Abstract

Fluorescence emission after two-photon absorption of coumarins and xanthenes in an alcoholic solution was measured in the tuning range of a femtosecond-pulsed titanium–sapphire laser (750–840 nm). Xanthenes, which have a low one-photon absorption in the near UV, show a higher fluorescence signal after two-photon absorption than the UV-excitable coumarins. When fluxes of 1028 photons/(cm2 s) are used, the two-photon absorption cross sections for xanthenes are 1 order of magnitude higher than the two-photon absorption cross sections of the coumarins. Absolute cross sections have been estimated for three coumarins and three xanthenes. For the xanthenes a significant wavelength-dependent departure from the expected fluorescence intensity square law was observed. The coumarins follow the square-law dependence. The consequences of the findings are discussed for analytic and diagnostic methods. An especially important result is that the resolution in two-photon microscopy of xanthenes is worse than expected.

© 1995 Optical Society of America

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1994 (1)

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

1993 (1)

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

1990 (1)

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

1986 (1)

S. M. Kennedy, F. E. Lytle, “p-bis(o-methylstyryl) as a power squared sensor for two-photon absorption measurements between 537 and 694 nm,” Anal. Chem. 58, 2643–2647 (1986).
[CrossRef]

1985 (1)

S. Speiser, N. Shakkour, “Photoquenching parameters for commonly used laser dyes,” Appl. Phys. B 38, 191–197 (1985).
[CrossRef]

1978 (1)

W. Falkenstein, A. Penzkofer, W. Kaiser, “Amplified spontaneous emission in Rhodamine dyes: generation of picosecond light pulses and determination of excited state absorption and relaxation,” Opt. Commun. 27, 151–156 (1978).
[CrossRef]

1974 (1)

K. H. Drexhage, G. A. Reynolds, “New highly efficient laser dyes,” IEEE J. Quantum Electron. QE-10, 695–696 (1974).
[CrossRef]

1972 (4)

J. P. Hermann, J. Ducuing, “Absolute measurements of two-photon cross sections,” Phys. Rev. A 5, 2557–2568 (1972).
[CrossRef]

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, T. Morrow, G. H. C. New, M. S. Petty, “Interactions of picosecond laser pulses with organic molecules I: two-photon fluorescence quenching and singlet excited state excitation in Rhodamine dyes,” Proc. R. Soc. London Ser. A 328, 97–121 (1972).
[CrossRef]

J. P. Hermann, J. Ducuing, “Dispersion of the two-photon cross section in Rhodamine dyes,” Opt. Commun. 6, 101–105 (1972).
[CrossRef]

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, “Interactions of picosecond laser pulses with organic molecules II: two-photon absorption cross sections,” Proc. R. Soc. London Ser. A 329, 105–119 (1972).
[CrossRef]

1971 (1)

W. M. McClain, “Excited state assignment through polarized two-photon absorption studies of fluids,” J. Chem. Phys. 55, 2789–2796 (1971).
[CrossRef]

1970 (1)

P. R. Monson, W. M. McClain, “Polarization dependence of the two-photon absorption of tumbling molecules with application to liquid 1-chloronaphtalene and benzene,” J. Chem. Phys. 53, 29–37 (1970).
[CrossRef]

1969 (1)

M. D. Galanin, B. P. Kirsanov, Z. A. Chizhikova, “Luminescence quenching of complex molecules in a strong laser field,” Sov. Phys. JETP Lett. 9, 304–306 (1969).

1966 (2)

F. P. Schäfer, W. Schmidt, “3C3-geometrical model and experimental verification of two-photon absorption in organic dye solutions,” IEEE J. Quantum Electron. QE-2, 357–360 (1966).
[CrossRef]

M. D. Galanin, Z. A. Chizhikova, “Effective cross sections of two-photon absorption in organic molecules,” Sov. Phys. JETP Lett. 4, 27–28 (1966).

1963 (1)

W. L. Peticolas, K. E. Rieckhoff, “Double-photon excitation of organic molecules in dilute solution,” J. Chem. Phys. 39, 1347–1348 (1963).
[CrossRef]

1962 (1)

1961 (1)

W. Kaiser, C. G. B. Garret, “Two-photon excitation in CaF2:Eu2+,” Phys. Rev. Lett. 7, 229–231 (1961).
[CrossRef]

1937 (1)

M. Göppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. 9, 273–294 (1937).

Arden-Jacob, J.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Bachteler, G.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

Bashford, C. L.

R. K. Poole, C. L. Bashford, “Spectra,” in Spectrophotometry & Spectrofluorimetry: a Practical Approach, D. A. Harris, C. L. Bashford, ed. (IRL Press, Oxford, 1987), p. 32.

Brackmann, U.

U. Brackmann, Lambdachrome Laser Dyes (Lamda Physik, Göttingen, Germany, 1986).

Bradley, D. J.

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, T. Morrow, G. H. C. New, M. S. Petty, “Interactions of picosecond laser pulses with organic molecules I: two-photon fluorescence quenching and singlet excited state excitation in Rhodamine dyes,” Proc. R. Soc. London Ser. A 328, 97–121 (1972).
[CrossRef]

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, “Interactions of picosecond laser pulses with organic molecules II: two-photon absorption cross sections,” Proc. R. Soc. London Ser. A 329, 105–119 (1972).
[CrossRef]

Chizhikova, Z. A.

M. D. Galanin, B. P. Kirsanov, Z. A. Chizhikova, “Luminescence quenching of complex molecules in a strong laser field,” Sov. Phys. JETP Lett. 9, 304–306 (1969).

M. D. Galanin, Z. A. Chizhikova, “Effective cross sections of two-photon absorption in organic molecules,” Sov. Phys. JETP Lett. 4, 27–28 (1966).

Deltau, G.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

Denk, W.

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

Drexhage, K. H.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

K. H. Drexhage, G. A. Reynolds, “New highly efficient laser dyes,” IEEE J. Quantum Electron. QE-10, 695–696 (1974).
[CrossRef]

K. H. Drexhage, “Structure and properties of laser dyes,” in Dye Lasers, F. P. Schäfer, ed., Vol. 1 of Springer Series in Topics in Applied Physics (Springer-Verlag, Berlin, 1990), p. 179 ff.

K. H. Drexhage, “Structure and properties of laser dyes,” in Dye Lasers, F. P. Schäfer, ed., Vol. 1 of Springer Series in Topics in Applied Physics (Springer-Verlag, Berlin, 1990), p. 160 ff.

Drexhage, K.-H.

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Ducuing, J.

J. P. Hermann, J. Ducuing, “Absolute measurements of two-photon cross sections,” Phys. Rev. A 5, 2557–2568 (1972).
[CrossRef]

J. P. Hermann, J. Ducuing, “Dispersion of the two-photon cross section in Rhodamine dyes,” Opt. Commun. 6, 101–105 (1972).
[CrossRef]

Falkenstein, W.

W. Falkenstein, A. Penzkofer, W. Kaiser, “Amplified spontaneous emission in Rhodamine dyes: generation of picosecond light pulses and determination of excited state absorption and relaxation,” Opt. Commun. 27, 151–156 (1978).
[CrossRef]

Galanin, M. D.

M. D. Galanin, B. P. Kirsanov, Z. A. Chizhikova, “Luminescence quenching of complex molecules in a strong laser field,” Sov. Phys. JETP Lett. 9, 304–306 (1969).

M. D. Galanin, Z. A. Chizhikova, “Effective cross sections of two-photon absorption in organic molecules,” Sov. Phys. JETP Lett. 4, 27–28 (1966).

Galla, K.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Garret, C. G. B.

W. Kaiser, C. G. B. Garret, “Two-photon excitation in CaF2:Eu2+,” Phys. Rev. Lett. 7, 229–231 (1961).
[CrossRef]

Göppert-Mayer, M.

M. Göppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. 9, 273–294 (1937).

Han, K.-T.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Hell, S.

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Hermann, J. P.

J. P. Hermann, J. Ducuing, “Absolute measurements of two-photon cross sections,” Phys. Rev. A 5, 2557–2568 (1972).
[CrossRef]

J. P. Hermann, J. Ducuing, “Dispersion of the two-photon cross section in Rhodamine dyes,” Opt. Commun. 6, 101–105 (1972).
[CrossRef]

Hutchinson, M. H. R.

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, “Interactions of picosecond laser pulses with organic molecules II: two-photon absorption cross sections,” Proc. R. Soc. London Ser. A 329, 105–119 (1972).
[CrossRef]

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, T. Morrow, G. H. C. New, M. S. Petty, “Interactions of picosecond laser pulses with organic molecules I: two-photon fluorescence quenching and singlet excited state excitation in Rhodamine dyes,” Proc. R. Soc. London Ser. A 328, 97–121 (1972).
[CrossRef]

Kaiser, W.

W. Falkenstein, A. Penzkofer, W. Kaiser, “Amplified spontaneous emission in Rhodamine dyes: generation of picosecond light pulses and determination of excited state absorption and relaxation,” Opt. Commun. 27, 151–156 (1978).
[CrossRef]

W. Kaiser, C. G. B. Garret, “Two-photon excitation in CaF2:Eu2+,” Phys. Rev. Lett. 7, 229–231 (1961).
[CrossRef]

Kennedy, S. M.

S. M. Kennedy, F. E. Lytle, “p-bis(o-methylstyryl) as a power squared sensor for two-photon absorption measurements between 537 and 694 nm,” Anal. Chem. 58, 2643–2647 (1986).
[CrossRef]

Kirsanov, B. P.

M. D. Galanin, B. P. Kirsanov, Z. A. Chizhikova, “Luminescence quenching of complex molecules in a strong laser field,” Sov. Phys. JETP Lett. 9, 304–306 (1969).

Koetser, H.

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, T. Morrow, G. H. C. New, M. S. Petty, “Interactions of picosecond laser pulses with organic molecules I: two-photon fluorescence quenching and singlet excited state excitation in Rhodamine dyes,” Proc. R. Soc. London Ser. A 328, 97–121 (1972).
[CrossRef]

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, “Interactions of picosecond laser pulses with organic molecules II: two-photon absorption cross sections,” Proc. R. Soc. London Ser. A 329, 105–119 (1972).
[CrossRef]

Köllner, M.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Lakowicz, J. R.

J. R. Lakowicz. Principles of Fluorescence Spectroscopy (Plenum, New York, 1983), p. 37.

Lytle, F. E.

S. M. Kennedy, F. E. Lytle, “p-bis(o-methylstyryl) as a power squared sensor for two-photon absorption measurements between 537 and 694 nm,” Anal. Chem. 58, 2643–2647 (1986).
[CrossRef]

Martin, M.

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Marx, N.

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

McClain, W. M.

W. M. McClain, “Excited state assignment through polarized two-photon absorption studies of fluids,” J. Chem. Phys. 55, 2789–2796 (1971).
[CrossRef]

P. R. Monson, W. M. McClain, “Polarization dependence of the two-photon absorption of tumbling molecules with application to liquid 1-chloronaphtalene and benzene,” J. Chem. Phys. 53, 29–37 (1970).
[CrossRef]

Meluish, W. H.

Monson, P. R.

P. R. Monson, W. M. McClain, “Polarization dependence of the two-photon absorption of tumbling molecules with application to liquid 1-chloronaphtalene and benzene,” J. Chem. Phys. 53, 29–37 (1970).
[CrossRef]

Morrow, T.

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, T. Morrow, G. H. C. New, M. S. Petty, “Interactions of picosecond laser pulses with organic molecules I: two-photon fluorescence quenching and singlet excited state excitation in Rhodamine dyes,” Proc. R. Soc. London Ser. A 328, 97–121 (1972).
[CrossRef]

Müller, R.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

New, G. H. C.

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, T. Morrow, G. H. C. New, M. S. Petty, “Interactions of picosecond laser pulses with organic molecules I: two-photon fluorescence quenching and singlet excited state excitation in Rhodamine dyes,” Proc. R. Soc. London Ser. A 328, 97–121 (1972).
[CrossRef]

Penzkofer, A.

W. Falkenstein, A. Penzkofer, W. Kaiser, “Amplified spontaneous emission in Rhodamine dyes: generation of picosecond light pulses and determination of excited state absorption and relaxation,” Opt. Commun. 27, 151–156 (1978).
[CrossRef]

Peticolas, W. L.

W. L. Peticolas, K. E. Rieckhoff, “Double-photon excitation of organic molecules in dilute solution,” J. Chem. Phys. 39, 1347–1348 (1963).
[CrossRef]

Petty, M. S.

D. J. Bradley, M. H. R. Hutchinson, H. Koetser, T. Morrow, G. H. C. New, M. S. Petty, “Interactions of picosecond laser pulses with organic molecules I: two-photon fluorescence quenching and singlet excited state excitation in Rhodamine dyes,” Proc. R. Soc. London Ser. A 328, 97–121 (1972).
[CrossRef]

Pick, R.

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Poole, R. K.

R. K. Poole, C. L. Bashford, “Spectra,” in Spectrophotometry & Spectrofluorimetry: a Practical Approach, D. A. Harris, C. L. Bashford, ed. (IRL Press, Oxford, 1987), p. 32.

Reynolds, G. A.

K. H. Drexhage, G. A. Reynolds, “New highly efficient laser dyes,” IEEE J. Quantum Electron. QE-10, 695–696 (1974).
[CrossRef]

Rieckhoff, K. E.

W. L. Peticolas, K. E. Rieckhoff, “Double-photon excitation of organic molecules in dilute solution,” J. Chem. Phys. 39, 1347–1348 (1963).
[CrossRef]

Ritter, G.

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Rumphorst, A.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

Salmon, N.

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Sauer, M.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Schäfer, F. P.

F. P. Schäfer, W. Schmidt, “3C3-geometrical model and experimental verification of two-photon absorption in organic dye solutions,” IEEE J. Quantum Electron. QE-2, 357–360 (1966).
[CrossRef]

F. P. Schäfer, “Principles of dye laser operation,” in Dye Lasers, F. P. Schäfer, ed., Vol. 1 of Springer Series in Topics in Applied Physics (Springer-Verlag, Berlin, 1990), p. 27 ff.

Schmidt, W.

F. P. Schäfer, W. Schmidt, “3C3-geometrical model and experimental verification of two-photon absorption in organic dye solutions,” IEEE J. Quantum Electron. QE-2, 357–360 (1966).
[CrossRef]

Schulz, A.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

Seeger, S.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Shakkour, N.

S. Speiser, N. Shakkour, “Photoquenching parameters for commonly used laser dyes,” Appl. Phys. B 38, 191–197 (1985).
[CrossRef]

Speiser, S.

S. Speiser, N. Shakkour, “Photoquenching parameters for commonly used laser dyes,” Appl. Phys. B 38, 191–197 (1985).
[CrossRef]

Stelzer, E. H. K.

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Storz, C.

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Stricker, R.

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[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]

Webb, W. W.

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

Wolfram, S.

S. Wolfram, Mathematica, 2nd ed. (Addison-Wesley, New York, 1991).

Wolfrum, J.

S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

Anal. Chem. (1)

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[CrossRef]

Ann. Phys. (1)

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[CrossRef]

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S. Seeger, G. Bachteler, K. H. Drexhage, G. Deltau, J. Arden-Jacob, K. Galla, K.-T. Han, M. Köllner, R. Müller, A. Rumphorst, M. Sauer, A. Schulz, J. Wolfrum, “Biodiagnostics and polymer identification with multiplex dyes,” Ber. Bunsenges. Phys. Chem. 97, 1542–1546 (1993).
[CrossRef]

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[CrossRef]

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[CrossRef]

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[CrossRef]

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[CrossRef]

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[CrossRef]

E. H. K. Stelzer, S. Hell, R. Stricker, R. Pick, C. Storz, G. Ritter, N. Salmon, “Nonlinear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

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[CrossRef]

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K. H. Drexhage, “Structure and properties of laser dyes,” in Dye Lasers, F. P. Schäfer, ed., Vol. 1 of Springer Series in Topics in Applied Physics (Springer-Verlag, Berlin, 1990), p. 179 ff.

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Photomultiplier Tubes (Hamamatsu Photonics, Herrsching, Germany, 1990).

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S. Wolfram, Mathematica, 2nd ed. (Addison-Wesley, New York, 1991).

S. Seeger, J. Arden-Jacob, N. Marx, K.-H. Drexhage, K. Galla, M. Martin, K.-T. Han, M. Köllner, R. Müller, M. Sauer, J. Wolfrum, “Biodiagnostics with multiplex dyes,” in Biochemical Diagnostic Instrumentation, R. F. Bonner, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2136, 75–86 (1994).

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Figures (10)

Fig. 1
Fig. 1

Experimental arrangement for the measurement of fluorescence spectra after one- and two-photon absorption. The same sample can be excited with either the light from an argon–ion laser (476, 488, or 514 nm) or the light from a titanium–sapphire laser (750–850 nm). The spectra are recorded under computer control and are available as a digital data set. HV, high voltage.

Fig. 2
Fig. 2

Jablonski diagram for two-photon absorption of near-infrared light (750–850 nm) in xanthenes, whose first singlet state S1 absorbs around 500 nm.

Fig. 3
Fig. 3

Normalized fluorescence signals after two-photon absorption of the coumarins versus the peak flux of the titanium–sapphire laser for the excitation wavelength 770 nm. A power function was fitted to the experimental values. The measurements agree with a slope of 2.

Fig. 4
Fig. 4

Normalized fluorescence signals after two-photon absorption of the xanthenes versus the peak flux of the titanium–sapphire laser for the two different excitation wavelengths, 770 and 825 nm. The xanthenes show a departure from the usually expected intensity square law. The sum N1(t) + N2(t) of the model (13) was fitted with σ2n as a parameter and the constant values τ21 = 1 ps for the relaxation time and τ = 100 fs for the pulse duration. The resulting values for σ2n are presented in Table 4. For the excitation wavelength of 825 nm, the fit with the simple model (13) shows a significant deviation at lower light fluxes.

Fig. 5
Fig. 5

Fluorescence after two-photon absorption of the 10−5-M solutions in ethanol: C, coumarin; F, fluorescein; R, Rhodamine for different excitation wavelengths. The fluorescence spectra are corrected for self-absorption and sensitivity of the PMT. The spectra are normalized by the square of the cw power of the titanium–sapphire laser.

Fig. 6
Fig. 6

Comparison between fluorescence after two-photon absorption of the 10−5-M solutions in ethanol and the fluorescence after one-photon absorption of the 10−5-M solution of Rhodamine 6G in ethanol excited with the 514.5-nm line of the argon–ion laser used for calibration. The spectra are corrected for self-absorption and sensitivity of the PMT. Identical scales are used for both plots. The excitation wavelength at two-photon absorption was 784 nm at a cw power of 0.8 W and a pulse duration of 100 fs. The excitation wavelength for one-photon absorption was the 514.5-nm line of the argon–ion laser at a cw power of 3.4 × 10−6 W. The spectra were recorded with the same optical setup described in Fig. 1. C, coumarin; F, fluorescein; R, Rhodamine.

Fig. 7
Fig. 7

Fluorescence after one-photon absorption of the 10−5-M solutions in ethanol. C, coumarin; F, fluorescein; R, Rhodamine for different excitation wavelengths recorded with the fluorimeter. These spectra cannot be compared quantitatively with the results shown in Fig. 5. The fluorescence spectra are corrected for self-absorption and sensitivity of the PMT.

Fig. 8
Fig. 8

One-photon absorption cross sections calculated with the literature values of ɛ in Table 1 and the measured optical densities of the 10−5-M solutions in ethanol with σ = 3.824 × 10−21 OD(λ)/c, with c in moles/liter and σ in squared centimeters. Note that different scales are used for coumarins and xanthenes. The precision of the spectrophotometer was 4 mAbs. Because the 10−5-M solutions had an optical density of OD ≈ 1, the error in the determination of the one-photon absorption cross section was approximately 1.5 × 10−18 cm2.

Fig. 9
Fig. 9

Quenching factors for the fluorescence emission after two-photon absorption Δ calculated according to Eq. (15) for three measured excited-state absorption coefficients σ2n. The constant values τ21 = 1 ps for the relaxation time and τ = 100 fs for the pulse duration were assumed. The three curves belong to the following excited-state absorption coefficients: 1, σ2n = 2 × 10−16 cm2; 2, σ2n = 5 × 10−16 cm2; 3, σ2n = 8 × 10−16 cm2. For measurements of the two-photon absorption coefficient (Fig. 10, below) fluxes of (2.7 ± 1) × 1028 photons/cm2 s were used. The quenching factors of the fluorescence after two-photon absorption for the xanthenes are therefore around 1.8.

Fig. 10
Fig. 10

Estimated values of the two-photon absorption coefficient δ with Eq. (16) and the fluorescence spectra presented in Figs. 5 and 6 measured in cm4 s (see also Table 4). Note that different scales are used for coumarins and xanthenes. The errors in determining δ for the xanthenes are higher because the fluorescence signals of the xanthenes after two-photon absorption were quenched. δ was calculated with Eq. (16). For all xanthenes we assumed a quenching factor of 1.8 (see also Table 6). ○, experimental values; ×, probably not correct.

Tables (6)

Tables Icon

Table 1 Relative Fluorescence Signals after Two-Photon Absorption of the 10−3-M Solutions in Methanola

Tables Icon

Table 2 Quantum Efficiencies (QE’s) Relative to Rhodamine 6G for Excitation Wavelengths between 375 and 425 nma

Tables Icon

Table 3 Ratio of the Quantum Efficiencies for Excitation Wavelengths 375–425 nm and 514.5 nm Compared with Values Found by Other Authors

Tables Icon

Table 4 Estimation of the Two-Photon Absorption Cross Section for the Solutions in Ethanol Excited with the Titanium–Sapphire Lasera

Tables Icon

Table 5 Values for the Two-Photon Absorption Cross Section Taken from the Literaturea

Tables Icon

Table 6 Estimated Excited-State Absorption Coefficients σ2nof the Xanthenes for Excitation Wavelengths 770 and 825 nma

Equations (16)

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F l = λ k 1 ( λ ) S ( λ ) 10 OD ( λ ) / 2 d λ .
F l 1 = K Φ 1 n 1 σ l F 1 ,
F l 2 = K Φ 2 2 n 2 δ l F 2 2 ,
δ = 2 F l 2 Φ 1 n 1 σ F 1 F l 1 Φ 2 n 2 F 2 2 .
F l 2 = K Φ 2 2 n 2 δ l w + F 2 ( t ) d t = K Φ 2 2 n 2 δ l w + [ F max sech 2 ( 1 . 76 t τ ) ] 2 d t = 4 3 K Φ 2 2 n 2 δ l w τ 1 . 76 F max 2 ,
Δ x = 1 . 22 λ NA = 2 . 44 λ f d .
F = I λ h c = P λ π ( Δ x ) 2 h c ,
F cw = F max w + sech 2 ( 1 . 76 t τ ) d t F max = F cw 0 . 88 w τ ,
δ = 20 . 3 h c π Φ 1 F l 2 n 2 P cw , 1 d 1 2 λ 2 2 Φ 2 F l 2 n 2 P cw , 2 2 d 2 4 λ 1 f 2 w τσ .
F l i = F l i ( λ ) d λ = K Φ i ( λ i ) F 0 ( λ 1 ) exp [ n σ i ( λ 1 ) x ] n i σ i ( λ 1 ) l Φ i ( λ 1 ) = 1 K F l i exp [ n σ i ( λ 1 ) x ] F 0 ( λ 1 ) n i σ i ( λ 1 ) l .
Φ relative ( λ 1 ) = Φ a ( λ 1 ) Φ b ( λ 1 ) = F l a exp [ n a σ a ( λ 1 ) x ] n b σ b ( λ 1 ) F l b exp [ n b σ b ( λ 1 ) x ] n a σ a ( λ 1 ) .
d N 2 d t = N 0 δ 02 F 2 N 2 σ 2 n F N 2 τ 21 , d N 1 d t = N 2 τ 21 .
N 1 ( t ) = N 0 δ 02 F 2 { t τ 21 + τ 21 exp [ t ( F σ 2 n 1 / τ 21 ) ] + F σ 2 n τ 21 t } ( 1 + F σ 2 n τ 21 ) 2 , N 2 ( t ) = N 0 δ 02 F 2 τ 21 { 1 exp [ t ( F σ 2 n 1 / τ 21 ) ] } 1 + F σ 2 n τ 21 .
N ( t ) = N 0 δ 02 F 2 t .
Δ ( λ ) = N ( t ) N 1 ( t ) + N 2 ( t ) .
δ = 20 . 3 h c π Φ 1 F l 2 n 1 P cw , 1 d 1 2 λ 2 2 Φ 2 F l 1 n 2 P cw , 2 2 d 2 4 λ 1 f 2 w τσ Δ ( λ 2 ) .

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