Abstract

We designed a widefield frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM)setup, which is based on a Single Plane Illumination Microscope (SPIM). A SPIM provides an inherent optical sectioning capability and reduces photobleaching compared to conventional widefield and confocal fluorescence microscopes. The lifetime precision of the FLIM was characterized with Rhodamine 6G solutions of different quencher concentrations [KI]. We demonstrate the high spatial resolution of the SPIM-FLIM combination in the intensity domain as well as in the lifetime domain with latex bead samples and multiple recordings of three-dimensional live Madine-Darby Canine Kidney (MDCK) cysts. We estimate that the bleaching rate after 600 images have been recorded is below 5%.

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  1. A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
    [CrossRef]
  2. T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48(2), 221–239 (1993).
    [CrossRef]
  3. H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
    [CrossRef] [PubMed]
  4. S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
    [CrossRef]
  5. M. J. Booth and T. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocal microscopy,” J. Microsc. 214(1), 36–42 (2004).
    [CrossRef] [PubMed]
  6. R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
    [CrossRef] [PubMed]
  7. D. M. Grant, J. McGinty, E. J. McGhee, T. D. Bunney, D. M. Owen, C. B. Talbot, W. Zhang, S. Kumar, I. Munro, P. M. Lanigan, G. T. Kennedy, C. Dunsby, A. I. Magee, P. Courtney, M. Katan, M. A. Neil, and P. M. French, “High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events,” Opt. Express 15(24), 15656–15673 (2007).
    [CrossRef] [PubMed]
  8. A. Deniset-Besseau, S. Lévêque-Fort, M. P. Fontaine-Aupart, G. Roger, and P. Georges, “Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells,” Appl. Opt. 46(33), 8045–8051 (2007).
    [CrossRef] [PubMed]
  9. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
  10. K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
    [CrossRef] [PubMed]
  11. A. Elder, S. Schlachter, and C. F. Kaminski, “Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy,” J. Opt. Soc. Am. A 25(2), 452–462 (2008).
    [CrossRef] [PubMed]
  12. A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
    [CrossRef] [PubMed]
  13. Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
    [CrossRef] [PubMed]
  14. P. J. Keller, F. Pampaloni, and E. H. K. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
    [CrossRef] [PubMed]
  15. E. G. Reynaud, U. Krzic, K. Greger, and E. H. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J 2(5), 266–275 (2008).
    [CrossRef] [PubMed]

2008

A. Elder, S. Schlachter, and C. F. Kaminski, “Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy,” J. Opt. Soc. Am. A 25(2), 452–462 (2008).
[CrossRef] [PubMed]

E. G. Reynaud, U. Krzic, K. Greger, and E. H. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J 2(5), 266–275 (2008).
[CrossRef] [PubMed]

2007

2006

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

2005

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

2004

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

M. J. Booth and T. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocal microscopy,” J. Microsc. 214(1), 36–42 (2004).
[CrossRef] [PubMed]

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
[CrossRef] [PubMed]

2002

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

2001

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

1996

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

1993

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Apps, D. K.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

Bergmann, A.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
[CrossRef] [PubMed]

Booth, M. J.

M. J. Booth and T. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocal microscopy,” J. Microsc. 214(1), 36–42 (2004).
[CrossRef] [PubMed]

Bunney, T. D.

Clegg, R. M.

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Cole, M. J.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Courtney, P.

Cousin, M. A.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
[CrossRef] [PubMed]

Dai, X.

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Deniset-Besseau, A.

Dowling, K.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Duncan, R. R.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
[CrossRef] [PubMed]

Dunsby, C.

Elder, A.

Elder, A. D.

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Fontaine-Aupart, M. P.

Frank, J. H.

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

French, P. M.

French, P. M. W.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Gadella, T. W. J.

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Georges, P.

Gordon, G. W.

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Grant, D. M.

Greger, K.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J 2(5), 266–275 (2008).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

Gu, Y.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Hanley, Q. S.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

Herman, B.

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Jones, R.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Jovin, T. M.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Juškaitis, R.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Kaminski, C. F.

A. Elder, S. Schlachter, and C. F. Kaminski, “Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy,” J. Opt. Soc. Am. A 25(2), 452–462 (2008).
[CrossRef] [PubMed]

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Katan, M.

Keller, P. J.

P. J. Keller, F. Pampaloni, and E. H. K. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

Kennedy, G. T.

Krzic, U.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J 2(5), 266–275 (2008).
[CrossRef] [PubMed]

Kumar, S.

Kwon, S.

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Lanigan, P. M.

Lévêque-Fort, S.

A. Deniset-Besseau, S. Lévêque-Fort, M. P. Fontaine-Aupart, G. Roger, and P. Georges, “Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells,” Appl. Opt. 46(33), 8045–8051 (2007).
[CrossRef] [PubMed]

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Lever, M. J.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Magee, A. I.

McGhee, E. J.

McGinty, J.

Munro, I.

Neil, M. A.

Neil, M. A. A.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Owen, D. M.

Pampaloni, F.

P. J. Keller, F. Pampaloni, and E. H. K. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

Periasamy, A.

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Reynaud, E. G.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J 2(5), 266–275 (2008).
[CrossRef] [PubMed]

Roger, G.

Schlachter, S.

Shipston, M. J.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
[CrossRef] [PubMed]

Siegel, J.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Stelzer, E. H.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J 2(5), 266–275 (2008).
[CrossRef] [PubMed]

Stelzer, E. H. K.

P. J. Keller, F. Pampaloni, and E. H. K. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Subramaniam, V.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

Sucharov, L. O. D.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Swartling, J.

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Swoger, J.

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Talbot, C. B.

Wallrabe, H.

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

Wang, X. F.

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Webb, S. E. D.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Wilson, T.

M. J. Booth and T. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocal microscopy,” J. Microsc. 214(1), 36–42 (2004).
[CrossRef] [PubMed]

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Wodnicki, P.

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Zhang, W.

Appl. Opt.

Biophys. Chem.

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Curr. Opin. Biotechnol.

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

Cytometry

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

HFSP J

E. G. Reynaud, U. Krzic, K. Greger, and E. H. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J 2(5), 266–275 (2008).
[CrossRef] [PubMed]

J. Microsc.

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

M. J. Booth and T. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocal microscopy,” J. Microsc. 214(1), 36–42 (2004).
[CrossRef] [PubMed]

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Nat. Methods

P. J. Keller, F. Pampaloni, and E. H. K. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

Opt. Express

Rev. Sci. Instrum.

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

Science

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

In the SPIM-FLIM setup a laser diode illuminates the sample with a modulation frequency in the MHz range. The beam expander (BE) allows together with the cylindrical lens (f = 50mm) to form the light sheet. The detection lens dips directly into the medium of the sample chamber and no additional glass surfaces are between the sample and the detection lens. The fluorescence emission signal passes through the infinity corrected space (ICS), is spectrally filtered and enters the gated image intensifier. The final signal is integrated by the camera. The gain of the gated image intensifier and, therefore, the sensitivity of the detection system are modulated with the same frequency as the excitation signal. The sample is mounted on precision stages, which allow rotation and translation. A standard PC is used to control the hardware, acquire the images and calculate lifetime images for each plane.

Fig. 2
Fig. 2

The lifetimes for aqueous 1mM Rhodamine 6G solutions with seven different concentrations of the quencher potassium iodide [KI] are shown. The linear fit of the Stern-Volmer-Plot verifies the expected mono-exponential behavior of the dye solution.

Fig. 3
Fig. 3

Interpolation of the lifetime values for different sized beads in the lateral plane (left) and along the z-axis (right). For simplicity only the lifetime according to the phase shift is shown. The lifetimes of the different beads can be distinguished clearly along all three axes. An intensity threshold was applied to exclude the lifetime values corresponding to the background form the area outside the beads.

Fig. 4
Fig. 4

Lifetime images of a MDCK cyst expressing EGFP E-Cadherin (60 MHz, 6 phase shifts, binning 2, 100 planes with 500nm spacing). The lifetime images are an overlay of the intensity images (normalization per plane) with a color coded lifetime map. From top to bottom 251x251 pixels cutouts of the planes 11, 66 and 82 are shown.

Tables (2)

Tables Icon

Table 1 Lifetimes of aqueous Rhodamine 6G solutions as a function [KI].

Tables Icon

Table 2 Lifetimes of E-GFP fused to E-Cadherin in MDCK cysts

Equations (5)

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τm= 1 ω [ ( mref mem ) 2 ( 1+ ω 2 τre f 2 )1 ] 1/2
τφ= 1 ω tan[ φrefφem+ tan 1 ( ωτref ) ]
ωopt,m= 2 /τ
ωopt,φ=1/τ
1/τ=kq[Q]+1/τ0,

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