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

A major challenge for fluorescence imaging of living mammalian cells is mai ntaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster em bryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm ) while maintaining blastocyst, and even fetal, developmental competence. I n contrast, confocal imaging for only 8 h inhibits development, even withou t fluorophore excitation, Photo-induced production of H2O2 may account, in part, for this inhibition, Thus, two-photon microscopy, but not confocal mi croscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens s uch as mammalian embryos.