Fig. 1. The basic principle of digital motion analysis and measurement of blood cell concentration. (A) Blood cells moving through a vessel can easily be detected by their motion. (B) A difference image obtained by subtracting the two fields of one video frame (the odd and the even frames), showing several moving erythrocytes. Insert: enlargement of one erythrocyte image showing the direction of movement (arrow) (C,D) A schematic drawing showing moving erythrocytes and the subsequent summation of these differences, ending up with a complete cast of the vasculature. In the region of interest all diameters along the vessel were measured in 0.3 µm steps. The volume of a single erythrocyte cross section is calculated as: r²0.3 (µm³). The sum of all these sub-volumes along the vessel (white and black lines in D) gives in the vascular volume. The number of detected cells in B divided by this volume gives the red blood cell concentration in the area of interest (green box). See Materials and methods for further explanation.