Abstract

We report a fluorescence lifetime imaging technique that uses the time integrated response to a periodic optical excitation, eliminating the need for time resolution in detection. A Dirac pulse train of variable period is used to probe the frequency response of the total fluorescence per pulse leading to a frequency roll-off that is dependent on the relaxation rate of the fluorophores. The technique is validated by demonstrating wide-field, realtime, lifetime imaging of the endocytosis of inorganic quantum dots by a cancer cell line. Surface charging of the dots in the intra-cellular environment produces a switch in the fluorescence lifetime from ~ 40 ns to < 10 ns. A temporal resolution of half the excitation period is possible which in this instance is 15 ns. This stroboscopic technique offers lifetime based imaging at video rates with standard CCD cameras and has application in probing millisecond cell dynamics and in high throughput imaging assays.

© 2009 Optical Society of America

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  1. F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
    [CrossRef] [PubMed]
  2. K. Suhling, P. M. W. French, and D. Phillips, "Time resolved fluorescence microscopy," Photochem. Photobiol. Sci. 4, 13-22 (2005).
    [CrossRef]
  3. R. Cubeddu, P. Taroni, and G. Valentini, "Time-gated imaging system for tumor diagnosis," Opt. Eng. 32,320-324 (1993).
    [CrossRef]
  4. C. G. Morgan, A. C. Mitchell, and J. G. Murray, "Nanosecond Time-Resolved Fluorescence Microscopy: Principles and Practice," Proc.R. Microsc. Soc. 1, 463-466 (1990).
  5. E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
    [CrossRef]
  6. I. Bugiel, K. Konig, and H. Wabnitz, "Investigation of cells by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers Life Sci. 3, 47-53 (1989).
  7. X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A 2-dimensional fluorescence lifetime imaging-system using a gated image intensifier," Appl Spectrosc. 45, 360-366 (1991).
    [CrossRef]
  8. K. Dowling, M. J. Dayel, M. J. Lever, P. M. W. French, J. D. Hares, and A. K. L. Dymoke-Bradshaw, "Fluorescence lifetime imaging with picosecond resolution for biomedical applications," Opt. Lett. 23, 810-812 (1998).
    [CrossRef]
  9. J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
    [CrossRef] [PubMed]
  10. R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).
  11. D. R. Matthews, H. D. Summers, K. Njoh, R. J. Errington, P. J. Smith, P. Barber, S. Ameer-Beg, and B. Vojnovic, "Technique for measurement of fluorescence lifetime by use of stroboscopic excitation and continuous-wave detection," Appl. Opt. 45, 2115-2123 (2006).
    [CrossRef] [PubMed]
  12. Y. Sakai and S. Hirayama, "A fast deconvolution method to analyze fluorescence decays when the excitation pulse repetition period is less than the decay times," J. Lumin. 39, 145-151 (1988).
    [CrossRef]
  13. M. Müller, R. Ghauharali, K. Visscher, and G. Brakenhoff, "Double-pulse fluorescence lifetime imaging in confocal microscopy," J. Microsc. 177, 171-179 (1994).
    [CrossRef]
  14. H. Nyquist, "Certain topics in telegraph transmission theory," Trans. Am. Inst. Electr. Eng. 47, 617-644 (1928).
    [CrossRef]
  15. T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
    [CrossRef] [PubMed]
  16. O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, "Fluorescence lifetime imaging (FLI) in real time - a new technique in photosynthesis research," Photosynthetica 38, 581-599 (2000).
    [CrossRef]
  17. A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
    [CrossRef]
  18. D. J. Stephens and V. J. Allan, "Light microscopy techniques for live cell imaging," Science 300, 82-86 (2003).
    [CrossRef] [PubMed]
  19. B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
    [CrossRef] [PubMed]
  20. J. K. Jaiswal, H. Mattoussi, J. M. Mauro, and S. M. Simon, "Long-term multiple color imaging of live cells using quantum dot bioconjugates," Nature Biotech. 21, 47-51 (2002).
    [CrossRef]
  21. A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, "Applications of T-lymphoma labelled with fluorescent quantum dots to cell tracing markers in mouse body," Biochem. and Biophys. Res. Commun. 314, 46-53 (2003).
    [CrossRef]
  22. Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).
  23. Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
    [CrossRef]
  24. S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
    [CrossRef] [PubMed]
  25. S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, "Photo-activated luminescence of CdSe Quantum dot monolayers," J. Phys. Chem. 104, 12137-12142 (2000).
    [CrossRef]
  26. N. E. Korunska, M. Dybiec, L. Zhukov, S. Ostapenko, and T. Zhukov, "Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots," Semicond. Sci. Technol. 20, 876-881 (2005).
    [CrossRef]
  27. M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
    [CrossRef]
  28. C. D. McGuinness, K. Sagoo, D. McLoskey, and D. J. S. Birch, "A new sub-nanosecond LED at 280nm: application to protein fluorescence," Meas. Sci. Technol. 15, L19-L22 (2004).
    [CrossRef]

2007 (1)

Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).

2006 (3)

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
[CrossRef]

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

D. R. Matthews, H. D. Summers, K. Njoh, R. J. Errington, P. J. Smith, P. Barber, S. Ameer-Beg, and B. Vojnovic, "Technique for measurement of fluorescence lifetime by use of stroboscopic excitation and continuous-wave detection," Appl. Opt. 45, 2115-2123 (2006).
[CrossRef] [PubMed]

2005 (2)

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

N. E. Korunska, M. Dybiec, L. Zhukov, S. Ostapenko, and T. Zhukov, "Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots," Semicond. Sci. Technol. 20, 876-881 (2005).
[CrossRef]

2004 (1)

C. D. McGuinness, K. Sagoo, D. McLoskey, and D. J. S. Birch, "A new sub-nanosecond LED at 280nm: application to protein fluorescence," Meas. Sci. Technol. 15, L19-L22 (2004).
[CrossRef]

2003 (3)

A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, "Applications of T-lymphoma labelled with fluorescent quantum dots to cell tracing markers in mouse body," Biochem. and Biophys. Res. Commun. 314, 46-53 (2003).
[CrossRef]

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

D. J. Stephens and V. J. Allan, "Light microscopy techniques for live cell imaging," Science 300, 82-86 (2003).
[CrossRef] [PubMed]

2002 (2)

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
[CrossRef] [PubMed]

J. K. Jaiswal, H. Mattoussi, J. M. Mauro, and S. M. Simon, "Long-term multiple color imaging of live cells using quantum dot bioconjugates," Nature Biotech. 21, 47-51 (2002).
[CrossRef]

2001 (1)

F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

2000 (2)

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, "Fluorescence lifetime imaging (FLI) in real time - a new technique in photosynthesis research," Photosynthetica 38, 581-599 (2000).
[CrossRef]

S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, "Photo-activated luminescence of CdSe Quantum dot monolayers," J. Phys. Chem. 104, 12137-12142 (2000).
[CrossRef]

1999 (1)

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

1998 (1)

1996 (1)

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

1994 (1)

M. Müller, R. Ghauharali, K. Visscher, and G. Brakenhoff, "Double-pulse fluorescence lifetime imaging in confocal microscopy," J. Microsc. 177, 171-179 (1994).
[CrossRef]

1993 (1)

R. Cubeddu, P. Taroni, and G. Valentini, "Time-gated imaging system for tumor diagnosis," Opt. Eng. 32,320-324 (1993).
[CrossRef]

1992 (2)

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
[CrossRef] [PubMed]

1991 (1)

X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A 2-dimensional fluorescence lifetime imaging-system using a gated image intensifier," Appl Spectrosc. 45, 360-366 (1991).
[CrossRef]

1990 (2)

R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).

C. G. Morgan, A. C. Mitchell, and J. G. Murray, "Nanosecond Time-Resolved Fluorescence Microscopy: Principles and Practice," Proc.R. Microsc. Soc. 1, 463-466 (1990).

1989 (1)

I. Bugiel, K. Konig, and H. Wabnitz, "Investigation of cells by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers Life Sci. 3, 47-53 (1989).

1988 (1)

Y. Sakai and S. Hirayama, "A fast deconvolution method to analyze fluorescence decays when the excitation pulse repetition period is less than the decay times," J. Lumin. 39, 145-151 (1988).
[CrossRef]

1928 (1)

H. Nyquist, "Certain topics in telegraph transmission theory," Trans. Am. Inst. Electr. Eng. 47, 617-644 (1928).
[CrossRef]

Agronskaia, A. V.

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

Allan, V. J.

D. J. Stephens and V. J. Allan, "Light microscopy techniques for live cell imaging," Science 300, 82-86 (2003).
[CrossRef] [PubMed]

Ameer-Beg, S.

Barber, P.

Barnes, D.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Bastiaens, P. I.

F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Bawendi, M. G.

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

Berndta, K. W.

J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
[CrossRef] [PubMed]

Birch, D. J. S.

C. D. McGuinness, K. Sagoo, D. McLoskey, and D. J. S. Birch, "A new sub-nanosecond LED at 280nm: application to protein fluorescence," Meas. Sci. Technol. 15, L19-L22 (2004).
[CrossRef]

Bornancin, F.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Bradforth, S. E.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

Brakenhoff, G.

M. Müller, R. Ghauharali, K. Visscher, and G. Brakenhoff, "Double-pulse fluorescence lifetime imaging in confocal microscopy," J. Microsc. 177, 171-179 (1994).
[CrossRef]

Brivanlou, A. H.

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
[CrossRef] [PubMed]

Brus, L. E.

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

Bugiel, I.

I. Bugiel, K. Konig, and H. Wabnitz, "Investigation of cells by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers Life Sci. 3, 47-53 (1989).

Buratto, S. K.

S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, "Photo-activated luminescence of CdSe Quantum dot monolayers," J. Phys. Chem. 104, 12137-12142 (2000).
[CrossRef]

Buurman, E. P.

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

Carson, P. J.

S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, "Photo-activated luminescence of CdSe Quantum dot monolayers," J. Phys. Chem. 104, 12137-12142 (2000).
[CrossRef]

Chong, S. Y.

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
[CrossRef]

Chong, S. Y. C.

Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).

Clarke, S. J.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

Clegg, R. M.

R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).

Coleman, D. M.

X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A 2-dimensional fluorescence lifetime imaging-system using a gated image intensifier," Appl Spectrosc. 45, 360-366 (1991).
[CrossRef]

Cordero, S. R.

S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, "Photo-activated luminescence of CdSe Quantum dot monolayers," J. Phys. Chem. 104, 12137-12142 (2000).
[CrossRef]

Cubeddu, R.

R. Cubeddu, P. Taroni, and G. Valentini, "Time-gated imaging system for tumor diagnosis," Opt. Eng. 32,320-324 (1993).
[CrossRef]

Dabbousi, B. O.

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

Dayel, M. J.

Dimitrijevic, N. M.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

Dowling, K.

Draaijer, A.

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

Dubertret, B.

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
[CrossRef] [PubMed]

Dybiec, M.

N. E. Korunska, M. Dybiec, L. Zhukov, S. Ostapenko, and T. Zhukov, "Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots," Semicond. Sci. Technol. 20, 876-881 (2005).
[CrossRef]

Dymoke-Bradshaw, A. K. L.

Errington, R. J.

Estabrook, R. A.

S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, "Photo-activated luminescence of CdSe Quantum dot monolayers," J. Phys. Chem. 104, 12137-12142 (2000).
[CrossRef]

Feddersen, B. A

R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).

French, P. M. W.

Gerritsen, H. C.

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

Gertitsen, H. C.

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

Ghauharali, R.

M. Müller, R. Ghauharali, K. Visscher, and G. Brakenhoff, "Double-pulse fluorescence lifetime imaging in confocal microscopy," J. Microsc. 177, 171-179 (1994).
[CrossRef]

Gohlke, C.

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, "Fluorescence lifetime imaging (FLI) in real time - a new technique in photosynthesis research," Photosynthetica 38, 581-599 (2000).
[CrossRef]

Gratton, E.

R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).

Gundersen, M. A.

Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
[CrossRef]

Hanaki, K.

A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, "Applications of T-lymphoma labelled with fluorescent quantum dots to cell tracing markers in mouse body," Biochem. and Biophys. Res. Commun. 314, 46-53 (2003).
[CrossRef]

Hanby, A.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Hansra, G.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Hares, J. D.

Harris, T. D.

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

Harris, W.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Hirayama, S.

Y. Sakai and S. Hirayama, "A fast deconvolution method to analyze fluorescence decays when the excitation pulse repetition period is less than the decay times," J. Lumin. 39, 145-151 (1988).
[CrossRef]

Hollmann, C. A.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

Holub, O.

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, "Fluorescence lifetime imaging (FLI) in real time - a new technique in photosynthesis research," Photosynthetica 38, 581-599 (2000).
[CrossRef]

Hoshino, A.

A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, "Applications of T-lymphoma labelled with fluorescent quantum dots to cell tracing markers in mouse body," Biochem. and Biophys. Res. Commun. 314, 46-53 (2003).
[CrossRef]

Houtpt, P. M.

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

Jaiswal, J. K.

J. K. Jaiswal, H. Mattoussi, J. M. Mauro, and S. M. Simon, "Long-term multiple color imaging of live cells using quantum dot bioconjugates," Nature Biotech. 21, 47-51 (2002).
[CrossRef]

Johnson, M.

J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
[CrossRef] [PubMed]

Jovin, T. M.

R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).

Konig, K.

I. Bugiel, K. Konig, and H. Wabnitz, "Investigation of cells by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers Life Sci. 3, 47-53 (1989).

Korunska, N. E.

N. E. Korunska, M. Dybiec, L. Zhukov, S. Ostapenko, and T. Zhukov, "Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots," Semicond. Sci. Technol. 20, 876-881 (2005).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
[CrossRef] [PubMed]

Lever, M. J.

Levine, Y. K.

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

Liang, C. H.

Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
[CrossRef]

Libchaber, A.

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
[CrossRef] [PubMed]

Liu, Y. S.

Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
[CrossRef]

Macklin, J. J.

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

Marcu, L.

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
[CrossRef]

Marriott, G.

R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).

Matthews, D. R.

Mattoussi, H.

J. K. Jaiswal, H. Mattoussi, J. M. Mauro, and S. M. Simon, "Long-term multiple color imaging of live cells using quantum dot bioconjugates," Nature Biotech. 21, 47-51 (2002).
[CrossRef]

Mauro, J. M.

J. K. Jaiswal, H. Mattoussi, J. M. Mauro, and S. M. Simon, "Long-term multiple color imaging of live cells using quantum dot bioconjugates," Nature Biotech. 21, 47-51 (2002).
[CrossRef]

McGuinness, C. D.

C. D. McGuinness, K. Sagoo, D. McLoskey, and D. J. S. Birch, "A new sub-nanosecond LED at 280nm: application to protein fluorescence," Meas. Sci. Technol. 15, L19-L22 (2004).
[CrossRef]

McLoskey, D.

C. D. McGuinness, K. Sagoo, D. McLoskey, and D. J. S. Birch, "A new sub-nanosecond LED at 280nm: application to protein fluorescence," Meas. Sci. Technol. 15, L19-L22 (2004).
[CrossRef]

Mellor, H.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Minami, S.

X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A 2-dimensional fluorescence lifetime imaging-system using a gated image intensifier," Appl Spectrosc. 45, 360-366 (1991).
[CrossRef]

Minarik, W. G.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
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Mitchell, A. C.

C. G. Morgan, A. C. Mitchell, and J. G. Murray, "Nanosecond Time-Resolved Fluorescence Microscopy: Principles and Practice," Proc.R. Microsc. Soc. 1, 463-466 (1990).

Morgan, C. G.

C. G. Morgan, A. C. Mitchell, and J. G. Murray, "Nanosecond Time-Resolved Fluorescence Microscopy: Principles and Practice," Proc.R. Microsc. Soc. 1, 463-466 (1990).

Müller, M.

M. Müller, R. Ghauharali, K. Visscher, and G. Brakenhoff, "Double-pulse fluorescence lifetime imaging in confocal microscopy," J. Microsc. 177, 171-179 (1994).
[CrossRef]

Murray, J. G.

C. G. Morgan, A. C. Mitchell, and J. G. Murray, "Nanosecond Time-Resolved Fluorescence Microscopy: Principles and Practice," Proc.R. Microsc. Soc. 1, 463-466 (1990).

Nadeau, J. L.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

Ng, T.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Nirmal, M.

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

Njoh, K.

Noireaux, V.

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
[CrossRef] [PubMed]

Norris, D. J.

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
[CrossRef] [PubMed]

Nowaczyka, K.

J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
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Nyquist, H.

H. Nyquist, "Certain topics in telegraph transmission theory," Trans. Am. Inst. Electr. Eng. 47, 617-644 (1928).
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Ostapenko, S.

N. E. Korunska, M. Dybiec, L. Zhukov, S. Ostapenko, and T. Zhukov, "Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots," Semicond. Sci. Technol. 20, 876-881 (2005).
[CrossRef]

Parker, P. J.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
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Phillips, D.

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

Prevostel, C.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Sagoo, K.

C. D. McGuinness, K. Sagoo, D. McLoskey, and D. J. S. Birch, "A new sub-nanosecond LED at 280nm: application to protein fluorescence," Meas. Sci. Technol. 15, L19-L22 (2004).
[CrossRef]

Sakai, Y.

Y. Sakai and S. Hirayama, "A fast deconvolution method to analyze fluorescence decays when the excitation pulse repetition period is less than the decay times," J. Lumin. 39, 145-151 (1988).
[CrossRef]

Sanders, P.

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

Schmidt, S.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

Seufferheld, M. J.

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, "Fluorescence lifetime imaging (FLI) in real time - a new technique in photosynthesis research," Photosynthetica 38, 581-599 (2000).
[CrossRef]

Simon, S. M.

J. K. Jaiswal, H. Mattoussi, J. M. Mauro, and S. M. Simon, "Long-term multiple color imaging of live cells using quantum dot bioconjugates," Nature Biotech. 21, 47-51 (2002).
[CrossRef]

Skourides, P.

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, "In vivo imaging of quantum dots encapsulated in phospholipid micelles," Science 298, 1759-1762 (2002).
[CrossRef] [PubMed]

Smith, P. J.

Squire, A.

T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker, "Imaging protein kinase Cα activation in cells," Science 283, 2085-2089 (1999).
[CrossRef] [PubMed]

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D. J. Stephens and V. J. Allan, "Light microscopy techniques for live cell imaging," Science 300, 82-86 (2003).
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Strouse, G. F.

S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, "Photo-activated luminescence of CdSe Quantum dot monolayers," J. Phys. Chem. 104, 12137-12142 (2000).
[CrossRef]

Suffern, D.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

Suhling, K.

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

Summers, H. D.

Sun, Y. H.

Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
[CrossRef]

Suzuki, K.

A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, "Applications of T-lymphoma labelled with fluorescent quantum dots to cell tracing markers in mouse body," Biochem. and Biophys. Res. Commun. 314, 46-53 (2003).
[CrossRef]

Szmacinskia, H.

J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
[CrossRef] [PubMed]

Taroni, P.

R. Cubeddu, P. Taroni, and G. Valentini, "Time-gated imaging system for tumor diagnosis," Opt. Eng. 32,320-324 (1993).
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A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
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Trautman, J. K.

M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus, "Fluorescence intermittency in single cadmium selenide nanocrystals," Nature 383, 802-804 (1996).
[CrossRef]

Uchida, T.

X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A 2-dimensional fluorescence lifetime imaging-system using a gated image intensifier," Appl Spectrosc. 45, 360-366 (1991).
[CrossRef]

Valentini, G.

R. Cubeddu, P. Taroni, and G. Valentini, "Time-gated imaging system for tumor diagnosis," Opt. Eng. 32,320-324 (1993).
[CrossRef]

van Veen, J. J. F.

E. P. Buurman, P. Sanders, A. Draaijer, H. C. Gertitsen, J. J. F. van Veen, P. M. Houtpt, and Y. K. Levine, "Fluorescence lifetime imaging using a confocal laser scanning microscope," Scanning 14, 155-159 (1992).
[CrossRef]

Vernier, P. T.

Y. S. Liu, Y. H. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, "pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells," J. Phys. Chem. 111, 2872-2878 (2007).

Y. H. Sun, Y. S. Liu, P. T. Vernier, C. H. Liang, S. Y. Chong, L. Marcu, and M. A. Gundersen, "Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells," Nanotechnology 17, 4469-4476 (2006).
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Verveer, P. J.

F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

Visscher, K.

M. Müller, R. Ghauharali, K. Visscher, and G. Brakenhoff, "Double-pulse fluorescence lifetime imaging in confocal microscopy," J. Microsc. 177, 171-179 (1994).
[CrossRef]

Vojnovic, B.

Wabnitz, H.

I. Bugiel, K. Konig, and H. Wabnitz, "Investigation of cells by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers Life Sci. 3, 47-53 (1989).

Wang, X. F.

X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A 2-dimensional fluorescence lifetime imaging-system using a gated image intensifier," Appl Spectrosc. 45, 360-366 (1991).
[CrossRef]

Wouters, F. S.

F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

Yamamoto, K.

A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, "Applications of T-lymphoma labelled with fluorescent quantum dots to cell tracing markers in mouse body," Biochem. and Biophys. Res. Commun. 314, 46-53 (2003).
[CrossRef]

Zhang, Z.

S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, and J. L. Nadeau, "Photophysics of dopamine-modified quantum dots and effects on biological systems," Nature Materials 5, 409-417 (2006).
[CrossRef] [PubMed]

Zhukov, L.

N. E. Korunska, M. Dybiec, L. Zhukov, S. Ostapenko, and T. Zhukov, "Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots," Semicond. Sci. Technol. 20, 876-881 (2005).
[CrossRef]

Zhukov, T.

N. E. Korunska, M. Dybiec, L. Zhukov, S. Ostapenko, and T. Zhukov, "Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots," Semicond. Sci. Technol. 20, 876-881 (2005).
[CrossRef]

Anal. Biochem. (1)

J. R. Lakowicz, H. Szmacinskia, K. Nowaczyka, K. W. Berndta, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
[CrossRef] [PubMed]

Appl Spectrosc. (1)

X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A 2-dimensional fluorescence lifetime imaging-system using a gated image intensifier," Appl Spectrosc. 45, 360-366 (1991).
[CrossRef]

Appl. Opt. (1)

Biochem. and Biophys. Res. Commun. (1)

A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, "Applications of T-lymphoma labelled with fluorescent quantum dots to cell tracing markers in mouse body," Biochem. and Biophys. Res. Commun. 314, 46-53 (2003).
[CrossRef]

Biophys. J. (1)

R. M. Clegg, G. Marriott, B. A Feddersen, E. Gratton, and T. M. Jovin, "Sensitive and rapid determinations of fluorescence lifetimes in the frequency domain in a light-microscope," Biophys. J. 57,A375-A375 (1990).

J. Lumin. (1)

Y. Sakai and S. Hirayama, "A fast deconvolution method to analyze fluorescence decays when the excitation pulse repetition period is less than the decay times," J. Lumin. 39, 145-151 (1988).
[CrossRef]

J. Microsc. (1)

M. Müller, R. Ghauharali, K. Visscher, and G. Brakenhoff, "Double-pulse fluorescence lifetime imaging in confocal microscopy," J. Microsc. 177, 171-179 (1994).
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Supplementary Material (1)

» Media 1: AVI (2169 KB)     

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Figures (6)

Fig. 1.
Fig. 1.

Schematic of the stroboscopic excitation technique in time and frequency domains.

Fig. 2.
Fig. 2.

(a) Streak camera data for excitation frequencies of 1 MHz (blue trace), 10 MHz (black trace) and 30 MHz (red trace) illustrating the inhibited fluorescence decay of 705 nm quantum dots in response to a periodic impulse; (b) a single-exponential fit to the complete inter-pulse recovery of the 705 nm quantum dots under 1 MHz excitation and (c) the frequency roll-off of integrated fluorescence per pulse as the inter-pulse period is reduced to less than the quantum dot lifetime. The solid line is a best-fit to the data using equation 1, the parameter values of which are shown.

Fig. 3.
Fig. 3.

(a) Experimental setup illustrating the laser input at the upper lamp input-port and the spectral filters used for imaging; (b) 50 × 50 μm, fluorescence intensity images of 705 nm QDs and Cy-5 dye and (c) 705 nm and 611 nm QDs; (d) ratio images derived from intensity images taken at 1 MHz and 30 MHz excitation frequencies for 705 nm QDs and Cy-5 and (e) 705 nm and 611 nm QDs; (f) histograms of pixel-to-pixel variation in R for 705 nm QDs and Cy-5 and (g) 705 nm and 611 nm QDs. In both cases the distribution is bi-modal illustrating the ability of stroboscopic imaging to distinguish the fluorophore pairs from their lifetime.

Fig. 4.
Fig. 4.

(a) schematic of the QD uptake and localisation within intra-cellular vesicles via endocytosis; (b) typical 50 × 50 μm, transmission and (c) fluorescence intensity images of a single cell; (d) 50 × 30 μm intensity image of a cell, fixed in PBS 24 hours following QD uptake and (e) intensity image of a cell fixed in Prolong® Gold, anti-fade reagent 24 hours following QD uptake; (f) corresponding ratio images for cells fixed in PBS and (g) anti-fade reagent.

Fig. 5.
Fig. 5.

(Media 1) shows 140 seconds of imaging time at x10 speed. From left to right, the first two images are the raw data taken at 1 MHz and 30 MHz repetition rates respectively and the final image is the frequency ratio, R. Rapid motion of the QD loaded vesicles can be seen and this temporal resolution is maintained in the ratio image.

Fig. 6.
Fig. 6.

(a) 30 × 30 μm, Ratio images of live cells at 24 hours post QD loading, (b) 48 hours post QD loading and (c) 120 hours post QD loading; (d) the histograms of the R variation, the symbols on the insets of fig. 6a–c relate each trace to the corresponding acquisition time. The three histograms show a systematic reduction as the QDs are degraded within the cells leading to a reduced QY and a shorter t as non-radiative relaxation routes become active.

Equations (9)

Equations on this page are rendered with MathJax. Learn more.

S c = κ [ e 2 αP 1 e 2 αP e γ f ex ] ( 1 e γ f ex )
d N 2 dt = I p ( N 1 N 2 ) N 2 τ
d N 1 dt = I p ( N 1 N 2 ) + N 2 τ
dn dt = I p ( 1 2 n ) n τ
i . e . n 1 n u 1 ( 1 2 n ) dn = I p 0 T p dt
and n u n l 1 n dn = 0 T 1 τ dt
Δ n = 1 2 ( e 2 I p T p 1 ) e I p T p e T τ { 1 e T τ }
I p T p = η ex E p E q σ A
S c = κ ( e 2 αP 1 ) ( e 2 αP e γ f ex ) { 1 e γ f ex }

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