Evaluating Preimplantation Embryo Metabolism Through Imaging of Cellular Autofluorescence

Optical imaging offers a non-invasive approach for assessing embryo viability. This may be achieved through capturing fluorescence from endogenous fluorophores that are associated with cellular metabolism. By recording and analysing natural autofluorescence emitted by molecules involved in metabolism, such as NADH and FAD, this technique offers crucial insights into the metabolic state of embryos, in the absence of exogenous labels. This approach is particularly valuable for preimplantation embryos developed in vitro, where the ability to identify those with high developmental potential may lead to improved outcomes in a clinical setting. Light sheet microscopy has recently emerged as a powerful imaging modality suitable for recording autofluorescence from the developing embryo. In contrast to standard point-scanning (confocal) approaches, it only illuminates the plane of interest, minimising light exposure and photodamage while enabling rapid 3D image acquisition. These aspects make light sheet microscopy particularly well-suited for metabolic imaging of live preimplantation embryos. In this chapter, we describe a general procedure to perform metabolic imaging of live mammalian embryos using light sheet microscopy. This protocol enables direct observation of metabolic changes within embryos in a spatial manner and provides guidelines for optimising imaging parameters to ensure accuracy and reproducibility.