Abstract

Two-photon excitation of the ultraviolet-absorbing fluorescent calcium indicator Indo-1 in laser scanning microscopy makes possible a quantitative, three-dimensional recording of intracellular free calcium activity ([Ca2+]i) distributions and dynamics with low background and minimal photobleaching. We have constructed a simple optical system that facilitates collection of the 400–500-nm Indo-1 fluorescence without the use of a confocal spatial filter. Instead of the fluorescence being descanned as is normally required in confocal microscopy, the fluorescence is deflected by a dichroic mirror into a separate detection pathway. Images of [Ca2+]i distributions with three-dimensional submicrometer resolution and 10% precision are obtained at 100-μM Indo-1 concentration and 3-s recording time for 384 × 512 pixels. Data on [Ca2+]i in tumor mast cells and cardiac myocytes illustrate the capabilities of this technique.

© 1994 Optical Society of America

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  2. K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging in laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
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  4. M. J. Berridge, R. F. Irvine, “Inositol phosphates and cell signaling,” Nature (London) 341, 197–205 (1989).
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  5. J. A. Connor, W. J. Wadman, P. E. Hockberger, R. K. S. Wong, “Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons,” Science 240, 649–653 (1988).
    [CrossRef] [PubMed]
  6. R. Llinas, M. Sugimori, R. B. Silver, “Microdomains of high calcium concentration in a presynaptic terminal,” Science 256, 677–679(1992).
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  7. M. Cannell, J. Berlin, W. J. Lederer, “Effect of membrane potential changes in single rat cardiac muscle cells,” Science 236, 1419–1423 (1987).
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    [PubMed]
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    [CrossRef]
  29. J. A. Ridsdale, W. W. Webb, “The viability of cultured cells under two-photon laser scanning microscopy,” Biophys. J. 63, A109 (1993).
  30. J. Thomas, W. W. Webb, “Fluorescence photobleaching recovery: a probe of membrane dynamics,” in Non-Invasive Techniques in Cell Biology, S. Grinstein, K. Foskett, eds. (Wiley-Liss, New York, 1990), pp. 129–152.
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1993 (2)

C. Bliton, J. Lechleiter, D. E. Clapham, “Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet- and visible-wavelength light,” J. Microsc. 169, 15–26(1993).
[CrossRef]

J. A. Ridsdale, W. W. Webb, “The viability of cultured cells under two-photon laser scanning microscopy,” Biophys. J. 63, A109 (1993).

1992 (2)

S. J. Sollott, B. D. Ziman, E. G. Lakatta, “Novel technique to load Indo-1 free acid into single adult cardiac myocytes to assess cytosolic calcium,” Am. J. Physiol. 262, H1941–H1949 (1992).
[PubMed]

R. Llinas, M. Sugimori, R. B. Silver, “Microdomains of high calcium concentration in a presynaptic terminal,” Science 256, 677–679(1992).
[CrossRef] [PubMed]

1991 (4)

T. Meyer, L. Styer, “Calcium spiking,” Ann. Rev. Biophys. Biophys. Chem. 20, 153–174 (1991).
[CrossRef]

J. Lechleiter, S. Girard, E. Peralta, D. E. Clapham, “Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes,” Science 252, 123–126 (1991).
[CrossRef] [PubMed]

A. T. Harootunian, J. P. Y. Kao, S. Paranjape, R. Y. Tsien, “Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3,” Science 251, 75–78 (1991).
[CrossRef] [PubMed]

B. Ulfhake, K. Carlsson, K. Mossberg, U. Arvidsson, P. J. Helm, “Imaging of fluorescent neurons labeled with fluorogold and fluorescent axon terminals labeled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope,” J. Neurosci. Methods 40, 39–48 (1991).
[CrossRef] [PubMed]

1990 (4)

E. Niggli, W. J. Lederer, “Real-time confocal microscopy of heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution,” Cell Calcium 11, 121–130 (1990).
[CrossRef] [PubMed]

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

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

A. H. C. Bell, S. M. Finkbeiner, M. S. Cooper, S. J. Smith, “Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling,” Science 247, 470–473 (1990).
[CrossRef]

1989 (5)

J. Berlin, M. Cannell, W. J. Lederer, “ITI in single rat cardiac ventricular cells: relationship to fluctuations in intracellular calcium,” Circ. Res. 65, 115–126 (1989).
[CrossRef] [PubMed]

A. Minta, J. P. Y. Kao, R. Y. Tsien, “Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores,” J. Biol. Chem. 264, 8171–8178 (1989).
[PubMed]

M. J. Berridge, R. F. Irvine, “Inositol phosphates and cell signaling,” Nature (London) 341, 197–205 (1989).
[CrossRef]

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

P. J. Millard, T. A. Ryan, W. W. Webb, C. Fewtrell, “Immunoglobulin E receptor cross-linking induces oscillations in [Ca2+]i in individual tumor mast cells,” J. Biol. Chem. 264, 19,730–19,739 (1989).

1988 (1)

J. A. Connor, W. J. Wadman, P. E. Hockberger, R. K. S. Wong, “Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons,” Science 240, 649–653 (1988).
[CrossRef] [PubMed]

1987 (1)

M. Cannell, J. Berlin, W. J. Lederer, “Effect of membrane potential changes in single rat cardiac muscle cells,” Science 236, 1419–1423 (1987).
[CrossRef]

1986 (2)

R. R. Birge, “Two-photon spectroscopy of protein-bound chromophores,” Ace. Chem. Res. 19, 138–146 (1986).
[CrossRef]

S. M. Kennedy, F. E. Lytle, “p-Bis(o-methylstyryl)benzene as a power-squared sensor for two-photon absorption measurements,” Anal. Chem. 58, 2643–2653 (1986).
[CrossRef]

1985 (1)

G. Grynkiewicz, M. Poenie, R. Y. Tsien, “A new generation of indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).
[PubMed]

1980 (1)

D. M. Friedrich, W. M. McClain, “Two-photon molecular electronic spectroscopy,” Ann. Rev. Phys. Chem. 31, 559–577 (1980).
[CrossRef]

1931 (1)

M. Goppert-Mayer, “Uber Elementarake mit zwei Quantensprungen,” Ann. Phys. (Berlin) 9, 273–294 (1931).
[CrossRef]

Arvidsson, U.

B. Ulfhake, K. Carlsson, K. Mossberg, U. Arvidsson, P. J. Helm, “Imaging of fluorescent neurons labeled with fluorogold and fluorescent axon terminals labeled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope,” J. Neurosci. Methods 40, 39–48 (1991).
[CrossRef] [PubMed]

Balaban, R. S.

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

Bell, A. H. C.

A. H. C. Bell, S. M. Finkbeiner, M. S. Cooper, S. J. Smith, “Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling,” Science 247, 470–473 (1990).
[CrossRef]

Berlin, J.

J. Berlin, M. Cannell, W. J. Lederer, “ITI in single rat cardiac ventricular cells: relationship to fluctuations in intracellular calcium,” Circ. Res. 65, 115–126 (1989).
[CrossRef] [PubMed]

M. Cannell, J. Berlin, W. J. Lederer, “Effect of membrane potential changes in single rat cardiac muscle cells,” Science 236, 1419–1423 (1987).
[CrossRef]

Berridge, M. J.

M. J. Berridge, R. F. Irvine, “Inositol phosphates and cell signaling,” Nature (London) 341, 197–205 (1989).
[CrossRef]

Bighouse, R.

R. Bighouse, “Measurement of the two-photon absorption spectra of several fluorescent dyes,” M.S. thesis (Cornell University, Ithaca, N.Y., 1991).

Birge, R. R.

R. R. Birge, “Two-photon spectroscopy of protein-bound chromophores,” Ace. Chem. Res. 19, 138–146 (1986).
[CrossRef]

Bliton, C.

C. Bliton, J. Lechleiter, D. E. Clapham, “Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet- and visible-wavelength light,” J. Microsc. 169, 15–26(1993).
[CrossRef]

Cannell, M.

J. Berlin, M. Cannell, W. J. Lederer, “ITI in single rat cardiac ventricular cells: relationship to fluctuations in intracellular calcium,” Circ. Res. 65, 115–126 (1989).
[CrossRef] [PubMed]

M. Cannell, J. Berlin, W. J. Lederer, “Effect of membrane potential changes in single rat cardiac muscle cells,” Science 236, 1419–1423 (1987).
[CrossRef]

Carlsson, K.

B. Ulfhake, K. Carlsson, K. Mossberg, U. Arvidsson, P. J. Helm, “Imaging of fluorescent neurons labeled with fluorogold and fluorescent axon terminals labeled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope,” J. Neurosci. Methods 40, 39–48 (1991).
[CrossRef] [PubMed]

Clapham, D. E.

C. Bliton, J. Lechleiter, D. E. Clapham, “Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet- and visible-wavelength light,” J. Microsc. 169, 15–26(1993).
[CrossRef]

J. Lechleiter, S. Girard, E. Peralta, D. E. Clapham, “Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes,” Science 252, 123–126 (1991).
[CrossRef] [PubMed]

Connor, J. A.

J. A. Connor, W. J. Wadman, P. E. Hockberger, R. K. S. Wong, “Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons,” Science 240, 649–653 (1988).
[CrossRef] [PubMed]

Cooper, M. S.

A. H. C. Bell, S. M. Finkbeiner, M. S. Cooper, S. J. Smith, “Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling,” Science 247, 470–473 (1990).
[CrossRef]

Denk, W.

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

Eng, J.

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

Fewtrell, C.

P. J. Millard, T. A. Ryan, W. W. Webb, C. Fewtrell, “Immunoglobulin E receptor cross-linking induces oscillations in [Ca2+]i in individual tumor mast cells,” J. Biol. Chem. 264, 19,730–19,739 (1989).

Finkbeiner, S. M.

A. H. C. Bell, S. M. Finkbeiner, M. S. Cooper, S. J. Smith, “Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling,” Science 247, 470–473 (1990).
[CrossRef]

Friedrich, D. M.

D. M. Friedrich, W. M. McClain, “Two-photon molecular electronic spectroscopy,” Ann. Rev. Phys. Chem. 31, 559–577 (1980).
[CrossRef]

Girard, S.

J. Lechleiter, S. Girard, E. Peralta, D. E. Clapham, “Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes,” Science 252, 123–126 (1991).
[CrossRef] [PubMed]

Goppert-Mayer, M.

M. Goppert-Mayer, “Uber Elementarake mit zwei Quantensprungen,” Ann. Phys. (Berlin) 9, 273–294 (1931).
[CrossRef]

Grynkiewicz, G.

G. Grynkiewicz, M. Poenie, R. Y. Tsien, “A new generation of indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).
[PubMed]

Gu, M.

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

Harootunian, A. T.

A. T. Harootunian, J. P. Y. Kao, S. Paranjape, R. Y. Tsien, “Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3,” Science 251, 75–78 (1991).
[CrossRef] [PubMed]

Haugland, R. G.

R. G. Haugland, Molecular Probes Handbook of Fluorescence Probes and Research Chemicals (Molecular Probes, Eugene, Ore., 1992), pp. 113–128.

Helm, P. J.

B. Ulfhake, K. Carlsson, K. Mossberg, U. Arvidsson, P. J. Helm, “Imaging of fluorescent neurons labeled with fluorogold and fluorescent axon terminals labeled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope,” J. Neurosci. Methods 40, 39–48 (1991).
[CrossRef] [PubMed]

Hockberger, P. E.

J. A. Connor, W. J. Wadman, P. E. Hockberger, R. K. S. Wong, “Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons,” Science 240, 649–653 (1988).
[CrossRef] [PubMed]

Irvine, R. F.

M. J. Berridge, R. F. Irvine, “Inositol phosphates and cell signaling,” Nature (London) 341, 197–205 (1989).
[CrossRef]

Kao, J. P. Y.

A. T. Harootunian, J. P. Y. Kao, S. Paranjape, R. Y. Tsien, “Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3,” Science 251, 75–78 (1991).
[CrossRef] [PubMed]

A. Minta, J. P. Y. Kao, R. Y. Tsien, “Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores,” J. Biol. Chem. 264, 8171–8178 (1989).
[PubMed]

Kapitza, H. G.

H. G. Kapitza, V. Wilke, “Applications of the microscope system LSM,” in Scanning Imaging, T. Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 173–179 (1988).

Kennedy, S. M.

S. M. Kennedy, F. E. Lytle, “p-Bis(o-methylstyryl)benzene as a power-squared sensor for two-photon absorption measurements,” Anal. Chem. 58, 2643–2653 (1986).
[CrossRef]

Lakatta, E. G.

S. J. Sollott, B. D. Ziman, E. G. Lakatta, “Novel technique to load Indo-1 free acid into single adult cardiac myocytes to assess cytosolic calcium,” Am. J. Physiol. 262, H1941–H1949 (1992).
[PubMed]

Lechleiter, J.

C. Bliton, J. Lechleiter, D. E. Clapham, “Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet- and visible-wavelength light,” J. Microsc. 169, 15–26(1993).
[CrossRef]

J. Lechleiter, S. Girard, E. Peralta, D. E. Clapham, “Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes,” Science 252, 123–126 (1991).
[CrossRef] [PubMed]

Lederer, W. J.

E. Niggli, W. J. Lederer, “Real-time confocal microscopy of heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution,” Cell Calcium 11, 121–130 (1990).
[CrossRef] [PubMed]

J. Berlin, M. Cannell, W. J. Lederer, “ITI in single rat cardiac ventricular cells: relationship to fluctuations in intracellular calcium,” Circ. Res. 65, 115–126 (1989).
[CrossRef] [PubMed]

M. Cannell, J. Berlin, W. J. Lederer, “Effect of membrane potential changes in single rat cardiac muscle cells,” Science 236, 1419–1423 (1987).
[CrossRef]

Llinas, R.

R. Llinas, M. Sugimori, R. B. Silver, “Microdomains of high calcium concentration in a presynaptic terminal,” Science 256, 677–679(1992).
[CrossRef] [PubMed]

Lynch, R. M.

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

Lytle, F. E.

S. M. Kennedy, F. E. Lytle, “p-Bis(o-methylstyryl)benzene as a power-squared sensor for two-photon absorption measurements,” Anal. Chem. 58, 2643–2653 (1986).
[CrossRef]

McClain, W. M.

D. M. Friedrich, W. M. McClain, “Two-photon molecular electronic spectroscopy,” Ann. Rev. Phys. Chem. 31, 559–577 (1980).
[CrossRef]

Meyer, T.

T. Meyer, L. Styer, “Calcium spiking,” Ann. Rev. Biophys. Biophys. Chem. 20, 153–174 (1991).
[CrossRef]

Millard, P. J.

P. J. Millard, T. A. Ryan, W. W. Webb, C. Fewtrell, “Immunoglobulin E receptor cross-linking induces oscillations in [Ca2+]i in individual tumor mast cells,” J. Biol. Chem. 264, 19,730–19,739 (1989).

Minta, A.

A. Minta, J. P. Y. Kao, R. Y. Tsien, “Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores,” J. Biol. Chem. 264, 8171–8178 (1989).
[PubMed]

Mossberg, K.

B. Ulfhake, K. Carlsson, K. Mossberg, U. Arvidsson, P. J. Helm, “Imaging of fluorescent neurons labeled with fluorogold and fluorescent axon terminals labeled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope,” J. Neurosci. Methods 40, 39–48 (1991).
[CrossRef] [PubMed]

Niggli, E.

E. Niggli, W. J. Lederer, “Real-time confocal microscopy of heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution,” Cell Calcium 11, 121–130 (1990).
[CrossRef] [PubMed]

Paranjape, S.

A. T. Harootunian, J. P. Y. Kao, S. Paranjape, R. Y. Tsien, “Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3,” Science 251, 75–78 (1991).
[CrossRef] [PubMed]

Peralta, E.

J. Lechleiter, S. Girard, E. Peralta, D. E. Clapham, “Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes,” Science 252, 123–126 (1991).
[CrossRef] [PubMed]

Piston, D. W.

D. R. Sandison, D. W. Piston, W. W. Webb, “Background rejection and optimization of signal-to-noise in confocal microscopy,” in Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, J. K. Stevens, L. R. Mills, J. E. Trogades, eds. (Academic, San Diego, Calif., 1993).

Poenie, M.

G. Grynkiewicz, M. Poenie, R. Y. Tsien, “A new generation of indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).
[PubMed]

Ridsdale, J. A.

J. A. Ridsdale, W. W. Webb, “The viability of cultured cells under two-photon laser scanning microscopy,” Biophys. J. 63, A109 (1993).

Ryan, T. A.

P. J. Millard, T. A. Ryan, W. W. Webb, C. Fewtrell, “Immunoglobulin E receptor cross-linking induces oscillations in [Ca2+]i in individual tumor mast cells,” J. Biol. Chem. 264, 19,730–19,739 (1989).

Sandison, D. R.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging in laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

D. R. Sandison, D. W. Piston, W. W. Webb, “Background rejection and optimization of signal-to-noise in confocal microscopy,” in Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, J. K. Stevens, L. R. Mills, J. E. Trogades, eds. (Academic, San Diego, Calif., 1993).

Sheppard, C. J. R.

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

Silver, R. B.

R. Llinas, M. Sugimori, R. B. Silver, “Microdomains of high calcium concentration in a presynaptic terminal,” Science 256, 677–679(1992).
[CrossRef] [PubMed]

Smith, S. J.

A. H. C. Bell, S. M. Finkbeiner, M. S. Cooper, S. J. Smith, “Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling,” Science 247, 470–473 (1990).
[CrossRef]

Sollott, S. J.

S. J. Sollott, B. D. Ziman, E. G. Lakatta, “Novel technique to load Indo-1 free acid into single adult cardiac myocytes to assess cytosolic calcium,” Am. J. Physiol. 262, H1941–H1949 (1992).
[PubMed]

Strickler, J.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging in laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[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]

Styer, L.

T. Meyer, L. Styer, “Calcium spiking,” Ann. Rev. Biophys. Biophys. Chem. 20, 153–174 (1991).
[CrossRef]

Sugimori, M.

R. Llinas, M. Sugimori, R. B. Silver, “Microdomains of high calcium concentration in a presynaptic terminal,” Science 256, 677–679(1992).
[CrossRef] [PubMed]

Thomas, J.

J. Thomas, W. W. Webb, “Fluorescence photobleaching recovery: a probe of membrane dynamics,” in Non-Invasive Techniques in Cell Biology, S. Grinstein, K. Foskett, eds. (Wiley-Liss, New York, 1990), pp. 129–152.

Tsien, R. Y.

A. T. Harootunian, J. P. Y. Kao, S. Paranjape, R. Y. Tsien, “Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3,” Science 251, 75–78 (1991).
[CrossRef] [PubMed]

A. Minta, J. P. Y. Kao, R. Y. Tsien, “Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores,” J. Biol. Chem. 264, 8171–8178 (1989).
[PubMed]

G. Grynkiewicz, M. Poenie, R. Y. Tsien, “A new generation of indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).
[PubMed]

Ulfhake, B.

B. Ulfhake, K. Carlsson, K. Mossberg, U. Arvidsson, P. J. Helm, “Imaging of fluorescent neurons labeled with fluorogold and fluorescent axon terminals labeled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope,” J. Neurosci. Methods 40, 39–48 (1991).
[CrossRef] [PubMed]

Wadman, W. J.

J. A. Connor, W. J. Wadman, P. E. Hockberger, R. K. S. Wong, “Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons,” Science 240, 649–653 (1988).
[CrossRef] [PubMed]

Webb, W. W.

J. A. Ridsdale, W. W. Webb, “The viability of cultured cells under two-photon laser scanning microscopy,” Biophys. J. 63, A109 (1993).

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

P. J. Millard, T. A. Ryan, W. W. Webb, C. Fewtrell, “Immunoglobulin E receptor cross-linking induces oscillations in [Ca2+]i in individual tumor mast cells,” J. Biol. Chem. 264, 19,730–19,739 (1989).

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging in laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

D. R. Sandison, D. W. Piston, W. W. Webb, “Background rejection and optimization of signal-to-noise in confocal microscopy,” in Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, J. K. Stevens, L. R. Mills, J. E. Trogades, eds. (Academic, San Diego, Calif., 1993).

J. Thomas, W. W. Webb, “Fluorescence photobleaching recovery: a probe of membrane dynamics,” in Non-Invasive Techniques in Cell Biology, S. Grinstein, K. Foskett, eds. (Wiley-Liss, New York, 1990), pp. 129–152.

Wells, K. S.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging in laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

Wilke, V.

H. G. Kapitza, V. Wilke, “Applications of the microscope system LSM,” in Scanning Imaging, T. Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 173–179 (1988).

Wong, R. K. S.

J. A. Connor, W. J. Wadman, P. E. Hockberger, R. K. S. Wong, “Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons,” Science 240, 649–653 (1988).
[CrossRef] [PubMed]

Woo, K.

K. Woo, “Photobleaching of biological fluorophores,” M.S. thesis (Cornell University, Ithaca, New York, 1991).

Ziman, B. D.

S. J. Sollott, B. D. Ziman, E. G. Lakatta, “Novel technique to load Indo-1 free acid into single adult cardiac myocytes to assess cytosolic calcium,” Am. J. Physiol. 262, H1941–H1949 (1992).
[PubMed]

Ace. Chem. Res. (1)

R. R. Birge, “Two-photon spectroscopy of protein-bound chromophores,” Ace. Chem. Res. 19, 138–146 (1986).
[CrossRef]

Am. J. Physiol. (1)

S. J. Sollott, B. D. Ziman, E. G. Lakatta, “Novel technique to load Indo-1 free acid into single adult cardiac myocytes to assess cytosolic calcium,” Am. J. Physiol. 262, H1941–H1949 (1992).
[PubMed]

Anal. Chem. (1)

S. M. Kennedy, F. E. Lytle, “p-Bis(o-methylstyryl)benzene as a power-squared sensor for two-photon absorption measurements,” Anal. Chem. 58, 2643–2653 (1986).
[CrossRef]

Ann. Phys. (Berlin) (1)

M. Goppert-Mayer, “Uber Elementarake mit zwei Quantensprungen,” Ann. Phys. (Berlin) 9, 273–294 (1931).
[CrossRef]

Ann. Rev. Biophys. Biophys. Chem. (1)

T. Meyer, L. Styer, “Calcium spiking,” Ann. Rev. Biophys. Biophys. Chem. 20, 153–174 (1991).
[CrossRef]

Ann. Rev. Phys. Chem. (1)

D. M. Friedrich, W. M. McClain, “Two-photon molecular electronic spectroscopy,” Ann. Rev. Phys. Chem. 31, 559–577 (1980).
[CrossRef]

Biophys. J. (2)

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

J. A. Ridsdale, W. W. Webb, “The viability of cultured cells under two-photon laser scanning microscopy,” Biophys. J. 63, A109 (1993).

Cell Calcium (1)

E. Niggli, W. J. Lederer, “Real-time confocal microscopy of heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution,” Cell Calcium 11, 121–130 (1990).
[CrossRef] [PubMed]

Circ. Res. (1)

J. Berlin, M. Cannell, W. J. Lederer, “ITI in single rat cardiac ventricular cells: relationship to fluctuations in intracellular calcium,” Circ. Res. 65, 115–126 (1989).
[CrossRef] [PubMed]

J. Biol. Chem. (3)

A. Minta, J. P. Y. Kao, R. Y. Tsien, “Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores,” J. Biol. Chem. 264, 8171–8178 (1989).
[PubMed]

G. Grynkiewicz, M. Poenie, R. Y. Tsien, “A new generation of indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).
[PubMed]

P. J. Millard, T. A. Ryan, W. W. Webb, C. Fewtrell, “Immunoglobulin E receptor cross-linking induces oscillations in [Ca2+]i in individual tumor mast cells,” J. Biol. Chem. 264, 19,730–19,739 (1989).

J. Microsc. (1)

C. Bliton, J. Lechleiter, D. E. Clapham, “Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet- and visible-wavelength light,” J. Microsc. 169, 15–26(1993).
[CrossRef]

J. Neurosci. Methods (1)

B. Ulfhake, K. Carlsson, K. Mossberg, U. Arvidsson, P. J. Helm, “Imaging of fluorescent neurons labeled with fluorogold and fluorescent axon terminals labeled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope,” J. Neurosci. Methods 40, 39–48 (1991).
[CrossRef] [PubMed]

Nature (London) (1)

M. J. Berridge, R. F. Irvine, “Inositol phosphates and cell signaling,” Nature (London) 341, 197–205 (1989).
[CrossRef]

Optik (1)

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

Science (7)

J. A. Connor, W. J. Wadman, P. E. Hockberger, R. K. S. Wong, “Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons,” Science 240, 649–653 (1988).
[CrossRef] [PubMed]

R. Llinas, M. Sugimori, R. B. Silver, “Microdomains of high calcium concentration in a presynaptic terminal,” Science 256, 677–679(1992).
[CrossRef] [PubMed]

M. Cannell, J. Berlin, W. J. Lederer, “Effect of membrane potential changes in single rat cardiac muscle cells,” Science 236, 1419–1423 (1987).
[CrossRef]

J. Lechleiter, S. Girard, E. Peralta, D. E. Clapham, “Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes,” Science 252, 123–126 (1991).
[CrossRef] [PubMed]

A. T. Harootunian, J. P. Y. Kao, S. Paranjape, R. Y. Tsien, “Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3,” Science 251, 75–78 (1991).
[CrossRef] [PubMed]

A. H. C. Bell, S. M. Finkbeiner, M. S. Cooper, S. J. Smith, “Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling,” Science 247, 470–473 (1990).
[CrossRef]

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

Other (7)

H. G. Kapitza, V. Wilke, “Applications of the microscope system LSM,” in Scanning Imaging, T. Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 173–179 (1988).

R. G. Haugland, Molecular Probes Handbook of Fluorescence Probes and Research Chemicals (Molecular Probes, Eugene, Ore., 1992), pp. 113–128.

D. R. Sandison, D. W. Piston, W. W. Webb, “Background rejection and optimization of signal-to-noise in confocal microscopy,” in Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, J. K. Stevens, L. R. Mills, J. E. Trogades, eds. (Academic, San Diego, Calif., 1993).

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging in laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

R. Bighouse, “Measurement of the two-photon absorption spectra of several fluorescent dyes,” M.S. thesis (Cornell University, Ithaca, N.Y., 1991).

J. Thomas, W. W. Webb, “Fluorescence photobleaching recovery: a probe of membrane dynamics,” in Non-Invasive Techniques in Cell Biology, S. Grinstein, K. Foskett, eds. (Wiley-Liss, New York, 1990), pp. 129–152.

K. Woo, “Photobleaching of biological fluorophores,” M.S. thesis (Cornell University, Ithaca, New York, 1991).

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Figures (6)

Fig. 1
Fig. 1

Schematic diagram of the two-photon laser scanning microscope with two-channel external detection. The incoming laser light is raster scanned (X−Y scanner), passes through the first dichroic mirror (DM 1, a 550DCLP from Omega Optical), and is focused onto the sample by the objective lens. The fluorescence returns down the same path until it is reflected into the external detection system by DM 1. The emitted signal is then refocused so that the back aperture of the objective is conjugate to the front face of the two photomultiplier tubes (PMT 1 and PMT 2). The signal is split by the second dichroic mirror (DM 2, a 450DCLP) and passes through a bandpass interference filter (BF 1, a 495DF20, or BF 2, a 405DF35) before reaching the PMT’s.

Fig. 2
Fig. 2

Preliminary measurements of two-photon excitation spectra of both free (open circles) and Ca2+-bound (filled circles) Indo-1. The spectra are normalized by calibration with bis-MSB, a known two-photon absorption standard. No points of the spectra were acquired between 650 and 690 nm, so this region of the spectra is represented by the dotted lines.

Fig. 3
Fig. 3

Estimates of the Indo-1 emission profiles for the Ca2+-bound and free forms for two-photon excitation with 705-nm light (solid curves) compared with published emission profiles for conventional excitation (scored curves). To approximate the two-photon excited emission profiles, the emission spectra of the bound and the free forms of one-photon-excited Indo-1 are rescaled by the relative excitation power at 705 nm taken from Fig. 2.

Fig. 4
Fig. 4

Images of EtBr uptake by Indo-1 loaded RBL cells after scanning with the two-photon-excitation beam. The top half of each of two frames was scanned continuously for 5 min. The results of irradiation at a typical excitation power used for [Ca2+] i measurements in RBL (~5 mW at the sample) are shown in frame a. It shows slight bleaching of the Indo-1 signal in the upper half of the image where the scanning took place but no difference in EtBr uptake between the upper and the lower halves of the image. The second frame, b, was scanned at full power and shows a dramatic decline of cell viability in the scanned region.

Fig. 5
Fig. 5

Images of stimulated RBL cells from two-photon-excited Indo-1 fluorescence. The two channels, a, ~405 nm and b, ~495 nm correspond to the expected Ca2+-bound and free peaks of Indo-1 with conventional excitation at ~355 nm. With two-photon excitation at 705 nm, image a is close to an isospestic point of the Indo-1 emission as a function of Ca2+. The calibrated ratio image of panels a and b is shown in c. Each image is 60 × 100 μm.

Fig. 6
Fig. 6

Fluorescence images of a rat cardiac myocyte acquired with two-photon excitation of Indo-1. Approximately half way through the scan the cell is stimulated, causing a transient Ca2+ rise. As in Fig. 5, the two channels, a, ~405 nm and b, ~495 nm correspond to the expected Ca2+-bound and free peaks of Indo-1 with conventional excitation at ~355 nm. The ratio image of panels a and b is shown in c. Each imaee is 100 × 100 μm.

Equations (1)

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[ Ca 2 + ] i = K d β ( R R min / R max R ) ,

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