The dataset consists of 810 microscopy images collected from CHO, U2OS, and RPE1 cell lines in fluorescent and brightfield modalities. Cell lines were grown in standard medium, seeded on plates, fixed with formaldehyde, stained with a fluorescent dye (Hoechst33342) and imaged with PerkinElmer Phenix high-throughput confocal microscope in fluorescence and brightfield modalities using a 20x objective. For the fluorescent modality, images were acquired in low-exposure (20 ms) and high-exposure (100 ms) modes. For the brightfield, we used a single exposure (100 ms). Higher exposure time usually translates into better image quality, alleviating Poisson noise, however, it leads to sample degradation, and lower measurement speed. In total, three wells on a plate were imaged, one well per cell line, each with nine fields of view. Each field of view was imaged across ten focal planes separated by 2 micrometers in z-stack, resulting in 270 microscopy images of size 2160 x 2160 pixels per exposure time per modality. We used the following folder structure: /{well}_{cell line}/{modality}_{exposure time}/fov_{field of view}_plane_{plane}.png We recommend to use the first two wells (CHO and U2OS, 180 images) as a training set, the first three fields of view from the last well (RPE1, 30 images) as a validation set, and the last six fields of view (RPE1, 60 images) as a test set.