Thank you to the laboratory of Rick Horwitz at the University of Virginia for providing the stable CHO-K1 cells expressing paxillin-EGFP. without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The Fosdagrocorat assays designed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity. Live cell imaging has become common practice across the physical, life and health sciences. In light of this, many high quality Fosdagrocorat reviews, procedures and protocols for live cell imaging have been published1,2,3,4,5,6,7,8,9,10. Fluorescent protein fusions and cellular markers are required to follow fundamental biological processes, visualize whole cells and/or proteins of interest. The very nature of the photo-physical process in the excitation of a fluorophore and emission of fluorescent light often leads to the secondary effects of photo-bleaching and photo-toxicity. However, a recent editorial piece highlighted how photo-toxicity has essentially been ignored by most researchers11. In fact, Carlton and are the relative amplitudes of each decay component and are the decay rates for each component. For easier comparison between experiments performed with different laser powers or different laser lines (e.g. 473?nm vs 488?nm), photo-bleaching rates were expressed in terms of the number of images collected and were normalized to the continuous illumination dataset within each experiment. For comparison between different lasers and different experimental settings the laser powers used for the different experiments are SPERT shown in Table 2. For the line scan experiments all of the decay curves were fit with high R squared values (R2? ?0.99). Fit values for rate constants were very reproducible with low standard deviations of 1C7% between experiments and ROIs. The offset (yo), or amount of fluorescence intensity that was not photo-bleached at the end of the experiment, was found to increase as the pixel dwell time decreased with 5C10% unbleached with pixel dwell time of 3C13?s and 15% unbleached with pixel dwell occasions of 0.8C1.6?s Mitochrondrial Morphology and TetraMethyl Rhodamine Methyl Ester (TMRM) CHO-K1 cells expressing paxillin-EGFP were stained with MitoTracker Red CMXRos (ThermoFisher Scientific, M-7512) using the manufacturers Fosdagrocorat protocol. Cells were exposed to 488?nm laser light for 100 continuous scans at 20% laser power. Exposure was conducted with one single slow line scan or 16 rapid line scans that were averaged. Mitochondrial morphology was imaged in 3D. The z-stack of images of the MitoTracker stain was collected with 1% laser power from a 2?mW-543?nm laser line. Approximately 20 images at 0.36?m apart were collected for each cell. Mitochondrial membrane potential was imaged using TMRM staining. TMRM is usually a cell-permeant, cationic, Fosdagrocorat red-orange fluorescent dye that is readily sequestered by active mitochondria. TMRM accumulates in the inner membrane of mitochondria in healthy cells, and is released into the cell cytosol when the membrane potential depolarizes during apoptosis28. Therefore, high TMRM is an indication of cell health and cell stress results in a decrease in TMRM intensity. TMRM experimental conditions were validated with 2?M carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; Sigma Aldrich, Milwaukee, WI) to depolarize cells and decrease TMRM staining or 2?g/mL oligomycin (Sigma Aldrich, Milwaukee, WI) to hyperpolarize the mitochondria and increase TMRM staining. TMRM solid powder (ThermoFisher Scientific, T-668) was dissolved in DMSO to make a stock answer of 5?mg/mL. The TMRM stock answer was then diluted to 20?M with complete DMEM cell culture media and applied to paxillin-EGFP expressing CHO-K1 cells in a 35?mm glass bottom dish (prepared as described above) at 37?C and allowed to adhere overnight. Cells were then washed two times with DMEM and left in 2?mL of fresh DMEM for live cell imaging. Cells were exposed to 488?nm laser light for 100 continuous scans at either 1% or 20% power. Exposure was conducted with one single slow line scan or 16 rapid line scans that were averaged. Both before and following exposure to 488?nm light 3D image-stacks of the TMRM staining were collected. Images of the TMRM were generated with 3% power from a 2?mW-543?nm laser.