title: Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye.
abstract: OBJECTIVE
  Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye.
  
  
  METHODS
  Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion.
  
  
  RESULTS
  All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Müller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina.
  
  
  CONCLUSIONS
  This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes.
authors.affiliation: The Institute of Optics, University of Rochester, Rochester, New York, United States 2Center for Visual Science, University of Rochester, Rochester, New York, United States.
authors.forename: Robin
authors.initials: R
authors.lastname: Sharma
authors.affiliation: The Institute of Optics, University of Rochester, Rochester, New York, United States 2Center for Visual Science, University of Rochester, Rochester, New York, United States 3Flaum Eye Institute, University of Rochester, Rochester, New York, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Williams
authors.affiliation: Polgenix, Inc., Cleveland, Ohio, United States.
authors.forename: Grazyna
authors.initials: G
authors.lastname: Palczewska
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
authors.affiliation: Center for Visual Science, University of Rochester, Rochester, New York, United States 3Flaum Eye Institute, University of Rochester, Rochester, New York, United States.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Hunter
mesh_terms: D000818:Animals; D063928:Ependymoglial Cells / Q000166:cytology; D005260:Female; D008252:Macaca fascicularis; D008253:Macaca mulatta; D008297:Male; D009887:Ophthalmoscopy / Q000379:methods; D061848:Optical Imaging / Q000379:methods*; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology; D012160:Retina / Q000166:cytology*; D055213:Retinal Pigment Epithelium / Q000166:cytology