Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy.
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.
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Department of Pharmacy, University of Sydney, NSW, 2006, Syndey, Australia. vadimd@pharm.usyd.edu.au
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D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005727:Ganglia, Spinal / Q000166:cytology; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000648:ultrastructure*; D009474:Neurons / Q000648:ultrastructure*; D051381:Rats; D017207:Rats, Sprague-Dawley
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine