Motion-Based Structure Separation for Label-Free High-Speed 3-D Cardiac Microscopy

Capturing the dynamics of individual structures in the embryonic heart is an essential step for studying its function and development. Label-free brightfield microscopy allows for higher acquisition frame-rates than techniques requiring molecular labeling, without interfering with embryo viability or needing complex equipment. However, since different structures contribute similarly to image contrast, label-free microscopy lacks specificity. Here, we mitigate this problem by separating a single-channel image series into multiple channels specific to different cardio-vascular structures, based only on their motion patterns. The technique combines images from multiple cardiac cycles and z-sections after nonuniform temporal registration to produce 3-D+time image volumes of one full cardiac cycle with separate channels for static, transient and periodically moving structures. The resulting data is well suited for velocity analysis and 3-D-visualization. We characterize the separating capabilities of our technique on a synthetic cardiac dataset and demonstrate its practical applicability, by reconstructing three-channel views of the beating embryonic zebrafish heart with an effective frame rate of 1000 volumes (256 × 256 × 20 voxels each) per second. This technique enables quantitative characterization of dynamic heart function during cardiogenesis.