Tracing and ablation of single cells in the mammalian blastocyst using fluorescent DNA staining and multi-photon laser microscopy

Comparing the vital DNA dyes Hoechst33342 and DAPI in their ability to visualise cell nuclei of the late rabbit blastocyst, both dyes were found to be equally suited despite differences in staining intensity in embryonic versus extraembryonic tissues and in nuclear versus cytoplasmic domains at the subcellular level: Both dyes stain all nuclei of a given cell layer (e.g. epiblast or hypoblast) evenly and provide satisfactory fluorescence contrast throughout the blastocyst, while not interfering with normal development up to 10 h in vitro. Using short period (60-300 min) irradiation experiments with either dye, single-photon (405 nm) and multi-photon (800 nm) laser excitation was compared in different areas of the same embryo and parameters of multi-photon microscopy were defined for gentle live imaging and tracing of single cells deep to the surface of the embryo. In addition, individual cells were ablated by reducing the “area of interest” to sub-nucleus-size, thus maximally increasing the density of laser energy brought into the tissue. Thickening of epiblast and hypoblast and increased numbers of dense cytoplasmic inclusions within the limits of irradiated areas were found in semithin histological sections in a dose-dependent manner. Ablated cells were found in a necrotic state while neighbouring cells remained apparently unscathed.