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.

Figure 1

Viability of embryos after long-term imaging. 514 nm LSCM (A and B) or 1,047 nm TPLSM (E and F) single optical sections of Mitotracker-labeled embryos at the start (A and E) and the end (B and F) (t = 8 h in B and t = 24 h in F) of an imaging sequence. Nomarski images of the TPLSM-imaged embryos (C and G) and their nonimaged stage controls (D and H) at the time of expected blastocoel formation (82 h PEA). (I) Graph depicts the percentage of embryos per replicate that developed to morulae and blastocysts following imaging under various conditions. Values above columns represent number of embryos, and each pair of columns represents at least three replicates. Imaging period is the total time over which the embryos were imaged. Embryos were either stained (+) or not stained (−) with mitochondrial label. Imaging frequency indicates time interval between z-series collection in minutes. Error bars represent one standard deviation from the mean of the replicates. Scale bar = 45 µm.

Figure 2

Production of reactive oxygen in imaged embryos. Two photon images of two-cell embryos (A–E) before and (F–J) following irradiation with: (F–H) 450–490 nm epifluorescence (5 s irradiation); (I) 1,047 nm TPLSM (every 15 min for 8 h); or (J) 514 nm LSCM (every 15 min for 8 h). All embryos, except (C) and (H), were labeled with DHF. Embryo in (B) and (G) was treated with catalase before staining and irradiation. (K) Quantitation of change in fluorescence intensity. Bars indicate the mean post-irradiation fluorescence of blastomeres relative to each blastomere’s fluorescence before irradiation. Values above bars are numbers of blastomeres analyzed. Error bars represent one standard deviation of the mean blastomere intensity value. Scale bar = 45 µm.

Figure 3

Fetal development following long-term imaging. Two photon images of mitochondria-labeled embryos at (A) the initiation of imaging (B) after 8.5 h of imaging, showing a mitotic spindle (arrow), and (C) the completion of a 24 h imaging sequence. (D) After imaging, embryos were cultured in the incubator until 82 h PEA (Nomarski) at which point they were transferred to a recipient female. (E) A black-eyed fetus that developed from one of these imaged embryos is shown next to an albino uterine mate. Scale bar for (A–D) = 45 µm. Scale bar for (E) = 4.75 mm.