Live and Dead Cells Counting from Microscopic Trypan Blue Staining Images using Thresholding and Morphological Operation Techniques

Su Mon Aung, Kanyanatt Kanokwiroon, Tonghathai Phairatana, Surapong Chatpun


Cell counting is a required procedure in biomedical experiments and drug testing. Manual cell counting performed with a hemocytometer is time consuming and individual dependence. This study reportedthe development of a computer-assisted program for trypan blue stained-cell counting using digital image analysis. Images of trypan blue-stained breast cancer cells line were obtained by a microscope with a digital camera. Undesired noise and debris were removed by applying a guided image filter. Color space HSV (Hue, Saturation and Value)conversion and grayscale conversion were performed for distinguishing between live and dead cellsImage thresholding and morphological operators were applied for image segmentation. Live and dead cells were counted after image segmentation and the results were compared with manual counting by three well-experienced counters. The computer-assisted cell counting from thirty-six trypan blue-stained microscopic images had a high correlation coefficient with the live cell results of the experts (r=0.99). The correlation coefficient of the number of dead cells comparing the computer-assisted count and the expertscount was 0.74. Our approach offers high accuracy (>85%)on counting live cells compared with the expertscounting. This automated cell counting approach can assist biomedical researchers for both live and dead cells counting.


Cell counting; Guide image filter; Image thresholding; Morphological operator; Trypan blue

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ISSN 2088-8708, e-ISSN 2722-2578