59-05-032 Proceeding

45 Proceedings of the Princess Maha Chakri Sirindhorn Congress II. Materials and Methods A. Experimental setup Neutrophilswere separated fromwhole bloodusing amicrofluidic technology developed previously [5]. In this experiment, neutrophils were treatedwith tumor necrosis factor alpha (TNF- α ), phorbol myristate acetate (PMA) and glucose to induce inflammation and disease conditions. Both treated and healthy neutrophils were then pumped into E-selectin coated microfluidic channel under physiological shear conditions (~2 dyne/cm 2 ).Thewidth ofmicrofluidic channel is 400 µm and the length is 2 cm. B. Image acquisition Time-lapse images of rolling neutrophils were taken using an inverted microscope (Nikon Eclipse Ti-E) under phase contrast every 0.5 second for 30 seconds. C. Image processing With MATLAB software, every image was processed in the following procedure: 1) Microchannel boundary removal is an important procedure to remove computational burden. 2) Top hat filtering is applied in order to increase contrast between high intensity area (rolling neutrophils) and low intensity area (fast moving cells in other flow layers). 3) Image thresholding by histogram segmentation is used to remove low intensity regions (flowing cells in higher layer) in image. 4) Labeling cell in image by using 4 connected pixel to determine centroid of each cells. After centroids of cells in every frame are acquired, the two sets of centroids from two close frames werematched by using K-nearest neighbors. As a result, the distance each cell traveled can be calculated and subsequently velocity of each cell can be acquired. In this paper, only the cells that rolled on microchannel were analyzed. For some cells that started to flow away (high transient velocity) were taken in consideration only the past period when they moved slowly. Figure 1 Image taken from Nikon microscope.