Abstract

We describe a novel whole-field fluorescence lifetime imaging system, based on a time-gated image intensifier and a solid-state laser oscillator–amplifier, that images lifetime differences of less than 10  ps. This system was successfully applied to discrimination between biological tissue constituents.

© 1998 Optical Society of America

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References

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  1. J. R. Lakowicz, ed., Techniques, Vol.??1 of Topics in Fluorescence Spectroscopy (Plenum, New York, 1991).
  2. H. Szmacinski, J. R. Lakowicz, and M. L. Johnson, Methods Enzymol. 240, 723 (1994).
    [CrossRef]
  3. R. Cubeddu, P. Taroni, and G. Valentini, Opt. Eng. 32, 320 (1993).
    [CrossRef]
  4. A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
    [CrossRef] [PubMed]
  5. K. Dowling, S. C. W. Hyde, J. C. Dainty, P. M. W. French, and J. D. Hares, Opt. Commun. 135, 27 (1997).
    [CrossRef]
  6. R. Mellish, N. P. Barry, S. C. W. Hyde, R. Jones, P. M. W. French, J. R. Taylor, C. J. Van der Poel, and A. Valster, Opt. Lett. 20, 2312 (1995).
    [CrossRef]
  7. The shorter fluorescence lifetime that we reported in Ref.??5 for Coumarin??314 was measured with a tightly focused pump beam that resulted in significant stimulated emission. For this measurement we reduced the pump intensity and eliminated this artifact. We are unaware of any published value for this lifetime.
  8. W. Sibbett and J. R. Taylor, J. Lumin. 28, 367 (1983).
    [CrossRef]
  9. B. B. Das, F. Liu, and R. R. Alfano, Rep. Prog. Phys. 60, 227 (1997).
    [CrossRef]
  10. S. Pongor, P. C. Ulrich, F. A. Bencsath, and A. Cerami, Proc. Nat. Acad. Sci. USA Sec. B 81, 2684 (1984).
    [CrossRef]

1997

K. Dowling, S. C. W. Hyde, J. C. Dainty, P. M. W. French, and J. D. Hares, Opt. Commun. 135, 27 (1997).
[CrossRef]

B. B. Das, F. Liu, and R. R. Alfano, Rep. Prog. Phys. 60, 227 (1997).
[CrossRef]

1996

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

1995

1994

H. Szmacinski, J. R. Lakowicz, and M. L. Johnson, Methods Enzymol. 240, 723 (1994).
[CrossRef]

1993

R. Cubeddu, P. Taroni, and G. Valentini, Opt. Eng. 32, 320 (1993).
[CrossRef]

1984

S. Pongor, P. C. Ulrich, F. A. Bencsath, and A. Cerami, Proc. Nat. Acad. Sci. USA Sec. B 81, 2684 (1984).
[CrossRef]

1983

W. Sibbett and J. R. Taylor, J. Lumin. 28, 367 (1983).
[CrossRef]

Alfano, R. R.

B. B. Das, F. Liu, and R. R. Alfano, Rep. Prog. Phys. 60, 227 (1997).
[CrossRef]

Barry, N. P.

Bencsath, F. A.

S. Pongor, P. C. Ulrich, F. A. Bencsath, and A. Cerami, Proc. Nat. Acad. Sci. USA Sec. B 81, 2684 (1984).
[CrossRef]

Botchway, S.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

Cerami, A.

S. Pongor, P. C. Ulrich, F. A. Bencsath, and A. Cerami, Proc. Nat. Acad. Sci. USA Sec. B 81, 2684 (1984).
[CrossRef]

Cubeddu, R.

R. Cubeddu, P. Taroni, and G. Valentini, Opt. Eng. 32, 320 (1993).
[CrossRef]

Dainty, J. C.

K. Dowling, S. C. W. Hyde, J. C. Dainty, P. M. W. French, and J. D. Hares, Opt. Commun. 135, 27 (1997).
[CrossRef]

Das, B. B.

B. B. Das, F. Liu, and R. R. Alfano, Rep. Prog. Phys. 60, 227 (1997).
[CrossRef]

Dowling, K.

K. Dowling, S. C. W. Hyde, J. C. Dainty, P. M. W. French, and J. D. Hares, Opt. Commun. 135, 27 (1997).
[CrossRef]

French, P. M. W.

Hares, J. D.

K. Dowling, S. C. W. Hyde, J. C. Dainty, P. M. W. French, and J. D. Hares, Opt. Commun. 135, 27 (1997).
[CrossRef]

Hyde, S. C. W.

Johnson, M. L.

H. Szmacinski, J. R. Lakowicz, and M. L. Johnson, Methods Enzymol. 240, 723 (1994).
[CrossRef]

Jones, R.

Lakowicz, J. R.

H. Szmacinski, J. R. Lakowicz, and M. L. Johnson, Methods Enzymol. 240, 723 (1994).
[CrossRef]

Liu, F.

B. B. Das, F. Liu, and R. R. Alfano, Rep. Prog. Phys. 60, 227 (1997).
[CrossRef]

MacRobert, A. J.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

Mellish, R.

O'Neill, P.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

Ostler, R. B.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

Parker, A. W.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

Phillips, D.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

Pongor, S.

S. Pongor, P. C. Ulrich, F. A. Bencsath, and A. Cerami, Proc. Nat. Acad. Sci. USA Sec. B 81, 2684 (1984).
[CrossRef]

Scully, A. D.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

Sibbett, W.

W. Sibbett and J. R. Taylor, J. Lumin. 28, 367 (1983).
[CrossRef]

Szmacinski, H.

H. Szmacinski, J. R. Lakowicz, and M. L. Johnson, Methods Enzymol. 240, 723 (1994).
[CrossRef]

Taroni, P.

R. Cubeddu, P. Taroni, and G. Valentini, Opt. Eng. 32, 320 (1993).
[CrossRef]

Taylor, J. R.

Ulrich, P. C.

S. Pongor, P. C. Ulrich, F. A. Bencsath, and A. Cerami, Proc. Nat. Acad. Sci. USA Sec. B 81, 2684 (1984).
[CrossRef]

Valentini, G.

R. Cubeddu, P. Taroni, and G. Valentini, Opt. Eng. 32, 320 (1993).
[CrossRef]

Valster, A.

Van der Poel, C. J.

J. Fluoresc.

A. D. Scully, A. J. MacRobert, S. Botchway, P. O'Neill, A. W. Parker, R. B. Ostler, and D. Phillips, J. Fluoresc. 6, 119 (1996).
[CrossRef] [PubMed]

J. Lumin.

W. Sibbett and J. R. Taylor, J. Lumin. 28, 367 (1983).
[CrossRef]

Methods Enzymol.

H. Szmacinski, J. R. Lakowicz, and M. L. Johnson, Methods Enzymol. 240, 723 (1994).
[CrossRef]

Opt. Commun.

K. Dowling, S. C. W. Hyde, J. C. Dainty, P. M. W. French, and J. D. Hares, Opt. Commun. 135, 27 (1997).
[CrossRef]

Opt. Eng.

R. Cubeddu, P. Taroni, and G. Valentini, Opt. Eng. 32, 320 (1993).
[CrossRef]

Opt. Lett.

Proc. Nat. Acad. Sci. USA Sec. B

S. Pongor, P. C. Ulrich, F. A. Bencsath, and A. Cerami, Proc. Nat. Acad. Sci. USA Sec. B 81, 2684 (1984).
[CrossRef]

Rep. Prog. Phys.

B. B. Das, F. Liu, and R. R. Alfano, Rep. Prog. Phys. 60, 227 (1997).
[CrossRef]

Other

The shorter fluorescence lifetime that we reported in Ref.??5 for Coumarin??314 was measured with a tightly focused pump beam that resulted in significant stimulated emission. For this measurement we reduced the pump intensity and eliminated this artifact. We are unaware of any published value for this lifetime.

J. R. Lakowicz, ed., Techniques, Vol.??1 of Topics in Fluorescence Spectroscopy (Plenum, New York, 1991).

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Figures (5)

Fig. 1
Fig. 1

Experimental setup.

Fig. 2
Fig. 2

Top, FLIM map of three samples of Coumarin 314 interlaced with samples of DASPI (note the nonlinear gray scale). Bottom, cross section through the FLIM map.

Fig. 3
Fig. 3

(a) Fluorescence lifetime map of samples of DASPI in solutions of, from left to right, 70:30, 66:34, 70:30, and 66:34 ethanol:glycerol. (b) Variation of spatially averaged lifetimes over time (the error bars represent the standard deviation of spatial variations across samples).

Fig. 4
Fig. 4

Fluorescence decay profiles of (a) collagen and (b) elastin. The top plots show the measured intensity values and the best-fitted single and double exponential decays. The lower plots show the residuals of the fits.

Fig. 5
Fig. 5

(a) Time-gated fluorescence image recorded immediately following excitation, along with FLIM maps of (b) the short-lived τ1 and (c) long-lived τ2 components of fluorescence decay. The samples are elastin (e1 and e2), aorta (a1 and a2), and collagen (c1).

Tables (1)

Tables Icon

Table 1 Observed Mean Lifetimes and Relative Amplitude |I1|/|I2| of Decay Components for the Samples Defined in Fig.  5

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