Dual-Channel Confocal Ratioing of Calcium Dynamics in Living Eggs and Oocytes

  • Stephen A. Stricker
Part of the Methods in Molecular Biology™ book series (MIMB, volume 254)


Changes in intracellular calcium concentrations ([Ca2+]i) have been shown to regulate a wide variety of developmental processes. In particular, eggs that have completed meiosis or oocytes that are still in the process of undergoing meiotic maturation invariably display some form of a [Ca2+]i elevation during fertilization (1, 2, 3). In order to image fertilization-induced calcium responses, unfertilized eggs and oocytes can be loaded with photoproteins, (e.g., aequorin) for subsequent detection of the luminescence that is generated when free calcium ions bind to the photoprotein (4). Alternatively, eggs may either be injected with or simply incubated in various calcium-sensitive fluorophores (5). Such calcium indicators can then be used in conjunction with fluorescence microscopy to track calcium dynamics during fertilization. The relative benefits and disadvantages of aequorin vs calcium-sensitive probes have been discussed previously (6), and both types of calcium indicators remain widely employed in imaging analyses that involves conventional microscopy.


Neutral Density Protamine Sulfate Calcium Indicator Jelly Coat Injection Buffer 
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  1. 1.
    Whitaker, M. and Swann, K. (1993) Lighting the fuse at fertilization. Development 117, 1–12.Google Scholar
  2. 2.
    Stricker, S. A. (1999). Comparative calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157–176.PubMedCrossRefGoogle Scholar
  3. 3.
    Williams, C. J. (2002) Signalling mechanisms of mammalian oocyte maturation. Hum. Reprod. Update 8, 313–321.PubMedCrossRefGoogle Scholar
  4. 4.
    Miller, A. L., Karplus, E., and Jaffe, L. F. Imaging [Ca2+]i with aequorin using a photon imaging detector, in A Practical Guide to the Study of Calcium in Living Cells. (Nuccitelli, R., ed.) Academic Press, San Diego, CA, 1994, pp. 305–338.CrossRefGoogle Scholar
  5. 5.
    Kao, J. P. Y. Practical aspects of measuring [Ca2+]i with fluorescent indicators, in A Practical Guide to the Study of Calcium in Living Cells. (Nuccitelli, R., ed.), Academic Press, San Diego, CA, 1994, pp. 155–181.CrossRefGoogle Scholar
  6. 6.
    Stricker, S. A.. and Whitaker, M. J. (1999). Confocal laser scanning microscopy of calcium dynamics in living cells. Microscopy Res. Tech. 46, 356–369.CrossRefGoogle Scholar
  7. 7.
    Majlof, L. and Forsgren, P.-O. Confocal microscopy: important considerations for accurate imaging, in Cell Biological Applications of Confocal Microscopy. (Matsumoto, B., ed.), Academic Press, San Diego, CA, 1993, pp. 79–95.CrossRefGoogle Scholar
  8. 8.
    Wright, S. J., Centonze, V. E., Stricker, S. A., DeVries, P. J., Paddock, S. W., and Schatten, G. An introduction to confocal microscopy and three-dimensional reconstruction, in Cell Biological Applications of Confocal Microscopy. (Matsumoto, B., ed.), Academic Press, San Diego, CA, 1993, pp. 1–45.CrossRefGoogle Scholar
  9. 9.
    Inoue, S. Foundations of confocal scanned imaging in light microscopy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Pawley, J.B., ed.), Plenum Press, New York, NY, 1995, pp. 1–17.Google Scholar
  10. 10.
    Bliton, A. C. and Lechleiter, J. D. Optical considerations at ultraviolet wavelengths in confocal microscopy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Pawley, J.B., ed.), Plenum Press, New York, NY, 1995, pp. 431–444.Google Scholar
  11. 11.
    Denk, W., Strickler, J., and Webb, W. W. (1990) Two-photon laser scanning fluorescence microscopy. Science 248, 73–76.PubMedCrossRefGoogle Scholar
  12. 12.
    Centonze, V. E. and White, J. G. Multiphoton excitation fluorescence microscopy, in Cells: A Laboratory Manual (Spector, D.L., Goldman, R.D., and Leinwand, L.A. eds.) Cold Spring Harbo Press, Cold Spring Harbor, NY, 1997.Google Scholar
  13. 13.
    Denk, W. and Svoboda, K. (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357.PubMedCrossRefGoogle Scholar
  14. 14.
    Pawley, J. B., ed. Handbook of Biological Confocal Microscopy, 2nd ed. Plenum Press, New York, NY, 1995.Google Scholar
  15. 15.
    Brownlee, C. (2000) Cellular calcium imaging: so, what’s new? Trends Biol. Sci 10, 451–457.Google Scholar
  16. 16.
    Stricker, S. A. (2000) Confocal microscopy of intracellular calcium dynamics during fertilization. BioTechniques 29, 492–498.PubMedGoogle Scholar
  17. 17.
    Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th Edition. Molecular Probes Inc., Eugene, OR, 1996.Google Scholar
  18. 18.
    Lipp, P. and Niggli, E. (1993) Ratiometric confocal Ca2+ measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium 14 359–372.PubMedCrossRefGoogle Scholar
  19. 19.
    Diliberto, P. A., Wang, X. F., and Herman, B. Confocal imaging of Ca2+ in cells, in A Practical Guide to the Study of Calcium in Living Cells. (Nuccitelli, R., ed.), Academic Press, San Diego, CA, 1994, pp. 243–262.CrossRefGoogle Scholar
  20. 20.
    Floto, R. A., Mahautsmith, M. P., Somasundaram, B., and Allen, J. M. (1995) IgG-induced Ca2+ oscillations in differentiated U937 cells: A study using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Cell Calcium 18, 377–389.PubMedCrossRefGoogle Scholar
  21. 21.
    McDougall, A. and Sardet, C. (1995) Function and characteristics of repetitive calcium waves associated with meiosis. Curr. Biol. 5, 318–328.PubMedCrossRefGoogle Scholar
  22. 22.
    Stricker, S. A. (1995) Time-lapse confocal imaging of calcium dynamics in starfish embryos. Dev. Biol. 170, 496–518.PubMedCrossRefGoogle Scholar
  23. 23.
    Chiba, K., Kado, R. T., and Jaffe, L. A. (1990) Development of calcium release mechanisms during starfish oocyte maturation. Dev. Biol. 140, 300–306.PubMedCrossRefGoogle Scholar
  24. 24.
    Schroeder, T. E. and Stricker, S. A. (1983). Morphological changes during maturation of starfish oocytes: surface ultrastructure and cortical actin. Dev. Biol. 98, 373–384.PubMedCrossRefGoogle Scholar
  25. 25.
    Strathmann, M. F., ed. Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast. University of Washington Press, Seattle, WA, 1987.Google Scholar
  26. 26.
    Finkbeiner, S. (1992) Calcium waves in astrocytes—filling in the gaps. Neuron 8, 1101–1108.PubMedCrossRefGoogle Scholar
  27. 27.
    Stricker, S. A., Centonze, V. E., Paddock, S. W., and Schatten, G. (1992) Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs. Dev. Biol. 149, 370–380.PubMedCrossRefGoogle Scholar
  28. 28.
    Stricker, S. A., Centonze, V. E., and Melendez, R. F. (1994) Calcium dynamics during starfish oocyte maturation and fertilization. Dev. Biol. 165, 33–58.Google Scholar
  29. 29.
    Stricker, S. A. and Smythe, T. L. (2001) 5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms. Development 128, 1415–1427.PubMedGoogle Scholar
  30. 30.
    Yuce, O., and Sadler, K.C. (2001) Postmeiotic unfertilized starfish eggs die by apoptosis. Dev. Biol. 237, 29–44.PubMedCrossRefGoogle Scholar
  31. 31.
    Voronina, E. and Wessel, G. M. (2001) Apoptosis in sea urchin oocytes, eggs, and early embryos. Mol. Reprod. Dev. 60, 553–561.PubMedCrossRefGoogle Scholar
  32. 32.
    Stricker, S. A. and Smythe, T. L. (2000). Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin. J. Exp. Zool. 287, 243–261.PubMedCrossRefGoogle Scholar
  33. 33.
    Sasaki, K. and Chiba, K. (2001) Fertilization blocks apoptosis of starfish eggs by inactivation of the MAP kinase pathway. Dev. Biol. 237, 18–28.PubMedCrossRefGoogle Scholar
  34. 34.
    Summers, R. G., Stricker, S. A., and Cameron, R. A. Applications of confocal microscopy to studies of sea urchin embryogenesis, in Cell Biological Applications of Confocal Microscopy (Matsumoto, B., ed.), Academic Press, San Diego, CA, 1993, pp. 266–287.Google Scholar
  35. 35.
    Kishimoto, T. Microinjection and cytoplasmic transfer in starfish oocytes, in Methods in Cell Biology, vol. 27 (Schroeder, T. E., ed.), Academic Press, Orlando, FL, 1986, pp. 379–394.Google Scholar
  36. 36.
    DePamphilis, M. L., Herman, S. A., Martinzez-Salas, E., Chalifour, L. E., Wirak, D. O., Cupo, D. Y., and Miranda, M. (1988) Microinjecting DNA into mouse ova to study DNA replication and gene expression and to produce transgenic animals. Biotechniques 7, 662–680.Google Scholar
  37. 37.
    Stricker, S. A. Confocal microscopy of living eggs and embryos, in Three Dimensional Confocal Microscopy (Stevens, J. K., Mills, L. R., and Trogadis, J. E., eds.) Academic Press, San Diego, CA, 1994, pp. 281–300.Google Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Stephen A. Stricker
    • 1
  1. 1.Department of BiologyUniversity of New MexicoAlbuquerque

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