Injection of Microbubbles into Isolated Mouse Embryos: A Technique to Deliver Microbubble Contrast Agents into the Vasculature of Living Murine Embryos

For microbubble delivery, begin with a uniform suspension of endothelial cell-targeted lipid-shelled microbubbles comprising a gaseous core in a syringe connected to the tubing.

Load this assembly into a syringe pump and attach a glass needle to the tubing. Now, transfer a chilled mouse embryo expressing specific endothelial cell receptors into a dish placed on the stage of a stereoscope.

Gently exteriorize the embryo from the yolk sac and amniotic membrane. Secure the sac and the placental edges. Rinse the embryo with a warm buffer to restore its blood flow. Cover the embryo with pre-warmed ultrasound gel for optimal ultrasound wave transmission. Supplement with buffer to maintain its physiological conditions.

Carefully inject the microbubble suspension at the desired flow rate into a suitable vein on the fetal membrane surface of the placental disc using the syringe pump. The injected microbubbles travel through the circulation and bind to the specific receptors on the endothelial cells lining the blood vessels.

Use an ultrasound transducer to transmit high-frequency ultrasound waves to the embryo. The microbubbles' highly compressible gas cores cause them to oscillate nonlinearly, scattering ultrasound waves with frequencies different from the incident waves. These signals and the tissue's linear signals return to the transducer.

The transducer relays them to an imaging system that differentiates the microbubble scattered signals from the tissue signals, producing contrast-enhanced ultrasound images of the embryonic blood vessels.