Determination of red blood cell velocity by video shuttering and image analysis

A novel modification of conventional video imaging techniques has been developed to determine the velocity of red blood cells (RBCs), which offers compatibility with existing video-based methods for determining blood oxygenation and hemoglobin concentration. Traditional frame-by-frame analysis of video recordings limits the maximum velocity that can be measured for individual cells in vivo to about 2 mm/s. We have extended this range to about 20 mm/s, by electronic shuttering of an intensified charge-coupled device camera to produce multiple images of a single RBC in the same video frame. RBCs were labeled with fluorescein isothiocyanate and the labeled cells (FRBCs) were used as probes to determine RBC velocities in microvessels of the hamster retractor muscle. Velocity was computed as the product of the distance between centroids of two consecutive image positions of a FRBC and the shuttering frequency of the camera intensifier. In vitro calibrations of the system using FRBC and Sephadex beads coated onto a rotating disk yielded an average coefficient of variation of about 6%. Flow conservation studies at bifurcations indicated that the maximum diameter of microvessels below which all the FRBCs in the lumen could be detected was 50 microm. The technique was used to estimate mean-FRBC velocity distributions in vessels with diameters ranging from 8 to 50 microm. The mean-FRBC velocity profiles were found to be blunter than would be expected for Poiseuille flow. Single FRBCs tracked along an unbranched arteriole exhibited significant temporal variations in velocity.