Ate mitochondrial contribution to disease and to perform drug toxicity and efficacy screening.Live IMAGING OF CELL-BASED READOUTS TO MEASURE MITOCHONDRIAL FUNCTIONSVarious experimental methodologies quantify mitochondrial dysfunction by focusing on activity measurements of distinct mitochondrial enzymes and/or pathways following tissue/cells homogenization and/or utilizing isolated mitochondria (Picard et al., 2011). By contrast, live-cell microscopy assays have the advantage to visualize and quantify functional and structural (sub)cellular (spatial dimension) elements in situ in living cells. Furthermore, microscopy uniquely permits for simultaneous time-lapse monitoring (temporal dimension) and (semi)quantitative measurements of various parameters by multispectral imaging (spectral dimension). In specific, developments in fluorescent reporter technologies tremendously boosted the use of light microscopy for cell biology research (Sbalzarini, 2016). A limitation of fluorescent microscopy is the possible induction of phototoxic tension, which may be caused by illumination in the reporter molecules. Furthermore, fluorophores themselves can perturb the physiological function of biomolecules and are subjected to photobleaching. Additionally, because of calibration limitations, quantification of cellular parameters working with single wavelength dyes is usually difficult and, in some cases, only relative and qualitative measurements are possible. The application of ratiometric dyes, when achievable, requires care of variable dye loading and extrusion responding using a (semi)quantitative transform in fluorescence upon target binding. A drawback of the ratiometric dyes is related to their portability to high-throughput exactly where doubling information dimension can produce acquisition, storage and processing difficulties. Implementing ratiometric dyes in multispectral assays can be also inconvenient as a result of the wavelength limitation. When mitochondrial contribution to illness is evaluated in living cells, we contemplate mitochondrial morphology and membrane possible, ROS, ATP and mitochondrial respiration vital indicators of mitochondrial well being status. Their compatibility with fluorescence microscopy assays is going to be presented in the next paragraphs and is summarized in Table 1.their spatiotemporal dynamics (Koopman et al., 2008). Various lipophilic cell-permeant, cationic and fluorescent molecules have already been presented, which diffuse across the plasma membrane of the cell and accumulate in the mitochondrial matrix inside a dependent manner. These molecules incorporate Aegeline Purity & Documentation tetramethylrhodamine methyl ester (TMRM), tetramethylrhodamine ethyl (TMRE) ester, rhodamine 123, DiOC6(three) (3,three – dihexyloxacarbocyanine iodide), JC-1 (5,5 ,six,6 tetrachloro-1,1 ,3,three -tetraethylbenzimidazolylcarbocyanine iodide), and also the MitoTracker family members. Amongst these molecules, TMRM was described to become the least toxic, the fastest in equilibrating across membranes, and displaying the lowest non-specific localization (Nicholls, 2012; Zorova et al., 2018). For that reason in our study we generally use TMRM to simultaneous analyze mitochondrial morphology and known as mitochondrial morphofunction (Koopman et al., 2008; Iannetti et al., 2016). The cell types, staining, imaging conditions and descriptors applicable for the evaluation of mitochondrial morphofunction have been previously reviewed (Iannetti et al., 2015; Zorova et al., 2018) and are summarized in our current study (Iannetti et al., 2016). To technically validate measurement.