Abstract

When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope. Fluorescence lifetime images from 16 individual wavelength components of the emission spectrum can be acquired with 10-nm resolution on a pixel-by-pixel basis. The instrument provides a unique visualization of cellular structures and processes through spectrally and temporally resolved information and may ultimately find applications in live cell and tissue imaging.

© 2004 Optical Society of America

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2004 (1)

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

2003 (3)

K. W. Eliceiri, C. H. Fan, G. A. Lyons, J. G. White, “Analysis of histology specimens using lifetime multiphoton microscopy,” J. Biomed. Opt. 8, 376–380 (2003).
[CrossRef] [PubMed]

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, S. E. Fraser, “Multiphoton excitation spectra in biological samples,” J. Biomed. Opt. 8, 329–338 (2003).
[CrossRef] [PubMed]

2002 (2)

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Q. S. Hanley, D. J. Arndt-Jovin, T. M. Jovin, “Spectrally resolved fluorescence lifetime imaging microscopy,” Appl. Spectrosc. 56, 155–166 (2002).
[CrossRef]

2001 (2)

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, S. E. Fraser, “Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy,” BioTechniques 31, 1272–1278 (2001).

2000 (2)

1999 (2)

P. I. Bastiaens, A. Squire, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9, 48–52 (1999).
[CrossRef] [PubMed]

J. M. Squirrell, D. L. Wokosin, J. G. White, B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17, 763–767 (1999).
[CrossRef] [PubMed]

1998 (3)

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (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]

G. A. Wagnieres, W. M. Star, B. C. Wilson, “In vivo fluorescence spectroscopy and imaging for oncological applications,” Photochem. Photobiol. 68, 603–632 (1998).
[PubMed]

1993 (1)

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48, 221–239 (1993).
[CrossRef]

1992 (2)

X. F. Wang, A. Periasamy, B. Herman, “Fluorescence lifetime imaging microscopy (FLIM): instrumentation and applications,” Crit. Rev. Anal. Chem. 23, 365–369 (1992).
[CrossRef]

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202, 316–330 (1992).
[CrossRef] [PubMed]

1990 (1)

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

Arndt-Jovin, D. J.

Bastiaens, P. I.

P. I. Bastiaens, A. Squire, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9, 48–52 (1999).
[CrossRef] [PubMed]

Bavister, B. D.

J. M. Squirrell, D. L. Wokosin, J. G. White, B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17, 763–767 (1999).
[CrossRef] [PubMed]

Bearman, G.

M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, S. E. Fraser, “Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy,” BioTechniques 31, 1272–1278 (2001).

Becker, W.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, “Multiwavelength TCSPC lifetime imaging,” in Multiphoton Microscopy in the Biomedical Sciences II, A. Periasamy, P. T. C. So, eds., Proc. SPIE4620, 74–84 (2002).
[CrossRef]

Benndorf, K.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, “Multiwavelength TCSPC lifetime imaging,” in Multiphoton Microscopy in the Biomedical Sciences II, A. Periasamy, P. T. C. So, eds., Proc. SPIE4620, 74–84 (2002).
[CrossRef]

Bergmann, A.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, “Multiwavelength TCSPC lifetime imaging,” in Multiphoton Microscopy in the Biomedical Sciences II, A. Periasamy, P. T. C. So, eds., Proc. SPIE4620, 74–84 (2002).
[CrossRef]

Berndt, K. W.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202, 316–330 (1992).
[CrossRef] [PubMed]

Biskup, C.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, “Multiwavelength TCSPC lifetime imaging,” in Multiphoton Microscopy in the Biomedical Sciences II, A. Periasamy, P. T. C. So, eds., Proc. SPIE4620, 74–84 (2002).
[CrossRef]

Bottinger, E. P.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Centonze, V. E.

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

Cermak, L.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Clegg, R. M.

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48, 221–239 (1993).
[CrossRef]

Cole, M. J.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Condeelis, J. S.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Dayel, M. J.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

deGrauw, K.

H. C. Gerritsen, K. deGrauw, “One and two-photon confocal fluorescence lifetime imaging and its applications,” in Methods in Cellular Imaging, A. Periasamy, ed. (Oxford U. Press, New York, 2001), pp. 309–323.
[CrossRef]

Denk, W.

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

Dickinson, M. E.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, S. E. Fraser, “Multiphoton excitation spectra in biological samples,” J. Biomed. Opt. 8, 329–338 (2003).
[CrossRef] [PubMed]

M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, S. E. Fraser, “Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy,” BioTechniques 31, 1272–1278 (2001).

Dowling, K.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Eliceiri, K. W.

K. W. Eliceiri, C. H. Fan, G. A. Lyons, J. G. White, “Analysis of histology specimens using lifetime multiphoton microscopy,” J. Biomed. Opt. 8, 376–380 (2003).
[CrossRef] [PubMed]

Fan, C. H.

K. W. Eliceiri, C. H. Fan, G. A. Lyons, J. G. White, “Analysis of histology specimens using lifetime multiphoton microscopy,” J. Biomed. Opt. 8, 376–380 (2003).
[CrossRef] [PubMed]

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, UK, 1993).

Fraser, S. E.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, S. E. Fraser, “Multiphoton excitation spectra in biological samples,” J. Biomed. Opt. 8, 329–338 (2003).
[CrossRef] [PubMed]

M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, S. E. Fraser, “Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy,” BioTechniques 31, 1272–1278 (2001).

French, P. M.

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

French, P. M. W.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Frohlich, V. C.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Gadella, T. W. J.

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48, 221–239 (1993).
[CrossRef]

Gerritsen, H. C.

H. C. Gerritsen, K. deGrauw, “One and two-photon confocal fluorescence lifetime imaging and its applications,” in Methods in Cellular Imaging, A. Periasamy, ed. (Oxford U. Press, New York, 2001), pp. 309–323.
[CrossRef]

Glatz, M.

Hanley, Q. S.

Hecht, E.

E. Hecht, Optics, 2nd ed. (Addison-Wesley, Reading, Mass., 1990).

Hell, S. W.

A. Schönle, M. Glatz, S. W. Hell, “Four-dimensional multiphoton microscopy with time-correlated single-photon counting,” Appl. Opt. 39, 6306–6311 (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]

Herman, B.

X. F. Wang, A. Periasamy, B. Herman, “Fluorescence lifetime imaging microscopy (FLIM): instrumentation and applications,” Crit. Rev. Anal. Chem. 23, 365–369 (1992).
[CrossRef]

Hink, M. A.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

Huttelmaier, S.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Johnson, M.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202, 316–330 (1992).
[CrossRef] [PubMed]

Jones, R.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Jovin, T. M.

Q. S. Hanley, D. J. Arndt-Jovin, T. M. Jovin, “Spectrally resolved fluorescence lifetime imaging microscopy,” Appl. Spectrosc. 56, 155–166 (2002).
[CrossRef]

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48, 221–239 (1993).
[CrossRef]

Juskaitis, R.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Klöcker, N.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, “Multiwavelength TCSPC lifetime imaging,” in Multiphoton Microscopy in the Biomedical Sciences II, A. Periasamy, P. T. C. So, eds., Proc. SPIE4620, 74–84 (2002).
[CrossRef]

König, K.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202, 316–330 (1992).
[CrossRef] [PubMed]

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1999).
[CrossRef]

Lalani, el-N.

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

Lansford, R.

M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, S. E. Fraser, “Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy,” BioTechniques 31, 1272–1278 (2001).

Lever, M. J.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Lyons, G. A.

K. W. Eliceiri, C. H. Fan, G. A. Lyons, J. G. White, “Analysis of histology specimens using lifetime multiphoton microscopy,” J. Biomed. Opt. 8, 376–380 (2003).
[CrossRef] [PubMed]

Neil, M. A. A.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Nowaczyk, K.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202, 316–330 (1992).
[CrossRef] [PubMed]

O’Connor, D. V.

D. V. O’Connor, D. Phillips, Time Correlated Single Photon Counting (Academic, London, 1984).

Oleynikov, Y.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Parsons-Karavassilis, D.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Periasamy, A.

X. F. Wang, A. Periasamy, B. Herman, “Fluorescence lifetime imaging microscopy (FLIM): instrumentation and applications,” Crit. Rev. Anal. Chem. 23, 365–369 (1992).
[CrossRef]

Phillips, D.

D. V. O’Connor, D. Phillips, Time Correlated Single Photon Counting (Academic, London, 1984).

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, UK, 1993).

Ramanujam, N.

N. Ramanujam, “Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,” Neoplasia 2, 89–117 (2000).
[CrossRef] [PubMed]

Schönle, A.

Segall, J. E.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Shousha, S.

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

Siegel, J.

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Simbuerger, E.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, S. E. Fraser, “Multiphoton excitation spectra in biological samples,” J. Biomed. Opt. 8, 329–338 (2003).
[CrossRef] [PubMed]

Singer, R. H.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Squire, A.

P. I. Bastiaens, A. Squire, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9, 48–52 (1999).
[CrossRef] [PubMed]

Squirrell, J. M.

J. M. Squirrell, D. L. Wokosin, J. G. White, B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17, 763–767 (1999).
[CrossRef] [PubMed]

Stamp, G. W.

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

Star, W. M.

G. A. Wagnieres, W. M. Star, B. C. Wilson, “In vivo fluorescence spectroscopy and imaging for oncological applications,” Photochem. Photobiol. 68, 603–632 (1998).
[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]

Sucharov, L. O. D.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Szmacinski, H.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202, 316–330 (1992).
[CrossRef] [PubMed]

Tadrous, P. J.

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

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, UK, 1993).

Tille, S.

M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, S. E. Fraser, “Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy,” BioTechniques 31, 1272–1278 (2001).

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, UK, 1993).

Wagnieres, G. A.

G. A. Wagnieres, W. M. Star, B. C. Wilson, “In vivo fluorescence spectroscopy and imaging for oncological applications,” Photochem. Photobiol. 68, 603–632 (1998).
[PubMed]

Wang, W.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Wang, X. F.

X. F. Wang, A. Periasamy, B. Herman, “Fluorescence lifetime imaging microscopy (FLIM): instrumentation and applications,” Crit. Rev. Anal. Chem. 23, 365–369 (1992).
[CrossRef]

Waters, C. W.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, S. E. Fraser, “Multiphoton excitation spectra in biological samples,” J. Biomed. Opt. 8, 329–338 (2003).
[CrossRef] [PubMed]

Webb, S. E. D.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Webb, W. W.

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

White, J. G.

K. W. Eliceiri, C. H. Fan, G. A. Lyons, J. G. White, “Analysis of histology specimens using lifetime multiphoton microscopy,” J. Biomed. Opt. 8, 376–380 (2003).
[CrossRef] [PubMed]

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

J. M. Squirrell, D. L. Wokosin, J. G. White, B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17, 763–767 (1999).
[CrossRef] [PubMed]

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

Wilson, B. C.

G. A. Wagnieres, W. M. Star, B. C. Wilson, “In vivo fluorescence spectroscopy and imaging for oncological applications,” Photochem. Photobiol. 68, 603–632 (1998).
[PubMed]

Wilson, T.

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

Wokosin, D. L.

J. M. Squirrell, D. L. Wokosin, J. G. White, B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17, 763–767 (1999).
[CrossRef] [PubMed]

Wyckoff, J. B.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Zavadil, J.

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Zimmer, T.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, “Multiwavelength TCSPC lifetime imaging,” in Multiphoton Microscopy in the Biomedical Sciences II, A. Periasamy, P. T. C. So, eds., Proc. SPIE4620, 74–84 (2002).
[CrossRef]

Zimmermann, B.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, S. E. Fraser, “Multiphoton excitation spectra in biological samples,” J. Biomed. Opt. 8, 329–338 (2003).
[CrossRef] [PubMed]

Anal. Biochem. (1)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202, 316–330 (1992).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Phys. Lett. (1)

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]

Appl. Spectrosc. (1)

Biophys. Chem. (1)

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48, 221–239 (1993).
[CrossRef]

Biophys. J. (1)

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

BioTechniques (1)

M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, S. E. Fraser, “Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy,” BioTechniques 31, 1272–1278 (2001).

Cancer Res. (1)

W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, J. E. Segall, J. S. Condeelis, “Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling,” Cancer Res. 62, 6278–6288 (2002).
[PubMed]

Crit. Rev. Anal. Chem. (1)

X. F. Wang, A. Periasamy, B. Herman, “Fluorescence lifetime imaging microscopy (FLIM): instrumentation and applications,” Crit. Rev. Anal. Chem. 23, 365–369 (1992).
[CrossRef]

J. Biomed. Opt. (2)

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, S. E. Fraser, “Multiphoton excitation spectra in biological samples,” J. Biomed. Opt. 8, 329–338 (2003).
[CrossRef] [PubMed]

K. W. Eliceiri, C. H. Fan, G. A. Lyons, J. G. White, “Analysis of histology specimens using lifetime multiphoton microscopy,” J. Biomed. Opt. 8, 376–380 (2003).
[CrossRef] [PubMed]

J. Microsc. (Oxford) (1)

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Time-domain whole-field fluorescence lifetime imaging with optical sectioning,” J. Microsc. (Oxford) 203, 246–257 (2001).
[CrossRef]

J. Pathol. (1)

P. J. Tadrous, J. Siegel, P. M. French, S. Shousha, el-N. Lalani, G. W. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63, 58–66 (2004).
[CrossRef]

Nat. Biotechnol. (1)

J. M. Squirrell, D. L. Wokosin, J. G. White, B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17, 763–767 (1999).
[CrossRef] [PubMed]

Neoplasia (1)

N. Ramanujam, “Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,” Neoplasia 2, 89–117 (2000).
[CrossRef] [PubMed]

Photochem. Photobiol. (1)

G. A. Wagnieres, W. M. Star, B. C. Wilson, “In vivo fluorescence spectroscopy and imaging for oncological applications,” Photochem. Photobiol. 68, 603–632 (1998).
[PubMed]

Science (1)

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

Trends Cell Biol. (1)

P. I. Bastiaens, A. Squire, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9, 48–52 (1999).
[CrossRef] [PubMed]

Other (9)

H. C. Gerritsen, K. deGrauw, “One and two-photon confocal fluorescence lifetime imaging and its applications,” in Methods in Cellular Imaging, A. Periasamy, ed. (Oxford U. Press, New York, 2001), pp. 309–323.
[CrossRef]

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1999).
[CrossRef]

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

E. Hecht, Optics, 2nd ed. (Addison-Wesley, Reading, Mass., 1990).

Hamamatsu Photonics K. K. Electron Tube Center, R5900U-L16 Series Data Sheet, http://www.hamamatsu.com (Hamamatsu Photonics, Hamamatsu City, Japan, 2003).

K. W. Eliceiri, “WiscScan: A DSP based Acquisition System for Laser Scanning Microscopes,” http://www.loci.wisc.edu/WiscScan/ .

D. V. O’Connor, D. Phillips, Time Correlated Single Photon Counting (Academic, London, 1984).

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, “Multiwavelength TCSPC lifetime imaging,” in Multiphoton Microscopy in the Biomedical Sciences II, A. Periasamy, P. T. C. So, eds., Proc. SPIE4620, 74–84 (2002).
[CrossRef]

Fluoresbrite Microparticles, Technical Data Sheet 431 (Polysciences Inc., Warrington, Pa., 2001).

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Figures (11)

Fig. 1
Fig. 1

Experimental setup of the combined two-photon spectral and lifetime microscope. O1, 1.4 NA oil 60× microscope objective; O2, 0.4 NA 10× microscope objective; D1, D2, dichromatic mirrors; E, eyepiece; L1, 160-mm transfer lens; BP1, BP2, bandpass filters.

Fig. 2
Fig. 2

Schematic diagram of the TCSPC spectrometer. Fluorescence collected by the multimode fiber is delivered to the concave diffraction grating G mounted on an xy translation stage. The 16-channel detector is positioned at the focus of grating G where reflected wavelength components impinge on the device such that λ1 < λ2. Anode pulses are amplified, encoded, and output through a routing channel C1–C16, corresponding to the cathode that detected the fluorescence photon.

Fig. 3
Fig. 3

Calibration of the TCSPC spectrometer. The green (532-nm) laser line is positioned on routing channel 4 resulting in the red (632.8-nm) laser line impinging on channel 14. Linear interpolation reveals a spectral resolution of approximately 10 nm.

Fig. 4
Fig. 4

Second-harmonic spectra recorded for incident illumination wavelengths of (a) 900 nm and (b) 860 nm. The harmonic peaks are at 450 and 430 nm, respectively.

Fig. 5
Fig. 5

System response function of the combined spectral–lifetime microscope to a SHG signal from a BBO crystal measured at routing channel 8 of the multianode photon-counting PMT. The FWHM of the curve is approximately 295 ps. Inset shows the measured system response at FWHM as a function of anode number in the PMT array.

Fig. 6
Fig. 6

Single-channel control lifetime image of a 10-μm-diameter fluorescent polymer microsphere imaged at 920 nm showing an approximately uniform 2.28 ± 0.1 ns lifetime distribution across the field.

Fig. 7
Fig. 7

(a) Recorded emission spectrum and (b) fluorescence lifetimes from a sample comprising a mix of two distinct species of fluorescent polymer microspheres with overlapping emission spectra imaged at 920 nm. Two peaks are observable in (a) at approximately 460 and 570 nm. Lifetime analysis of these wavelengths in (b) reveals two characteristic lifetimes, which can be used to identify the origin of any given wavelength component of the spectrum.

Fig. 8
Fig. 8

Two-photon fluorescence intensity images of a transverse section though the medulla of a Cynomolgus monkey kidney acquired at the (a) 480-, (b) 510-, (c) 550-, and (d) 580-nm wavelength components of the emission spectrum. The sample was stained with the methyl green fluorochrome and imaged with an illumination wavelength of 920 nm.

Fig. 9
Fig. 9

Fluorescence lifetime images of a transverse section though the medulla of a Cynomolgus monkey kidney acquired at the (a) 480-, (b) 510-, (c) 550-, and (d) 580-nm wavelength components of the emission spectrum. The sample was stained with the methyl green fluorochrome and imaged with an illumination wavelength of 920 nm.

Fig. 10
Fig. 10

Multiexponential best-fit analysis of a representative pixel (74, 65) for the (a) 480-, (b) 510-, (c) 550-, and (d) 580-nm wavelength components of the emission spectrum.

Fig. 11
Fig. 11

Fluorescence lifetime analysis across the 450–600-nm spectral range for a representative pixel (74, 65) on each lifetime image. The dotted and dashed curves depict the variation in the short (τ1) and long (τ2) lifetime components as a function of wavelength, respectively. The ratio of amplitudes (a 1/a 2) as a function of wavelength (solid curve) reveals the relative contribution of these components in the multiexponential sum.

Equations (1)

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It=i=0n ai exp-t/τi+c.

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