Problems	Possible causes
Z stacks and Time lapse
Fragmented/clumped mitochondria	Cells are not healthy. pH or temperature shock Mineral oil touching the cells High amount of lipid based transfecting agents used Transient transfection of the mitochondrial marker Effect of over-expression of the protein Immensely high scanning laser power Use of high energy/low wavelength laser (413/405nm etc)
Immotile mitochondria	Cells are not healthy for any of the reasons mentioned above. High speed of imaging
Projection of Z stacks is blur	Oversampling along Z Wide pinhole High scanning speed Saturated signals Low power objective
FRAP
No recovery	Dead cells Fragmented mitochondria Immotile mitochondria Bleach box covers more than 30% of the total signal Inappropriately low pinhole size Bleaching caused by high laser power
Bleached zone is more than the bleach box	Misalignment of the laser head and bleach box Slow bleaching
Not enough bleaching	Low laser power chosen for bleaching For RFP, 488, 458and 514 nm lasers not chosen
Poor recovery curve	Very long time intervals set for monitoring recovery phase Signal saturated in the beginning Scanning laser causing bleaching Not enough bleaching by the bleaching laser(s) Bleached molecule reverting to the fluorescent stage Movement of mitochondria interfering
Microirradiation
No loss of TMRE after microirradiation	No irradiation due to application of lower laser power Misalignment of the 2 photon laser Low iterations chosen for irradiation Low zoom factor chosen
Mitochondria shifting away during imaging Or Mitochondria fragmenting	Cell is swelling due to more than optimum irradiation
Increase in TMRE signal after microirradiation	TMRE loading is beyond quenching levels
Staining for potential
No staining	Mitochondria are absolutely depolarized for cells not being healthy (as mentioned above)
Poor resolution	Overstaining Inappropriate pinhole size lack of proper washing after staining
The oligomycin/FCCP treated samples not producing expected results as controls	Overload of TMRE beyond the quenching levels FRET between Mitotracker green and TMRE Bleed through from detector channel of Mitotracker green to that of TMRE
Loss of TMRE while scanning	Power of the scanning laser power is more than optimum
Fragmented mitochondria	Stained sample left for more than 30-45 minutes