Cardiac tissue and erythrocyte separation in bright-field microscopy images of the embryonic zebrafish heart for motion estimation

Bright-field (BF) microscopy enables imaging the beating embryonic zebrafish heart at high frame rates, thereby revealing motion of both tissues that form the heart and red blood cells (RBCs). However, single-channel BF images lack the specificity seen in multi-color fluorescence microscopy since all structures in the field of view contribute similarly to image contrast. We discuss an algorithm that overcomes this limitation by separating a BF sequence of the beating heart into two distinct image sequences: one showing only the heart and surrounding tissues and the other showing only the transient structures such as RBCs. These sequences can be analyzed separately to characterize heart wall and RBCs motion using common optical flow techniques (e.g. Lucas-Kanade method). We validate our technique on a synthetically generated image sequence and show its potential for facilitating quantitative characterization of heart function during cardiac morphogenesis by examining experimental BF images of the beating embryonic zebrafish heart.