This is the second 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 testing of mtFociCounter (no primary control; rho0 control; manual cell segmentation), as described in the manuscript. Samples were processed and imaged in parallel, on the same date, in biological replicates (different date). The dataset contains the 3t3 WT data from the following acquisition dates: no primary antibody (no1) vs. with primary antibody against dsDNA (nucleoids): 20221022_no1 vs. wt 20221025_no1 vs. wt 20221029_no1 vs. wt for 3t3 cells depleted of mtDNA (rho0) vs. normal 3t3 cells with mtDNA (wt): 20221223: sample_1 & sample_2 rho0 vs. sample_3 wt for 3t3 WT cells, same raw-data, manually segmented single cells on two independent days: 20221022 -> segmentation on 20221102 vs. segmentation on 20221103 20221029 -> segmentation on 20221102 vs. segmentation on 20221104 For further details of the exact experimental procedure, please refer to the accompanying manuscript, which will soon be made available on BioRxiv.