 3

Unmixing of two known triplet lifetimes with the help of the triplet population for a constant excitation at equilibrium P T eq.

Fig. 4
Fig. 4

Image processing steps. The triplet lifetime fitting is done pixel per pixel as discussed in our previous publication [1], involving knowledge of the singlet lifetime τS, the triplet yield ΦT as well as the excitation rate τex. The resulting triplet lifetime image is then averaged over small region of interests (ROI) corresponding to the respective cells in the image. This yields a mean and standard deviation for the triplet lifetime at the time-point t.

Fig. 5
Fig. 5

Triplet lifetime images of transferrin recycling (color encoded). (a) Beginning of the experiment, where the cells are visualized after complete internalization of transferrin (t=5min). Some cells show two different areas: an area with short lifetime (1) and an area of longer lifetime (2). (b) An image of cells after 20 min, where a large amount of the internalized TMR labeled transferrin is recycled back into the medium and (c) a typical image of the autofluorescence of CHO-cells excited at 532 nm. The calculation employs a singlet lifetime of τS = 2.4 ns for the TMR-labeled Transferrin (from fluorescence lifetime measurements) and ΦT = 0.3% (from triplet-lifetime and -yield fitting over ROI inside CHO-cells). (d–f) Corresponding fluorescence intensity images.

Fig. 6
Fig. 6

Assessment of transferrin recycling in CHO-cells for three different experiments compared to the autofluorescence within these cells. (a) Triplet lifetime, (b) proportional amount of the dye (α =100%) compared to autofluorescence (α =0%) as defined by Eq. (5), (c) fluorescence intensity and (d) relative quantity of transferrin inside the cell Q as defined by Eq. (6). For the control measurement, we have repeated every washing and incubation step as for the experiments, except that we did not incubate the cells with transferrin-DMEM but only DMEM. For the relative quantity calculations, we used τT, dye = 2.5μs and τT, autofluorescence = 5.5μs. Further on, the calculation employs a singlet lifetime of τS = 2.4 ns for the TMR-labeled Transferrin (from fluorescence lifetime measurements) and ΦT = 0.3% (from triplet-lifetime and -yield fitting over ROI of an intracellular region of CHO-cells).

Equations (6)

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d d t [ P 0 P 1 P T ] = [ P 1 τ S + P T τ T P 0 τ e x P 1 τ S P 1 τ isc + P 0 τ e x P 1 τ isc P T τ T ]
P T eq = Φ T τ T τ ex + τ S + Φ T τ T
Φ T = k isc k rad . + k non-rad + k isc τ S τ isc .
P T eq , mix = α P T eq , dye + ( 1 α ) P T eq , autofluorescence Φ T τ T , mix ( τ ex + τ S + Φ T τ T , mix ) = α Φ T τ T 1 ( τ ex + τ S + Φ T τ T 1 ) + ( 1 α ) Φ T τ T 2 ( τ ex + τ S + Φ T τ T 2 )
α = ( τ T 2 τ T , mix ) ( τ ex + τ S + τ T 1 Φ T ) ( τ T1 τ T 2 ) ( τ ex + τ S + τ T , mix Φ T )
Q ( 1 + I I BG I BG ) × α I I BG × α

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