Label-free photoacoustic imaging of glassfrog development

A primary focus of contemporary biology is to understand how internal molecules influence natural development. Many amphibians serve as highly effective model organisms for this research due to their rapid growth rates and transparent tissues, which facilitate high-resolution imaging. In our research, we utilized two complementary photoacoustic microscopy (PAM) configurations: hyperspectral PAM (HS-PAM) and ultrafast functional PAM (UFF-PAM). HS-PAM enabled us to achieve cellular-level resolution in vitro, while UFF-PAM allowed us to capture hemodynamic changes of adult specimens in vivo. We monitored the morphological changes in glassfrogs from neurulation to the tadpole stage by detecting a variety of intrinsic contrasts, including DNA/RNA, yolk proteins, lipids, hemoglobin, and melanin. The PAM images provided detailed depictions of anatomical development. To further explore the versatility of these systems, we also imaged tissue structures within the skeletal muscle, liver, and fat tissue of other treefrog species. Additionally, we monitored blood flow dynamics in two species of glassfrogs under both awake and under anesthesia. Overall, our findings demonstrate that PAM is a powerful and versatile method, that can be coupled with different species of amphibians to inform applications in developmental biology.