title: Third harmonic generation microscopy of a mouse retina.
abstract: OBJECTIVE
  To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities.
  
  
  METHODS
  A custom fiber laser and multiphoton microscope was constructed and optimized for simultaneous two-photon autofluorescence (TPAF) and THG retinal imaging. Imaging was performed using fixed-frozen sections of mouse eyes without the use of exogenous fluorescent dyes. In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei.
  
  
  RESULTS
  Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution. In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes. In the C57BL/6 strain, the THG signal from the RPE cells originates from the pigmented granules.
  
  
  CONCLUSIONS
  THG microscopy can be used to image structures of the mouse retina using contrast inherent to the tissue and without the use of a fluorescent dye or exogenously expressed recombinant protein.
authors.affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, CO.
authors.forename: Omid
authors.initials: O
authors.lastname: Masihzadeh
authors.affiliation: Department of Electrical Engineering, University of Colorado Denver, Denver, CO.
authors.forename: Tim C
authors.initials: TC
authors.lastname: Lei
authors.affiliation: Department of Electrical Engineering, Colorado State University, Fort Collins, CO.
authors.forename: Scott R
authors.initials: SR
authors.lastname: Domingue
authors.affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, CO.
authors.forename: Malik Y
authors.initials: MY
authors.lastname: Kahook
authors.affiliation: Department of Electrical Engineering, Colorado State University, Fort Collins, CO.
authors.forename: Randy A
authors.initials: RA
authors.lastname: Bartels
authors.affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, CO.
authors.forename: David A
authors.initials: DA
authors.lastname: Ammar
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D004867:Equipment Design; D006801:Humans; D050356:Lipid Metabolism; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D009893:Opossums; D061848:Optical Imaging / Q000295:instrumentation / Q000379:methods; D012160:Retina / Q000033:anatomy & histology* / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000033:anatomy & histology / Q000378:metabolism
chemical_list: D015415:Biomarkers; D008563:Membrane Lipids