Abstract

We demonstrate sensitive spatially resolved detection of physiological chromophores that emit in the ultraviolet (<330 nm). An atypical laser source (a visible wavelength femtosecond optical parametric oscillator), and an unconventional collection geometry (a lensless detector that detects the forward-emitted fluorescence) enable this detection. We report the excitation spectra of the catecholamines dopamine and norepinephrine, together with near-UV emitters serotonin and tryptophan, in the range of 550–595 nm. We estimate the molecular two-photon action cross section of dopamine, norepinephrine, and serotonin to be 1.2 mGM (1 GM, or Goppert Mayor, is equal to 10-58 m4 s-1 photon-1), 2 mGM, and 43 mGM, respectively, at 560 nm. The sensitivity achieved by this method holds promise for the microscopic imaging of vesicular catecholamines in live cells.

© 2004 Optical Society of America

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  1. M. Goeppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. (Leipzig) 9, 273–295 (1931).
  2. S. Maiti, J. B. Shear, W. W. Webb, “Multiphoton excitation of amino acids and neurotransmitters,” Biophys. J. 70, A-210 (1996).
  3. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73 (1990).
    [CrossRef] [PubMed]
  4. R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
    [PubMed]
  5. S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
    [CrossRef] [PubMed]
  6. R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
    [CrossRef] [PubMed]
  7. J. Balaji, S. K. Kaushalya, R. Desai, S. Maiti, “Single cell pharmacology: Quantitative imaging of serotonin in live neuronal cells,” Biophys. J. 84, 582a (2003).
  8. J. R. Cooper, F. E. Bloom, R. H. Roth, The Biochemical Basis of Neuropharmacology (Oxford U. Press, New York, 1996).
  9. E. R. Kandel, J. H. Schwartz, Principles of Neural Science (Elsevier, New York, 1983).
  10. J. W. M. L. Gostkowski, E. Okerberg, J. B. Shear, “Attomole electrophoretic analysis of catecholamines using copper-catalyzed intramolecular cyclization,” Anal. Biochem. 303, 199–202 (2002).
    [CrossRef] [PubMed]
  11. K. Valtteri, O. R. Juha, “Functional imaging studies of dopamine system and cognition in normal aging and Parkinson’s disease,” Neurosci. Biobehav. Rev. 26, 785–793 (2002).
    [CrossRef]
  12. M. Michiaki, S. Jun-ichi, O. Koichi, K. Yoichi, F. Shigeo, M. Kazumi, “Time-resolved fluorescence spectroscopy of dopamine in single cells,” in Advances in Fluorescence Sensing Technology, J. A. Lakowicz, R. B. Thompson, eds., Proc. SPIE4252, 140–148 (2001).
    [CrossRef]
  13. C. Xu, W. W. Webb, “Measurement of two photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
    [CrossRef]
  14. P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
    [CrossRef]
  15. J. Balaji, P. Sengupta, S. Maiti, “Probing diffusion and photochemical properties through localized photobleaching,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 329–335 (2001).
    [CrossRef]
  16. E. Okerberg, J. B. Shear, “Neuropeptide analysis using capillary electrophoresis with multiphoton-excited intrinsic fluorescence detection,” Anal. Biochem. 292, 311–313 (2001).
    [CrossRef] [PubMed]
  17. P. Sengupta, J. Balaji, S. Mukherjee, R. Philip, G. R. Kumar, S. Maiti, “Determination of the absolute two-photon absorption cross section of tryptophan,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 336–339 (2001).
    [CrossRef]
  18. A. A. Rehms, P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208, 276–282 (1993).
    [CrossRef]
  19. B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
    [CrossRef] [PubMed]
  20. D. Bruns, R. Jahn, “Real-time measurement of transmitter release from single synaptic vesicles,” Nature 377, 62–65 (1995).
    [CrossRef] [PubMed]

2003

J. Balaji, S. K. Kaushalya, R. Desai, S. Maiti, “Single cell pharmacology: Quantitative imaging of serotonin in live neuronal cells,” Biophys. J. 84, 582a (2003).

2002

J. W. M. L. Gostkowski, E. Okerberg, J. B. Shear, “Attomole electrophoretic analysis of catecholamines using copper-catalyzed intramolecular cyclization,” Anal. Biochem. 303, 199–202 (2002).
[CrossRef] [PubMed]

K. Valtteri, O. R. Juha, “Functional imaging studies of dopamine system and cognition in normal aging and Parkinson’s disease,” Neurosci. Biobehav. Rev. 26, 785–793 (2002).
[CrossRef]

2001

E. Okerberg, J. B. Shear, “Neuropeptide analysis using capillary electrophoresis with multiphoton-excited intrinsic fluorescence detection,” Anal. Biochem. 292, 311–313 (2001).
[CrossRef] [PubMed]

2000

P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
[CrossRef]

1999

R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
[CrossRef] [PubMed]

1997

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

1996

C. Xu, W. W. Webb, “Measurement of two photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
[CrossRef]

S. Maiti, J. B. Shear, W. W. Webb, “Multiphoton excitation of amino acids and neurotransmitters,” Biophys. J. 70, A-210 (1996).

1995

B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
[CrossRef] [PubMed]

D. Bruns, R. Jahn, “Real-time measurement of transmitter release from single synaptic vesicles,” Nature 377, 62–65 (1995).
[CrossRef] [PubMed]

1994

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

1993

A. A. Rehms, P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208, 276–282 (1993).
[CrossRef]

1990

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

1931

M. Goeppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. (Leipzig) 9, 273–295 (1931).

Balaji, J.

J. Balaji, S. K. Kaushalya, R. Desai, S. Maiti, “Single cell pharmacology: Quantitative imaging of serotonin in live neuronal cells,” Biophys. J. 84, 582a (2003).

P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
[CrossRef]

J. Balaji, P. Sengupta, S. Maiti, “Probing diffusion and photochemical properties through localized photobleaching,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 329–335 (2001).
[CrossRef]

P. Sengupta, J. Balaji, S. Mukherjee, R. Philip, G. R. Kumar, S. Maiti, “Determination of the absolute two-photon absorption cross section of tryptophan,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 336–339 (2001).
[CrossRef]

Banerjee, S.

P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
[CrossRef]

Bloom, F. E.

J. R. Cooper, F. E. Bloom, R. H. Roth, The Biochemical Basis of Neuropharmacology (Oxford U. Press, New York, 1996).

Bruns, D.

D. Bruns, R. Jahn, “Real-time measurement of transmitter release from single synaptic vesicles,” Nature 377, 62–65 (1995).
[CrossRef] [PubMed]

Bzowska, A.

B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
[CrossRef] [PubMed]

Callis, P. R.

A. A. Rehms, P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208, 276–282 (1993).
[CrossRef]

Cooper, J. R.

J. R. Cooper, F. E. Bloom, R. H. Roth, The Biochemical Basis of Neuropharmacology (Oxford U. Press, New York, 1996).

Denk, W.

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

Desai, R.

J. Balaji, S. K. Kaushalya, R. Desai, S. Maiti, “Single cell pharmacology: Quantitative imaging of serotonin in live neuronal cells,” Biophys. J. 84, 582a (2003).

Goeppert-Mayer, M.

M. Goeppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. (Leipzig) 9, 273–295 (1931).

Gostkowski, J. W. M. L.

J. W. M. L. Gostkowski, E. Okerberg, J. B. Shear, “Attomole electrophoretic analysis of catecholamines using copper-catalyzed intramolecular cyclization,” Anal. Biochem. 303, 199–202 (2002).
[CrossRef] [PubMed]

Gryczynski, I.

B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
[CrossRef] [PubMed]

Jahn, R.

D. Bruns, R. Jahn, “Real-time measurement of transmitter release from single synaptic vesicles,” Nature 377, 62–65 (1995).
[CrossRef] [PubMed]

Juha, O. R.

K. Valtteri, O. R. Juha, “Functional imaging studies of dopamine system and cognition in normal aging and Parkinson’s disease,” Neurosci. Biobehav. Rev. 26, 785–793 (2002).
[CrossRef]

Jun-ichi, S.

M. Michiaki, S. Jun-ichi, O. Koichi, K. Yoichi, F. Shigeo, M. Kazumi, “Time-resolved fluorescence spectroscopy of dopamine in single cells,” in Advances in Fluorescence Sensing Technology, J. A. Lakowicz, R. B. Thompson, eds., Proc. SPIE4252, 140–148 (2001).
[CrossRef]

Kandel, E. R.

E. R. Kandel, J. H. Schwartz, Principles of Neural Science (Elsevier, New York, 1983).

Kaushalya, S. K.

J. Balaji, S. K. Kaushalya, R. Desai, S. Maiti, “Single cell pharmacology: Quantitative imaging of serotonin in live neuronal cells,” Biophys. J. 84, 582a (2003).

Kazumi, M.

M. Michiaki, S. Jun-ichi, O. Koichi, K. Yoichi, F. Shigeo, M. Kazumi, “Time-resolved fluorescence spectroscopy of dopamine in single cells,” in Advances in Fluorescence Sensing Technology, J. A. Lakowicz, R. B. Thompson, eds., Proc. SPIE4252, 140–148 (2001).
[CrossRef]

Kierdaszuk, B.

B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
[CrossRef] [PubMed]

Koichi, O.

M. Michiaki, S. Jun-ichi, O. Koichi, K. Yoichi, F. Shigeo, M. Kazumi, “Time-resolved fluorescence spectroscopy of dopamine in single cells,” in Advances in Fluorescence Sensing Technology, J. A. Lakowicz, R. B. Thompson, eds., Proc. SPIE4252, 140–148 (2001).
[CrossRef]

Kumar, G. R.

P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
[CrossRef]

P. Sengupta, J. Balaji, S. Mukherjee, R. Philip, G. R. Kumar, S. Maiti, “Determination of the absolute two-photon absorption cross section of tryptophan,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 336–339 (2001).
[CrossRef]

Lakowicz, J. R.

B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
[CrossRef] [PubMed]

Maiti, S.

J. Balaji, S. K. Kaushalya, R. Desai, S. Maiti, “Single cell pharmacology: Quantitative imaging of serotonin in live neuronal cells,” Biophys. J. 84, 582a (2003).

P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
[CrossRef]

R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, W. W. Webb, “Multiphoton excitation of amino acids and neurotransmitters,” Biophys. J. 70, A-210 (1996).

P. Sengupta, J. Balaji, S. Mukherjee, R. Philip, G. R. Kumar, S. Maiti, “Determination of the absolute two-photon absorption cross section of tryptophan,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 336–339 (2001).
[CrossRef]

J. Balaji, P. Sengupta, S. Maiti, “Probing diffusion and photochemical properties through localized photobleaching,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 329–335 (2001).
[CrossRef]

Michiaki, M.

M. Michiaki, S. Jun-ichi, O. Koichi, K. Yoichi, F. Shigeo, M. Kazumi, “Time-resolved fluorescence spectroscopy of dopamine in single cells,” in Advances in Fluorescence Sensing Technology, J. A. Lakowicz, R. B. Thompson, eds., Proc. SPIE4252, 140–148 (2001).
[CrossRef]

Modrak-Wojcik, A.

B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
[CrossRef] [PubMed]

Mukherjee, S.

P. Sengupta, J. Balaji, S. Mukherjee, R. Philip, G. R. Kumar, S. Maiti, “Determination of the absolute two-photon absorption cross section of tryptophan,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 336–339 (2001).
[CrossRef]

Okerberg, E.

J. W. M. L. Gostkowski, E. Okerberg, J. B. Shear, “Attomole electrophoretic analysis of catecholamines using copper-catalyzed intramolecular cyclization,” Anal. Biochem. 303, 199–202 (2002).
[CrossRef] [PubMed]

E. Okerberg, J. B. Shear, “Neuropeptide analysis using capillary electrophoresis with multiphoton-excited intrinsic fluorescence detection,” Anal. Biochem. 292, 311–313 (2001).
[CrossRef] [PubMed]

Philip, R.

P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
[CrossRef]

P. Sengupta, J. Balaji, S. Mukherjee, R. Philip, G. R. Kumar, S. Maiti, “Determination of the absolute two-photon absorption cross section of tryptophan,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 336–339 (2001).
[CrossRef]

Piston, D. W.

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

Rehms, A. A.

A. A. Rehms, P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208, 276–282 (1993).
[CrossRef]

Roth, R. H.

J. R. Cooper, F. E. Bloom, R. H. Roth, The Biochemical Basis of Neuropharmacology (Oxford U. Press, New York, 1996).

Schwartz, J. H.

E. R. Kandel, J. H. Schwartz, Principles of Neural Science (Elsevier, New York, 1983).

Sengupta, P.

P. Sengupta, J. Balaji, S. Banerjee, R. Philip, G. R. Kumar, S. Maiti, “Sensitive measurement of absolute two-photon absorption cross sections,” J. Chem. Phys. 112, 9201–9205 (2000).
[CrossRef]

J. Balaji, P. Sengupta, S. Maiti, “Probing diffusion and photochemical properties through localized photobleaching,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 329–335 (2001).
[CrossRef]

P. Sengupta, J. Balaji, S. Mukherjee, R. Philip, G. R. Kumar, S. Maiti, “Determination of the absolute two-photon absorption cross section of tryptophan,” in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamu, P. T. So, eds., Proc. SPIE4262, 336–339 (2001).
[CrossRef]

Shear, J. B.

J. W. M. L. Gostkowski, E. Okerberg, J. B. Shear, “Attomole electrophoretic analysis of catecholamines using copper-catalyzed intramolecular cyclization,” Anal. Biochem. 303, 199–202 (2002).
[CrossRef] [PubMed]

E. Okerberg, J. B. Shear, “Neuropeptide analysis using capillary electrophoresis with multiphoton-excited intrinsic fluorescence detection,” Anal. Biochem. 292, 311–313 (2001).
[CrossRef] [PubMed]

R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, W. W. Webb, “Multiphoton excitation of amino acids and neurotransmitters,” Biophys. J. 70, A-210 (1996).

Shigeo, F.

M. Michiaki, S. Jun-ichi, O. Koichi, K. Yoichi, F. Shigeo, M. Kazumi, “Time-resolved fluorescence spectroscopy of dopamine in single cells,” in Advances in Fluorescence Sensing Technology, J. A. Lakowicz, R. B. Thompson, eds., Proc. SPIE4252, 140–148 (2001).
[CrossRef]

Shugar, D.

B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, J. R. Lakowicz, “Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation,” Photochem. Photobiol. 61, 319–324 (1995).
[CrossRef] [PubMed]

Strickler, J. H.

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

Valtteri, K.

K. Valtteri, O. R. Juha, “Functional imaging studies of dopamine system and cognition in normal aging and Parkinson’s disease,” Neurosci. Biobehav. Rev. 26, 785–793 (2002).
[CrossRef]

Webb, W. W.

R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, W. W. Webb, “Multiphoton excitation of amino acids and neurotransmitters,” Biophys. J. 70, A-210 (1996).

C. Xu, W. W. Webb, “Measurement of two photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
[CrossRef]

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

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

Williams, R. M.

R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

Xu, C.

Yoichi, K.

M. Michiaki, S. Jun-ichi, O. Koichi, K. Yoichi, F. Shigeo, M. Kazumi, “Time-resolved fluorescence spectroscopy of dopamine in single cells,” in Advances in Fluorescence Sensing Technology, J. A. Lakowicz, R. B. Thompson, eds., Proc. SPIE4252, 140–148 (2001).
[CrossRef]

Zipfel, W. R.

R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

Anal. Biochem.

J. W. M. L. Gostkowski, E. Okerberg, J. B. Shear, “Attomole electrophoretic analysis of catecholamines using copper-catalyzed intramolecular cyclization,” Anal. Biochem. 303, 199–202 (2002).
[CrossRef] [PubMed]

E. Okerberg, J. B. Shear, “Neuropeptide analysis using capillary electrophoresis with multiphoton-excited intrinsic fluorescence detection,” Anal. Biochem. 292, 311–313 (2001).
[CrossRef] [PubMed]

Ann. Phys. (Leipzig)

M. Goeppert-Mayer, “Über Elementarakte mit zwei Quantensprüngen,” Ann. Phys. (Leipzig) 9, 273–295 (1931).

Biophys. J.

S. Maiti, J. B. Shear, W. W. Webb, “Multiphoton excitation of amino acids and neurotransmitters,” Biophys. J. 70, A-210 (1996).

R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb, “Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence,” Biophys. J. 76, 1835–1846 (1999).
[CrossRef] [PubMed]

J. Balaji, S. K. Kaushalya, R. Desai, S. Maiti, “Single cell pharmacology: Quantitative imaging of serotonin in live neuronal cells,” Biophys. J. 84, 582a (2003).

Chem. Phys. Lett.

A. A. Rehms, P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208, 276–282 (1993).
[CrossRef]

FASEB J.

R. M. Williams, D. W. Piston, W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8, 804–813 (1994).
[PubMed]

J. Chem. Phys.

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Figures (3)

Fig. 1
Fig. 1

Schematic diagram of the experimental setup. Visible femtosecond pulses generated from the OPO induce fluorescence through two-photon excitation of specimens placed in a microscopic setup. Fluorescence is detected by a PMT placed in a straight geometry, i.e., facing the transmitted beam. Excitation light path is marked by arrows on bold lines; emission light is marked by arrows on thin dotted lines.

Fig. 2
Fig. 2

Fluorescence versus excitation power. Fluorescence count rates of tryptophan (circles), serotonin (inverted triangles) dopamine (triangles), and norepinephrine (diamonds) are plotted against average excitation power in a log-log plot. Dashed lines are straight-line fits to the lowest ten power points.

Fig. 3
Fig. 3

Two-photon action spectra of tryptophan (circles), serotonin (inverted triangles) dopamine (triangles), and norepinephrine (diamonds).

Tables (1)

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Table 1 Comparison of Collection Efficiencies between Different Collection Geometries and between Different Chromophores

Equations (3)

Equations on this page are rendered with MathJax. Learn more.

F=γηϕσ2CPave2,
log F=2 log Pave+logηγϕσ2C.
Ω=NAs2TsNAobj2TeTobjsin2tan-1D2dTsNAobj2TeTobj,

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