Abstract

We report on the implementation of fluorescence-lifetime imaging in multiphoton excitation microscopy that uses PC-compatible modules for time-correlated single-photon counting. Four-dimensional data stacks are produced with each pixel featuring fluorescence-decay curves that consist of as many as 4096 bins. Fluorescence lifetime(s) and their amplitude(s) are extracted by statistical methods at each pixel or in arbitrarily defined regions of interest. When employing an avalanche photodiode the width of the temporal response function is 420 ps. Although this response confines the temporal resolution to values greater than several hundreds of picoseconds, the lifetime precision is determined by the signal-to-noise ratio and can be in the range of tens of picosconds. Lifetime changes are visualized in pulsed-laser-deposited fluorescent layers as well as in cyan fluorescent proteins that transfer energy to yellow fluorescent proteins in live mammalian cells.

© 2000 Optical Society of America

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References

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  1. A. Draaijer, R. Sanders, H. C. Gerritsen, “Fluorescence lifetime imaging, a new tool in confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 491–505.
    [CrossRef]
  2. J. R. Lakowicz, H. Szmaczinski, K. Nowaczyk, “Fluorescence lifetime imaging,” Proc. Natl. Acad. Sci. USA 89, 1271–1275 (1992).
    [CrossRef]
  3. J. R. Lakowicz, K. W. Berndt, “Lifetime-selective fluorescence imaging using a rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).
  4. G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, “Time-resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging,” Biophys. J. 60, 1374–1387 (1991).
    [CrossRef] [PubMed]
  5. C. G. Morgan, A. C. Mitchell, C. G. Murray, “Prospects for confocal imaging based on nanosecond fluorescence decay time,” J. Microsc. 165, 49–60 (1991).
    [CrossRef]
  6. A. Squire, P. J. Verveer, P. I. H. Bastiaens, “Multiple frequency fluorescence imaging microscopy,” J. Microsc. 197, 136–149 (2000).
    [CrossRef] [PubMed]
  7. X. F. Wang, T. Uchida, D. M. Coleman, S. Minami, “A two-dimensional fluorescence lifetime imaging system using a gated image intensifier,” Appl. Spectrosc. 45, 360–366 (1991).
    [CrossRef]
  8. X. F. Wang, S. Kitajima, T. Uchida, D. M. Coleman, S. Minami, “Time-resolved fluorescence microscopy using multichannel photon counting,” Appl. Spectrosc. 44, 25–30 (1990).
    [CrossRef]
  9. M. Straub, S. W. Hell, “Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,” Appl. Phys. Lett. 73, 1769–1771 (1998).
    [CrossRef]
  10. M. Dyba, T. A. Klar, S. Jakobs, S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77, 597–599 (2000).
    [CrossRef]
  11. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  12. J. Sytsma, J. M. Vroom, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–42 (1998).
    [CrossRef]
  13. W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Vetterling, Numerical Recipes in C, 2nd. ed. (Cambridge U. Press, Cambridge, 1993).
  14. M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
    [CrossRef] [PubMed]
  15. W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
  16. S. W. Hell, A. Utz, P. E. Hänninen, E. Soini, “Pulsed laser fluorophore deposition: a method for measuring the axial resolution in two-photon fluorescence microscopy,” J. Opt. Soc. Am. A 12, 2072–2076 (1995).
    [CrossRef]
  17. P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

2000 (2)

A. Squire, P. J. Verveer, P. I. H. Bastiaens, “Multiple frequency fluorescence imaging microscopy,” J. Microsc. 197, 136–149 (2000).
[CrossRef] [PubMed]

M. Dyba, T. A. Klar, S. Jakobs, S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77, 597–599 (2000).
[CrossRef]

1999 (1)

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

1998 (3)

J. Sytsma, J. M. Vroom, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–42 (1998).
[CrossRef]

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

M. Straub, S. W. Hell, “Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,” Appl. Phys. Lett. 73, 1769–1771 (1998).
[CrossRef]

1996 (1)

P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

1995 (1)

1992 (1)

J. R. Lakowicz, H. Szmaczinski, K. Nowaczyk, “Fluorescence lifetime imaging,” Proc. Natl. Acad. Sci. USA 89, 1271–1275 (1992).
[CrossRef]

1991 (4)

J. R. Lakowicz, K. W. Berndt, “Lifetime-selective fluorescence imaging using a rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).

G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, “Time-resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging,” Biophys. J. 60, 1374–1387 (1991).
[CrossRef] [PubMed]

C. G. Morgan, A. C. Mitchell, C. G. Murray, “Prospects for confocal imaging based on nanosecond fluorescence decay time,” J. Microsc. 165, 49–60 (1991).
[CrossRef]

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

1990 (2)

Arndt-Jovin, D. J.

G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, “Time-resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging,” Biophys. J. 60, 1374–1387 (1991).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

A. Squire, P. J. Verveer, P. I. H. Bastiaens, “Multiple frequency fluorescence imaging microscopy,” J. Microsc. 197, 136–149 (2000).
[CrossRef] [PubMed]

P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

Becker, W.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Berndt, K. W.

J. R. Lakowicz, K. W. Berndt, “Lifetime-selective fluorescence imaging using a rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).

Clegg, R. M.

G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, “Time-resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging,” Biophys. J. 60, 1374–1387 (1991).
[CrossRef] [PubMed]

Coleman, D. M.

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Draaijer, A.

A. Draaijer, R. Sanders, H. C. Gerritsen, “Fluorescence lifetime imaging, a new tool in confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 491–505.
[CrossRef]

Drexhage, K. H.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Dyba, M.

M. Dyba, T. A. Klar, S. Jakobs, S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77, 597–599 (2000).
[CrossRef]

Flannery, B. P.

W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Vetterling, Numerical Recipes in C, 2nd. ed. (Cambridge U. Press, Cambridge, 1993).

Gerritsen, H. C.

J. Sytsma, J. M. Vroom, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–42 (1998).
[CrossRef]

A. Draaijer, R. Sanders, H. C. Gerritsen, “Fluorescence lifetime imaging, a new tool in confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 491–505.
[CrossRef]

Hänninen, P. E.

Hell, S. W.

M. Dyba, T. A. Klar, S. Jakobs, S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77, 597–599 (2000).
[CrossRef]

M. Straub, S. W. Hell, “Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,” Appl. Phys. Lett. 73, 1769–1771 (1998).
[CrossRef]

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

S. W. Hell, A. Utz, P. E. Hänninen, E. Soini, “Pulsed laser fluorophore deposition: a method for measuring the axial resolution in two-photon fluorescence microscopy,” J. Opt. Soc. Am. A 12, 2072–2076 (1995).
[CrossRef]

Hickl, H.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Hofmann, U. G.

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

Jakobs, S.

M. Dyba, T. A. Klar, S. Jakobs, S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77, 597–599 (2000).
[CrossRef]

Jovin, T. M.

P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, “Time-resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging,” Biophys. J. 60, 1374–1387 (1991).
[CrossRef] [PubMed]

Kitajima, S.

Klar, T. A.

M. Dyba, T. A. Klar, S. Jakobs, S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77, 597–599 (2000).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmaczinski, K. Nowaczyk, “Fluorescence lifetime imaging,” Proc. Natl. Acad. Sci. USA 89, 1271–1275 (1992).
[CrossRef]

J. R. Lakowicz, K. W. Berndt, “Lifetime-selective fluorescence imaging using a rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).

Majoul, I. V.

P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

Marriott, G.

G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, “Time-resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging,” Biophys. J. 60, 1374–1387 (1991).
[CrossRef] [PubMed]

Minami, S.

Mitchell, A. C.

C. G. Morgan, A. C. Mitchell, C. G. Murray, “Prospects for confocal imaging based on nanosecond fluorescence decay time,” J. Microsc. 165, 49–60 (1991).
[CrossRef]

Morgan, C. G.

C. G. Morgan, A. C. Mitchell, C. G. Murray, “Prospects for confocal imaging based on nanosecond fluorescence decay time,” J. Microsc. 165, 49–60 (1991).
[CrossRef]

Murray, C. G.

C. G. Morgan, A. C. Mitchell, C. G. Murray, “Prospects for confocal imaging based on nanosecond fluorescence decay time,” J. Microsc. 165, 49–60 (1991).
[CrossRef]

Nowaczyk, K.

J. R. Lakowicz, H. Szmaczinski, K. Nowaczyk, “Fluorescence lifetime imaging,” Proc. Natl. Acad. Sci. USA 89, 1271–1275 (1992).
[CrossRef]

Press, W. H.

W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Vetterling, Numerical Recipes in C, 2nd. ed. (Cambridge U. Press, Cambridge, 1993).

Sanders, R.

A. Draaijer, R. Sanders, H. C. Gerritsen, “Fluorescence lifetime imaging, a new tool in confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 491–505.
[CrossRef]

Sauer, M.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Schrader, M.

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

Siebert, S.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Soini, E.

Söling, H. D.

P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

Squire, A.

A. Squire, P. J. Verveer, P. I. H. Bastiaens, “Multiple frequency fluorescence imaging microscopy,” J. Microsc. 197, 136–149 (2000).
[CrossRef] [PubMed]

Straub, M.

M. Straub, S. W. Hell, “Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,” Appl. Phys. Lett. 73, 1769–1771 (1998).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Sytsma, J.

J. Sytsma, J. M. Vroom, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–42 (1998).
[CrossRef]

Szmaczinski, H.

J. R. Lakowicz, H. Szmaczinski, K. Nowaczyk, “Fluorescence lifetime imaging,” Proc. Natl. Acad. Sci. USA 89, 1271–1275 (1992).
[CrossRef]

Teukolsky, S. A.

W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Vetterling, Numerical Recipes in C, 2nd. ed. (Cambridge U. Press, Cambridge, 1993).

Uchida, T.

Utz, A.

Verveer, P. J.

A. Squire, P. J. Verveer, P. I. H. Bastiaens, “Multiple frequency fluorescence imaging microscopy,” J. Microsc. 197, 136–149 (2000).
[CrossRef] [PubMed]

P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

Vetterling, W. T.

W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Vetterling, Numerical Recipes in C, 2nd. ed. (Cambridge U. Press, Cambridge, 1993).

Vroom, J. M.

J. Sytsma, J. M. Vroom, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–42 (1998).
[CrossRef]

Wang, X. F.

Webb, W. W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Wolfrum, J.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Zander, C.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Appl. Phys. Lett. (2)

M. Straub, S. W. Hell, “Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,” Appl. Phys. Lett. 73, 1769–1771 (1998).
[CrossRef]

M. Dyba, T. A. Klar, S. Jakobs, S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77, 597–599 (2000).
[CrossRef]

Appl. Spectrosc. (2)

Biophys. J. (1)

G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, “Time-resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging,” Biophys. J. 60, 1374–1387 (1991).
[CrossRef] [PubMed]

EMBO (1)

P. I. H. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Söling, T. M. Jovin, “Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin,” EMBO 15, 4246–4253 (1996).

J. Microsc. (4)

J. Sytsma, J. M. Vroom, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–42 (1998).
[CrossRef]

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

C. G. Morgan, A. C. Mitchell, C. G. Murray, “Prospects for confocal imaging based on nanosecond fluorescence decay time,” J. Microsc. 165, 49–60 (1991).
[CrossRef]

A. Squire, P. J. Verveer, P. I. H. Bastiaens, “Multiple frequency fluorescence imaging microscopy,” J. Microsc. 197, 136–149 (2000).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (1)

Proc. Natl. Acad. Sci. USA (1)

J. R. Lakowicz, H. Szmaczinski, K. Nowaczyk, “Fluorescence lifetime imaging,” Proc. Natl. Acad. Sci. USA 89, 1271–1275 (1992).
[CrossRef]

Rev. Sci. Instrum. (2)

J. R. Lakowicz, K. W. Berndt, “Lifetime-selective fluorescence imaging using a rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).

Science (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Other (2)

W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Vetterling, Numerical Recipes in C, 2nd. ed. (Cambridge U. Press, Cambridge, 1993).

A. Draaijer, R. Sanders, H. C. Gerritsen, “Fluorescence lifetime imaging, a new tool in confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 491–505.
[CrossRef]

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Figures (7)

Fig. 1
Fig. 1

Schematic diagram of the fluorescence-lifetime microscope: A thin glass window (GL) reflects a portion of pulsed-laser light onto an external photodiode (PD) to provide a stop signal for the time-to-amplitude converter (TAC). The main beam passes through a laser-power controller (LPC) and is focused through a 30-µm pinhole (PH). A dichroic mirror (DC) reflects the excitation light into the objective lens. The collected fluorescence passes through the dichroic mirror and is focused onto a pinhole by the tube lens (TL); this fluorescence optionally passes through an emission filter (EF). Finally, the pinhole is imaged onto a single-photon-counting avalanche photodiode (APD). The counting event provides the start signal for the time-to-amplitude converter. The components within the box, i.e., the filter cube (FC) and the tube lens, can optionally be flipped into the optical path, and the sample can then be illuminated by a UV lamp and observed directly by eye. We used exchangeable filter cubes (Leica Microsystems, Wetzlar, Germany) that consist of excitation filters, dichroic beam splitters, and emission filters.

Fig. 2
Fig. 2

(a) Image of a 100-nm latex bead and (b) its intensity profile along the x axis through the center of the bead with a lateral FWHM of 300 nm.

Fig. 3
Fig. 3

(a) Image of a thin fluorescent layer with the optical axis oriented horizontally. (b) The extracted z profile that represents the z response of the system and is a measure of the axial resolution of a 3-D microscope. Its FWHM is 730 nm.

Fig. 4
Fig. 4

Instrument time response as measured by the recording of reflected laser light. Its FWHM is 420 ps.

Fig. 5
Fig. 5

Images of two distinct species of fluorescent beads that were imaged in the fluorescence-lifetime microscope: (a) An xy intensity section through the beads. (b) A display of the differences in the fluorescence lifetimes. (c) The histograms that were accumulated over all the pixels of boxes 1 and 2 in (a) and the results of a nonlinear curve fitting (in red). The lifetimes extracted by this method were 2.25 ns and 3.65 ns for the small and the large beads, respectively.

Fig. 6
Fig. 6

Lifetime analysis after PLFD. The 10 µm × 10 µm image is centered about a PLFD-generated spot: (a) An intensity image of the area. (b), (c) The spatial distributions of the signal amplitudes that belong to the two lifetime species. The upper outset on the right-hand side shows a semilogarithmic plot of the fluorescence decay in the boxed region, whereas the lower outset depicts the mean lifetime image of the whole area.

Fig. 7
Fig. 7

Part of the Golgi apparatus of a live monkey kidney cell imaged with MPE at 813 nm (a) without and (b) with an emission bandpass filter that suppresses the YFP fluorescence. The cell expresses CFP and YFP fusion transmembrane proteins that have been shown to act as donor and acceptor FRET pairs. The relative brightness of regions 1, 2, 6, 7, and 8 is reduced in (b), but the lifetime data indicate a reduced donor lifetime in only regions 6, 7, and 8.

Tables (1)

Tables Icon

Table 1 The Lifetimes τa and τ b Extracted from the Accumulated Histograms of Each Pixel in Regions 1–8 of Figs. 7(a) and 7(b), Respectivelya

Equations (2)

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It=i=0N ai exp-t/τi+C.
τ=Tlni=0N/2 nii=N/2N ni-1,

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