Mitochondria are fundamental and tiny structures (0,5 – 3 μm) of a cell whose mission is to assure, in part, the production of ATP for the well-functioning of our body. If mitochondria are altered, there is a burning issue because of this vital function that mitochondria provide. This could lead to multi-organ failure, and the patient’s life can even be compromised. These are called mitochondrial diseases. Their diagnosis is difficult and time-consuming. Therefore, we explored the capability of an optical microscope to detect differences in nano motions when extracted mitochondria and whole-cell fibroblasts are exposed to various stimuli. We have selected chemical stimuli that down-regulate the function of these mitochondria, either by inhibition in the pathway of the electron transport chain (case of rotenone) or by gradually exposing them to fatty acid (propionate) at a supra-physiological concentration that is noxious. The optical microscope has revealed itself to be efficient for demarcating average displacements performed by mitochondria exposed or not to rotenone and propionate. However, concerning the experiment where average displacements from healthy and Leigh Syndrome (LS) fibroblasts (prototype of a mitochondrial disease) exposed to increasing concentrations of propionate were compared, it seems to be more delicate. There was hardly any difference in the average displacements between those two groups. To conclude, optical microscopes could be an exciting alternative to conventional atomic force microscopes (AFMs) method for the detection of nano movements when manipulating extracted mitochondria.