Quantitative image-based cytometry (QIBC)

Automated multichannel wild-field microscopy for quantitative image-based cytometry was performed with the Olympus ScanR screening system equipped with an inverted motorized Olympus IX83 microscope, a motorized stage, IR-laser hardware autofocus, a fast emission filter wheel with single band emission filters, and a digital monochrome Hamamatsu ORCA-FLASH 4.0 V2 sCMOS camera (2048 × 2048 pixel, pixel size 6.5 μm x 6.5 μm, 12 bit dynamics) as previously described (Michelena et al., 2018). For each condition, a minimum of 1500 cells was acquired using the UPLSAPO 20x objective (NA 0.9). Images were taken under non-saturating conditions and identical settings were applied to all coverslips within the same experiment. Following acquisition, images were analyzed using the Olympus ScanR Image Analysis Software version 3.0.1. After a dynamic background correction was applied, image segmentation was performed based on the DAPI signal in order to identify cell nuclei as individual objects. Further, mitochondria were identified as associated objects using similar intensity-based segmentation based on ATP5a co-staining, within an area spaced minimally 1.6 μm and maximally 26 μm from the nuclear periphery. Mean fluorescence intensities within the nuclear or mitochondrial masks were quantified per cell and are displayed as cell population violin plots using GraphPad Prism 8.0. Untreated samples were arbitrarily set to 30 for visualization and normalization purposes and to allow statistical analyses across independent replicate experiments. Each QIBC-based ADP-ribosylation measurement was performed at least 3 times, and a representative graph from one experiment with ATP5a co-staining is shown. In addition, representative pictures, in which the individual channels have been adjusted for brightness and contrast to the same settings, were chosen to accompany the quantifications.