Deep learning has emerged as a breakthrough tool for the segmentation of images without supporting human experts. Here, we propose an automated approach that uses deep learning to generate pseudo-nuclear staining of cells from phase contrast images. Our proposed approach, which has the feature to generate pseudo-nuclear stained images by simple DNN, showed remarkable advantages over existing approaches in the cell-detection and the detection of the relative position of cells for various cell densities, as well as in counting the exact cell numbers. Pseudo-nuclear staining of cells by deep learning will improve the accuracy of automated cell counting in a label-free cellular population on phase contrast images. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.