Application of Higher Harmonic Generation Microscopy in Assisted reproductive technologies

It is known that the quality of the mammal oocytes and embryos greatly influence the outcomes of assisted reproductive technologies. Noninvasive imaging tools with high 3D resolution are thus needed to provide as much information about the embryos as possible. In previous studies, the images of in vitro cultured mouse oocytes and embryos were acquired by the harmonic generation microscopy (HGM). Various sub-cellular structures of the whole mouse oocytes and embryos were identified. In our presentation, we will report our study on application of harmonic generation microscopy in assisted reproductive technologies. We use Cr:forsterite laser as the excitation source to obtain the HGM images of mouse oocytes and embryos. First of all, several safety tests are performed to ensure the proper exposure doses of the embryos to the laser. Second, we try to figure out what characteristics in the HGM images of the oocytes and embryos are possibly related to their quality. As to our experimental setup, the HGM signals are collected in both the forward and the backward direction by high N.A. objectives. With limited available working distances, the thickness of the chamber containing the embryos must be within 2 mm. A homemade sterile glass-cover-and-bottom culture dish is designed to meet the need. The dish, containing the embryos, is then placed in a CO2 stage micro-incubator for HGM observation. Those embryos are later transferred to female mice for pregnancy. The viability of the mouse embryos will be investigated to see if the harmonic generation microscopy can play a role in embryo-selection.