title: Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.
abstract: OBJECTIVE
  To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques.
  
  
  METHODS
  Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm.
  
  
  RESULTS
  The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes.
  
  
  CONCLUSIONS
  CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.
authors.affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, Colorado, USA.
authors.forename: Omid
authors.initials: O
authors.lastname: Masihzadeh
authors.forename: David A
authors.initials: DA
authors.lastname: Ammar
authors.forename: Malik Y
authors.initials: MY
authors.lastname: Kahook
authors.forename: Tim C
authors.initials: TC
authors.lastname: Lei
mesh_terms: D000818:Animals; D003094:Collagen / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D036641:Microscopy, Fluorescence, Multiphoton; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology; D012160:Retina / Q000166:cytology*; D055213:Retinal Pigment Epithelium / Q000166:cytology; D012590:Sclera / Q000166:cytology / Q000378:metabolism; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D003094:Collagen
keywords: coherent anti-Stokes Raman scattering (CARS); multiphoton fluorescence microscopy; photoreceptors; retina; retinal pigment epithelium (RPE)