Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

Abstract

A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Viability of embryos after long-term imaging.
Figure 2: Production of reactive oxygen in imaged embryos.
Figure 3: Fetal development following long-term imaging.

References

  1. Terasaki, M. & Dailey, M.E. in Handbook of biological confocal microscopy (ed. Pawley, J.B.) 327–346 (Plenum, New York; 1995).

    Book  Google Scholar 

  2. Hillman, N. & Tasca, R. Ultrastructural and autoradiographic studies of mouse cleavage stages. Am. J. Anat. 126, 151–174 (1983).

    Article  Google Scholar 

  3. Batten, B.E., Albertini, D.F. & Ducibella, T. Patterns of organelle distribution in mouse embryos during preimplantation development. Am. J. Anat. 178, 204–213 (1987).

    CAS  Article  Google Scholar 

  4. Holy, J., Simerly, C., Paddock, S. & Schatten, G. Three-dimensional imaging of fertilization and early development. J. Electron Microsc. Technol. 17, 384–400 (1991).

    CAS  Article  Google Scholar 

  5. Capco, D.G., Gallicano, G.I., McGaughey, R.W., Downing, K.H., & Larabell, C.A. Cytoskeletal sheets of mammailian eggs and embryos: a lattice-like network of intermediate filaments. Cell Motil. Cytoskeleton 24, 85–99 (1993).

    CAS  Article  Google Scholar 

  6. Barnett, D., Kimura, J. & Bavister, B. Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal laser scanning microscopy. Dev. Dyn. 205, 64–72 (1996).

    CAS  Article  Google Scholar 

  7. Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D.W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).

    CAS  Article  Google Scholar 

  8. Takada, T. et al. Selective production of transgenic mice using green fluorescent protein as a marker. Nat. Biotechnol. 15, 458–461 (1997).

    CAS  Article  Google Scholar 

  9. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. & Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    CAS  Article  Google Scholar 

  10. Bavister, B. Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update 1, 91–148 (1995).

    CAS  Article  Google Scholar 

  11. Daniel, J.C. Cleavage of mammalian ova inhibited by visible light. Nature 201, 316–317 (1964).

    Article  Google Scholar 

  12. Hirao, Y. & Yanagimachi, R. Detrimental effect of visible light on meiosis of mammalian eggs in vitro. J. Exp. Zool. 206, 365–369 (1978).

    CAS  Article  Google Scholar 

  13. Hegele-Hartung, C., Schumacher, A. & Fischer, B. Effects of visible light and room temperature on the ultrastructure of preimplantation rabbit embryos: a time course study. Anat. Embryol. (Berl). 183, 559–571 (1991).

    CAS  PubMed  Google Scholar 

  14. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  Article  Google Scholar 

  15. Xu, C., Zipfel, W., Shear, J.B., Williams, R.M. & Webb, W.W. Multiphoton fluorescence excitation - new spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

    CAS  Article  Google Scholar 

  16. Wokosin, D. et al. All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging. Institute of Electrical and Electronics Engineering Journal of Selected Topics in Quantum Electronics. 2, 1051–1065 (1996).

    CAS  Article  Google Scholar 

  17. Konig, K., So, P.T.C., Mantulin, W. & Gratton, E. Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes. Optics Letters 22, 135–136 (1997).

    CAS  Article  Google Scholar 

  18. Williams, R.M., Piston, D.W. & Webb, W.W. Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. Faseb J. 8, 804–813 (1994).

    CAS  Article  Google Scholar 

  19. Konig, K., Simon, U. & Halbhuber, K.J. 3D resolved two photon fluorescence microscopy of living cells using a modified confocal laser scanning microscope. Cell. Mol. Biol. 42, 1181–1194 (1996).

    CAS  PubMed  Google Scholar 

  20. Barnett, D.K., Clayton, M.K., Kimura, J. & Bavister, B.D. Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol. Reprod. Dev. 48, 227–237 (1997).

    CAS  Article  Google Scholar 

  21. Wang, R.J. & Nixon, B.R. Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with "daylight" fluorescent light. In Vitro 14, 715–722 (1978).

    CAS  Article  Google Scholar 

  22. Konig, K.K. et al. Cell damage by UVA radiation of a mercury microscopy lamp probed by autofluroescence modifications, cloning assay, and comet assay. J. Biomed. Optics 1, 217–222 (1996).

    Article  Google Scholar 

  23. Hockberger, P.E. et al. Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. Proc. Natl. Acad. Sci. USA 96, 6255–6260 (1999).

    CAS  Article  Google Scholar 

  24. Nasr-Esfahani, M.H., Aitken, J.R. & Johnson, M.H. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development 109, 501–507 (1990).

    CAS  PubMed  Google Scholar 

  25. Nasr-Esfahani, M.M. & Johnson, M.H. The origin of reactive oxygen species in mouse embryos cultured in vitro. Development 113, 551–560 (1991).

    CAS  PubMed  Google Scholar 

  26. Nakayama, T., Noda, Y., Goto, Y. & Mori, T. Effects of visible light and other environmental faxtors on the production of oxygen radicals by hamster embryos. Theriogeneology. 41, 499–510 (1994).

    CAS  Article  Google Scholar 

  27. Rothe, G. & Valet, G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescein. J. Leukoc. Biol. 47, 440–448 (1990).

    CAS  Article  Google Scholar 

  28. LeBel, C.P., Ischiropoulos, H. & Bondy, S.C. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5, 227–231 (1992).

    CAS  Article  Google Scholar 

  29. Zhu, H., Bannenberg, G.L., Moldeus, P. & Shertzer, H.G. Oxidation pathways for the intracellular probe 2',7'-dichlorofluorescein. Arch. Toxicol. 68, 582–587 (1994).

    CAS  Article  Google Scholar 

  30. Haugland, R.P. Detecting enzymatic activity in cells using fluorogenic substrates. Biotechnics and Histochemistry. 70, 243–251 (1995).

    CAS  Article  Google Scholar 

  31. Hockberger, P.E. et al. in Optical diagnostics of living cells and biofluids, SPIE International Society for Optical Engineering, (eds Asakura, T., Farkas, D.L., Lief, R.C., Priezzhev, A.V. & Tromberg, B.J.) 129–140, Bellingham, WA, (1996).

    Book  Google Scholar 

  32. Aubin, J.E. Autofluorescence of viable cultured mammalian cells. J. Histochem. Cytochem. 27, 36–43 (1979).

    CAS  Article  Google Scholar 

  33. Cathcart, R., Schwiers, E. & Ames, B.N. Detection of picomole levels of lipid hydroperoxides using a dichlorofluorescein fluorescent assay. Methods Enzymol. 105, 352–358 (1984).

    CAS  Article  Google Scholar 

  34. Mohler, W. & Squirrell, J.M. in Imaging nerurons: a laboratory manual (eds Yuste, R., Lanni, K. & Konnerth, A.) (Cold Spring Harbor Press, Cold Spring Harbor, NY, in the press).

  35. Bavister, B.D. A minichamber device for maintaining a constant carbon dioxide in air atmosphere during prolonged culture of cells on the stage of an inverted microscope. In Vitro Cellular and Devolpment Biology. 24, 759–763 (1988).

    CAS  Article  Google Scholar 

  36. McKiernan, S. & Bavister, B. Pantothenate stimulates blastocyst formation in cultured one-cell hamster embryos. Theriogeneolgy. 49, 209 (1998).

    Article  Google Scholar 

  37. Wokosin, D.L. & White, J.G. in Three-dimensional microscopy: image acquisition and processing, SPIE International Socity for Optical Engineering (eds Cogswell, C.J., Conchello, J.-A. & Wilson, T.) 24–29, Bellingham, WA; 1997).

    Google Scholar 

  38. Ludwig, T.E., Lane, M. & Bavister, B.D. Increased fetal development after transfer of hamster embryos cultured with glucose. Biol. Reprod. 58, 167 (1998).

    Article  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Tenneille Ludwig for performing the embryo transfers, Kevin Eliceiri for technical assistance, Dr. Philip Hockberger for assistance with the peroxide study, and Drs. Jay Baltz, Victoria Centonze-Frohlich, Philip Hockberger, Keith Latham, Gary Lyons, Randall Prather, and Mark Westhusin for their comments on the manuscript. This work was supported by the NICHD National Cooperative Program on Non-Human In Vitro Preimplantation Embryo Development through grant HD22023 to BDB and the NIH grant RR00570 to the I.M.R.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jayne M. Squirrell.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Squirrell, J., Wokosin, D., White, J. et al. Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat Biotechnol 17, 763–767 (1999). https://doi.org/10.1038/11698

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/11698

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing