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