title: Live imaging of mammalian retina: rod outer segments are stained by conventional mitochondrial dyes.
abstract: The vertebrate retina is an array of "narrow-capture" photoreceptive elements of diverse cellular types that allow the fine spatial resolution characteristic of vision. Imaging of photoreceptors and of the whole retina has been previously reported; however, both were achieved exclusively after fixation. We report our development of a new technique for imaging live bovine retinas ex vivo. Using this technique, we conducted fluorescence confocal laser scanning microscopic imaging of bovine retinas. Eyecups were incubated with conventional fluorescent mitochondrial probes (MitoTracker and JC-1). Unexpectedly, we found that, besides the retinal mitochondria, the rod outer segments that are devoid of mitochondria were also stained. No other neuron was stained. Both protonophores, which decrease mitochondrial membrane potential, or inhibit electron transport strongly inhibited the selective association of dyes with both retinal rod outer segments and mitochondria. This is the first time that living rod outer segments were visualized by this technique. This finding may shed light on previous reports of the existence of a proton potential across the disk membranes and on the mechanism of the adenosine tri-phosphate (ATP) supply for phototransduction, which still requires investigation.
authors.affiliation: University of Genoa, Laboratory for Advanced, Microscopy, Bioimaging, and Spectroscopy (LAMBS), MicroSCoBiO Research Center, Department of Physics, Via Dodecaneso 33, 16146, Genoa, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bianchini
authors.forename: Daniela
authors.initials: D
authors.lastname: Calzia
authors.forename: Silvia
authors.initials: S
authors.lastname: Ravera
authors.forename: Giovanni
authors.initials: G
authors.lastname: Candiano
authors.forename: Angela
authors.initials: A
authors.lastname: Bachi
authors.forename: Alessandro
authors.initials: A
authors.lastname: Morelli
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bruschi
authors.forename: Isidoro M
authors.initials: IM
authors.lastname: Pepe
authors.forename: Alberto
authors.initials: A
authors.lastname: Diaspro
authors.forename: Isabella
authors.initials: I
authors.lastname: Panfoli
mesh_terms: D000447:Aldehydes; D000818:Animals; D001562:Benzimidazoles; D002232:Carbocyanines; D002417:Cattle; D003287:Contrast Media; D005456:Fluorescent Dyes; D007089:Image Enhancement / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D017948:Retinal Rod Photoreceptor Cells / Q000166:cytology*; D042262:Retinoscopy / Q000379:methods*
chemical_list: D000447:Aldehydes; D001562:Benzimidazoles; D002232:Carbocyanines; D003287:Contrast Media; D005456:Fluorescent Dyes; C111472:mitotracker green FM; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine