Multiscale 3D imaging of the embryonic heart

Biological systems, especially developing organs like the embryonic heart, are complex because their dynamic responses to environmental factors lead to significant anatomical and functional changes in the tissue. In this study, we introduce a multimodal imaging approach to visualize genetic expression, hemodynamics, and electrical activity in the heart in three dimensions. Our workflow starts with optical mapping using voltage-sensitive dyes to assess action potential propagation and conduction velocity (CV). We acquire a 3D volume of pre-fixation heart with Spectral-Domain OCT (SD-OCT), which serves as a reference for registering optical mapping and gene expression patterns. The fixed heart is then stained with Fluorescence In Situ Hybridization (FISH) probes and optically cleared for sequential fluorescence imaging. Through four rounds of imaging, we achieve high-resolution 3D mapping of 12 genes, including GJA5 (associated with fast conduction velocity) and HAS2 (related to cardiac cushion formation). This integrated imaging technique shows potential for comprehensive analysis of embryonic hearts by correlating gene expression patterns with functional maps of CV.