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Gridding cDNA microarray images is a critical step in gene expression analysis, since any errors in this stage are propagated in future steps in the analysis. We propose a fully automatic approach to detect the locations of the spots. The approach first detects and corrects rotations in the sub-grids by an affine transformation, followed by a polynomial-time optimal multi-level thresholding algorithm that finds the positions of the spots. Additionally, a new validity index is proposed in order to find the correct number of spots in each sub-grid, followed by a refinement procedure used to improve the performance of the method. Extensive experiments on real-life microarray images show that the proposed method performs these tasks automatically and with very high accuracy.