Non-invasive laser speckle contrast imaging (LSCI) of extra-embryonic blood vessels in intact avian eggs at early developmental stages

Imaging blood vessels in early-stage avian embryos has a wide range of practical applications for developmental biology studies, drug and vaccine testing, and early sex determination. Optical imaging, such as brightfield transmission imaging, offers a compelling solution due to its safe non-ionizing radiation, and operational benefits. However, it comes with challenges, such as eggshell opacity and light scattering. To address these, we have revisited an approach based on laser speckle contrast imaging (LSCI) and demonstrated a high-quality, comprehensive, and non-invasive visualization of blood vessels in few-days-old chicken eggs, with blood vessels as small as 100 µm in diameter (with LSCI profile full-width-at-half-maximum of 275 µm). We present its non-invasive use for monitoring blood flow, measuring the embryo’s heartbeat, and determining the embryo’s developmental stages using machine learning with 85% accuracy from stage HH15 to HH22. This method can potentially be used for non-invasive longitudinal studies of cardiovascular development and angiogenesis, as well as egg screening for the poultry industry.