In this work we investigated the relative merits of conventional single-photon confocal laser scanning fluorescence microscopy (CLSM) and two-photon laser scanning fluorescence microscopy (2p-LSM) for the study of mitochondria in living neurons. Dorsal root ganglion neurons were loaded with the mitochondrion-specific fluorescent dye JC-1, the ratio between red (J-aggregates) and green (monomer) fluorescence of which reflects mitochondrial membrane potential. Cells were illuminated at 488 nm for single-photon excitation or at 870 nm for two-photon excitation. In both modalities we found that mitochondria showed: (i) similar appearance; (ii) similar fluorescence ratio values over both whole cell bodies and individual mitochondria; and (iii) similar responses to mitochondrial uncoupler, which dropped the ratio values by 50%. However, 2p-LSM exhibited several advantages over CLSM: (i) better signal/noise ratio in the green emission channel; (ii) less phototoxicity upon repetitive scanning in the focal plane; and (iii) no significant loss of image quality upon repetitive scans in the z direction. We conclude that, while both techniques enable visualisation of individual mitochondria in living cells, 2p-LSM has significant advantages for physiological work requiring time-lapse experiments or four-dimensional reconstructions of mitochondria.