A computerized method for determination of microvascular density

Currently accepted stereological methods for vascular density measurements involve manual counting of labeled vessels with a grid image overlay and determination of vessel-grid intersections. This method both is tedious and may be prone to error; therefore, our laboratory has developed a method for computer-automated determination of microvascular density using digital image processing techniques. An image of the microvasculature is acquired using computer videomicroscopy. The image is processed in three general steps, involving (1) background correction, (2) thresholding of the gray level image to create a binary image, and (3) processing of the binary image using erosion, dilation, and skeletonization algorithms. Testing of this procedure was performed on 328 typical images of skeletal muscle tissue sections taken from the hindlimb of Sprague-Dawley rats and quantitated by both traditional measures and our new computer method. Results from this comparison reveal that the automated vessel counting is highly correlated (r2 = 0.71) and decreased analysis time from 15 min/image to 30 sec/image when compared with manual counting methods. Our computer-based method also appears to be superior to the traditional method due to the unbiased and nonsubjective nature of determining vessel-grid intersections.