The purpose of this study was to develop a more efficient fluorescent microsphere method to facilitate the rapid use of the histological technique and to enable its use in large tissue regions. Using fluorescent plate/slide imaging technology and automated detection and analysis software, we were able to rapidly image, detect, and count 3 separate microsphere colors in 200 microm thick tissue sections from piglet brain. In resting newborn piglets (n=6) on isoflurane anesthesia, we measured a median total cerebral blood flow (CBF) of 105 ml/min/100g (range 27-206 ml/min/100 g). Compared with other FM analysis methods, our method reduces the time required to determine blood flow, improves accuracy in lipid-rich tissues and large tissue regions and, unlike the radiolabeled microsphere method, can be combined with histological analysis.