title: New wrinkles in retinal densitometry.
abstract: OBJECTIVE
  Retinal densitometry provides objective information about retinal function. But, a number of factors, including retinal reflectance changes that are not directly related to photopigment depletion, complicate its interpretation. We explore these factors and suggest a method to minimize their impact.
  
  
  METHODS
  An adaptive optics scanning light ophthalmoscope (AOSLO) was used to measure changes in photoreceptor reflectance in monkeys before and after photopigment bleaching with 514-nm light. Reflectance measurements at 514 nm and 794 nm were recorded simultaneously. Several methods of normalization to extract the apparent optical density of the photopigment were compared.
  
  
  RESULTS
  We identified stimulus-related fluctuations in 794-nm reflectance that are not associated with photopigment absorptance and occur in both rods and cones. These changes had a magnitude approaching those associated directly with pigment depletion, precluding the use of infrared reflectance for normalization. We used a spatial normalization method instead, which avoided the fluctuations in the near infrared, as well as a confocal AOSLO designed to minimize light from layers other than the receptors. However, these methods produced a surprisingly low estimate of the apparent rhodopsin density (animal 1: 0.073 ± 0.006, animal 2: 0.032 ± 0.003).
  
  
  CONCLUSIONS
  These results confirm earlier observations that changes in photopigment absorption are not the only source of retinal reflectance change during dark adaptation. It appears that the stray light that has historically reduced the apparent density of cone photopigment in retinal densitometry arises predominantly from layers near the photoreceptors themselves. Despite these complications, this method provides a valuable, objective measure of retinal function.
authors.affiliation: The Institute of Optics, University of Rochester, Rochester, New York, United States Center for Visual Science, University of Rochester, Rochester, New York, United States.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Masella
authors.affiliation: Center for Visual Science, University of Rochester, Rochester, New York, United States Flaum Eye Institute, University of Rochester, Rochester, New York, United States.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Hunter
authors.affiliation: The Institute of Optics, University of Rochester, Rochester, New York, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Williams
mesh_terms: D000818:Animals; D003623:Dark Adaptation / Q000502:physiology*; D003720:Densitometry / Q000379:methods*; D005260:Female; D008251:Macaca; D008297:Male; D009887:Ophthalmoscopy; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*
chemical_list: D012243:Rhodopsin
keywords: adaptive optics; dark adaptation; densitometry; intrinsic signals; rhodopsin regeneration