Digital motion analysis as a tool for analysing the shape and performance of the circulatory system in transparent animals

Abstract

The analysis of perfusion parameters using the frame-to-frame technique and the observation of small blood vessels in transparent animals using video microscopy can be tedious and very difficult because of the poor contrast of the images. Injection of a fluorescent probe (fluorescein isothiocynate, FITC) bound to a high-molecular-mass dextran improved the visibility of blood vessels, but the gray-scale histogram showed blurring at the edges of the vessels. Furthermore, injection of the fluorescent probe into the ventricle of small zebrafish (Danio rerio) embryos (body mass approximately 1 mg) often resulted in reduced cardiac activity. Digital motion analysis, however, proved to be a very effective tool for analysing the shape and performance of the circulatory system in transparent animals and tissues. By subtracting the two fields of a video frame (the odd and the even frame), any movement that occurred within the 20 ms necessary for the acquisition of one field could be visualised. The length of the shifting vector generated by this subtraction, represented a direct measure of the velocity of a moving particle, i.e. an erythrocyte in the vascular system. By accumulating shifting vectors generated from several consecutive video frames, a complete trace of the routes over which erythrocytes moved could be obtained. Thus, a cast of the vascular system, except for those tiny vessels that are not entered by erythrocytes, could be obtained. Because the gray-scale value of any given pixel or any given group of pixels increased with the number of erythrocytes passing it, digital motion analysis could also be used to visualise the distribution of blood cells in transparent tissues. This method was used to describe the development of the peripheral vascular system in zebrafish larvae up to 8 days post-fertilisation. At this stage, food intake resulted in a clear redistribution of blood between muscle tissue and the gut, and alpha-adrenergic control of peripheral blood flow was established.