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Non-invasive imaging of blood cell concentration and blood distribution in zebrafish Danio rerio incubated in hypoxic conditions in vivo

Thorsten Schwerte, Dietmar Überbacher and Bernd Pelster

Institute for Zoology and Limnology, University of Innsbruck, Austria

View larger version (28K): [in a new window]   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.

View larger version (47K): [in a new window]   Fig. 2. Method for determination of red blood cell concentration. (A) Vascular cast of a 4 d.p.f. zebrafish larva obtained by digital vascular contrasting method. (B) Edge detection using colour thresholding method. The frame in the tail indicates the region of interest (ROI). (C) Close up of the ROI. White circles indicate motion detected single blood cells. The vascular volume in the ROI is calculated from the geometric data of the vessel shown in B.

View larger version (14K): [in a new window]   Fig. 3. Developmental changes in red blood cell concentration in zebrafish under chronic hypoxic and normoxic conditions. Values are means ± S.E.M., N=15; *significant difference (P<0.05). 1 mmHg = 1.333 Pa.

View larger version (111K): [in a new window]   Fig. 4. Typical changes in red blood cell perfusion in zebrafish under chronic hypoxic (A,C) and normoxic (B,D) conditions. Red blood cells were counted per minute at every position of the tissue, and represented in colour according to the calibration bar on the right. d.p.f., days post fertilization; a, anastomosis; an, anus; b, brain; da, dorsal artery; dv, dorsal vein; e, eye; g, gut; h, heart; iv, intersegmental vessel; sb, swimbladder.

View larger version (18K): [in a new window]   Fig. 5. Blood perfusion changes in zebrafish tissues under chronic hypoxic and normoxic conditions. Values are means ± S.E.M., N=15; *significant difference (P<0.05).