Combined Optoacoustic and high-frequency ultrasound imaging of live mouse embryos

The cell differentiation and proliferation of the central nervous system (CNS) are closely related to vascular development. An imaging protocol that integrated optoacoustics (OA) with high-frequency ultrasound (HFU) was developed for in vivo imaging of brain ventricles and vasculature in mouse embryos. A 40-MHz, co-polymer, 5-element annular-array transducer with a geometric focus of 12 mm was modified to accommodate free-beam, coaxial illumination. Three-dimensional (3-D) data sets were acquired by raster scanning the transducer-optics assembly in 50-mu m increments. A single intact conceptus from an anesthetized mouse was surgically exposed into PBS-filled Petri-dish. An 800-mu m spot illumination from a pulsed, 532-nm, Nd-YAG laser was synchronized with a high-voltage impulse excitation of the central array element to facilitate simultaneous and spatially co-registered OA and HFU data acquisition. The resulting OA and HFU signals from each scan location were recorded on all five array channels and post-processed using a synthetic-focusing algorithm to enhance the depth of field (DOF). Dual-modality images were acquired from mouse embryos at E11.5, E12.5, and E13.5 days of gestation. The extended DOF allowed morphologically accurate visualization of the embryonic head. The brain ventricles were segmented from the HFU data and rendered in 3-D. The OA data provided visualization of the vascular plexus as well as individual blood vessels. Feasibility of spatially co-registered, low-cost dual-modality in vivo imaging of mouse embryos was demonstrated.