Software (KEIO-IS2) for automatically tracking red blood cells (RBCs) with calculation of individual RBC velocities in single capillaries of rat brain

Abstract

Confocal fluorescence microscopy offers the potential to visualize cells at high resolution in living animals. However, studies of tissue microcirculation in vivo are limited by inability to follow individual red blood cells (RBCs) moving heterogeneously in space and time. Here we present our software (KEIO-IS2), which, in combination with high-speed (125-1000 sec-1 shutter speed) confocal fluorescent microscopy, can quantitatively treat RBC flow in single capillaries. Urethane-anesthetized Wistar rats (n=26) received intravenous injection of FITC-RBCs so that bright particles were observable flowing on a dark background with the confocal microscope (Seylaz et al. JCBFM 19:863,1999). Images were recorded directly to computer in uncompressed AVI file format. The dimensions of the measurement region were 250 micron 300 micron, with a layer thickness of ca. 20 micron, and the cortex could be scanned vertically down to 100 micron in depth. The recording period was usually 10 sec at a frame rate of 1/250 sec. The recorded movies were analyzed with MATLAB® and KEIO-IS2 software, which can track individual, rapidly moving RBCs on the acquired confocal images. The program exploits the light intensity difference between the dark background and the light RBCs in the visual field of the microscope to detect the RBCs automatically. It is possible to specify several parameters, such as the scale, the minimal RBC size and flow velocity, and the intensity threshold value, to customize the software. The algorithm for RBC tracking is as follows. First a gray level thresholding is applied to each frame to differentiate RBCs from background brightness, then all particles having at least 8 connected pixels are recognized as RBCs. Displacement of the particles in subsequent frames is analyzed, and the software builds up the tracks of the moving RBCs frame-by-frame and calculates the velocities of individual RBCs (displacement over frame interval).The results can be exported for further statistical analysis. Some results of RBC tracking through single capillaries are shown in Fig. 1a (RBC velocities ranging from 0.3–7.0 mm/s) and 1b (RBC trackings, see also M. Tomita et al. (Brain'05)). Fig. 2 shows an RBC shower in an artery of 100 micron sliced at 20 micron thickness longitudinally at its center. We could calculate intravascular velocity profiles and wall shear rates. This software would also be applicable to labeled platelets and white blood cells, providing a powerful tool for hemorrheological studies in the brain microvasculature.