This is the first dataset associated with the updated manuscript on mtFociCounter. Images are Spinning Disk Confocal Images of unsorted 3t3 NIH mouse fibroblasts stably expressing mitochondrially targeted dsRed, immunofluorescence against dsDNA (mitochondrial nucleoids) and AlexaFluor 647, and stained nuclei with Hoechst. It contains all data necessary to reproduce the analysis of 3t3 WT sampling (sample-to-sample comparison), as described in the manuscript. "samples" from the same day are 13mm coverslips processed in parallel (seeding of cells, fixation, immunofluorescence) and mounted on the same glass slide (#1 on left, #2 centre, #3 right). The dataset contains the 3t3 WT data from the following acquisition dates: 20220603_sample2 20220603_sample3 20220706_sample2 20220706_sample3 20220708_sample1 20220708_sample2 20220708_sample3 20221022_sample2 20221022_sample3 For details of the sampling procedure, please refer to the accompanying manuscript, which will soon be made available on BioRxiv.