A clonogenic assay is a biological technique for calculating the Surviving Fraction (SF) that quantifies the anti-proliferative effect of treatments on cell cultures: this evaluation is often performed via manual counting of cell colony-forming units. Unfortunately, this procedure is error-prone and strongly affected by operator dependence. Besides, conventional assessment does not deal with the colony size, which is generally correlated with the delivered radiation dose or administered

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... c agent. Relying upon the direct proportional relationship between the Area Covered by Colony (ACC) and the colony count and size, along with the growth rate, we propose MF2C3, a novel computational method leveraging spatial Fuzzy C-Means clustering on multiple local features (i.e., entropy and standard deviation extracted from the input color images acquired by a general-purpose flat-bed scanner) for ACC-based SF quantification, by considering only the covering percentage. To evaluate the accuracy of the proposed fully automatic approach, we compared the SFs obtained by MF2C3 against the conventional counting procedure on four different cell lines. The achieved results revealed a high correlation with the ground-truth measurements based on colony counting, by outperforming our previously validated method using local thresholding on L*u*v* color well images. In conclusion, the proposed multi-feature approach, which inherently leverages the concept of symmetry in the pixel local distributions, might be reliably used in biological studies.