**4. Discussion**

As a model biological system, the zebrafish possesses numerous advantages: rapid embryonic development, fully sequenced genome, low cost, etc. This organism also has a unique collection of features at pre-larval developmental stages: large size, optical transparency of the embryo's interior and relatively slow embryo movements inside the shell. This makes zebrafish an attractive model forin vivo study of the formation and functioning of its cardiovascular system using optical imaging techniques.

In this study, we have demonstrated that time-lapse bright-field microscopy and digital signal processing allow blood vessel imaging of a living embryo as well as heartbeat and blood flow velocity measurements without any anesthesia. In contrast to straightforward approaches based on image subtraction, the described pre-processing procedure enables the compensation of shifts and rotations between images and their pixel-to-pixel matching necessary for accurate quantitative characterization of cardiac activity.

The proposed algorithm is applicable for processing the images obtained by various microscope setups widely used for in vivo studies of zebrafish embryos. It can complement optical coherence tomography, acoustic microscopy and other imaging techniques by adding cardiac activity mapping capability.
