OSA's Digital Library

Applied Optics

Applied Optics


  • Vol. 33, Iss. 4 — Feb. 1, 1994
  • pp: 662–669

Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity

David W. Piston, Mark S. Kirby, Heping Cheng, W. J. Lederer, and Watt W. Webb  »View Author Affiliations

Applied Optics, Vol. 33, Issue 4, pp. 662-669 (1994)

View Full Text Article

Enhanced HTML    Acrobat PDF (1441 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



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

Original Manuscript: April 8, 1993
Revised Manuscript: August 2, 1993
Published: February 1, 1994

David W. Piston, Mark S. Kirby, Heping Cheng, W. J. Lederer, and Watt W. Webb, "Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity," Appl. Opt. 33, 662-669 (1994)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. 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).
  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. [CrossRef]
  3. T. Meyer, L. Styer, “Calcium spiking,” Ann. Rev. Biophys. Biophys. Chem. 20, 153–174 (1991). [CrossRef]
  4. M. J. Berridge, R. F. Irvine, “Inositol phosphates and cell signaling,” Nature (London) 341, 197–205 (1989). [CrossRef]
  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). [CrossRef] [PubMed]
  7. M. Cannell, J. Berlin, W. J. Lederer, “Effect of membrane potential changes in single rat cardiac muscle cells,” Science 236, 1419–1423 (1987). [CrossRef]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. R. G. Haugland, Molecular Probes Handbook of Fluorescence Probes and Research Chemicals (Molecular Probes, Eugene, Ore., 1992), pp. 113–128.
  14. 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]
  15. 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]
  16. 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).
  17. 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]
  18. 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]
  19. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990). [CrossRef] [PubMed]
  20. C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).
  21. M. Goppert-Mayer, “Uber Elementarake mit zwei Quantensprungen,” Ann. Phys. (Berlin) 9, 273–294 (1931). [CrossRef]
  22. D. M. Friedrich, W. M. McClain, “Two-photon molecular electronic spectroscopy,” Ann. Rev. Phys. Chem. 31, 559–577 (1980). [CrossRef]
  23. R. R. Birge, “Two-photon spectroscopy of protein-bound chromophores,” Ace. Chem. Res. 19, 138–146 (1986). [CrossRef]
  24. 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).
  25. 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]
  26. 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]
  27. R. Bighouse, “Measurement of the two-photon absorption spectra of several fluorescent dyes,” M.S. thesis (Cornell University, Ithaca, N.Y., 1991).
  28. 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]
  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.
  31. K. Woo, “Photobleaching of biological fluorophores,” M.S. thesis (Cornell University, Ithaca, New York, 1991).

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited