title: Endogenous fluorophores enable two-photon imaging of the primate eye.
abstract: OBJECTIVE
  Noninvasive two-photon imaging of a living mammalian eye can reveal details of molecular processes in the retina and RPE. Retinyl esters and all-trans-retinal condensation products are two types of retinoid fluorophores present in these tissues. We measured the content of these two types of retinoids in monkey and human eyes to validate the potential of two-photon imaging for monitoring retinoid changes in human eyes.
  
  
  METHODS
  Two-photon microscopy (TPM) was used to visualize excised retina from monkey eyes. Retinoid composition and content in human and monkey eyes were quantified by HPLC and mass spectrometry (MS).
  
  
  RESULTS
  Clear images of inner and outer segments of rods and cones were obtained in primate eyes at different eccentricities. Fluorescence spectra from outer segments revealed a maximum emission at 480 nm indicative of retinols and their esters. In cynomolgus monkey and human retinal extracts, retinyl esters existed predominantly in the 11-cis configuration along with notable levels of 11-cis-retinol, a characteristic of cone-enriched retinas. Average amounts of di-retinoid-pyridinium-ethanolamine (A2E) in primate and human eyes were 160 and 225 pmol/eye, respectively.
  
  
  CONCLUSIONS
  These data show that human retina contains sufficient amounts of retinoids for two-photon excitation imaging. Greater amounts of 11-cis-retinyl esters relative to rodent retinas contribute to the fluorescence signal from both monkey and human eyes. These observations indicate that TPM imaging found effective in mice could detect early age- and disease-related changes in human retina.
authors: Polgenix, Inc., Cleveland, Ohio, United States.
authors: Grazyna
authors: G
authors: Palczewska
authors: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States.
authors: Marcin
authors: M
authors: Golczak
authors: Center for Visual Science, University of Rochester, Rochester, New York, United States The Institute of Optics, University of Rochester, Rochester, New York, United States.
authors: David R
authors: DR
authors: Williams
authors: Center for Visual Science, University of Rochester, Rochester, New York, United States Flaum Eye Institute, University of Rochester, Rochester, New York, United States Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States.
authors: Jennifer J
authors: JJ
authors: Hunter
authors: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States.
authors: Krzysztof
authors: K
authors: Palczewski
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D015353:Eye Enucleation; D005453:Fluorescence; D006801:Humans; D008252:Macaca fascicularis; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D012160:Retina / Q000378:metabolism*; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D012176:Retinoids / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
chemical_list: D012176:Retinoids; D014801:Vitamin A
keywords: cone photoreceptors; primate retina; retinoid cycle; rod photoreceptors; two-photon microscopy