Masella Benjamin D, Hunter Jennifer J, Williams David R
The Institute of Optics, University of Rochester, Rochester, New York, United States Center for Visual Science, University of Rochester, Rochester, New York, United States.
Center for Visual Science, University of Rochester, Rochester, New York, United States Flaum Eye Institute, University of Rochester, Rochester, New York, United States.
Invest Ophthalmol Vis Sci. 2014 Oct 14;55(11):7525-34. doi: 10.1167/iovs.13-13795.
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.
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.
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).
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.
视网膜密度测定法可提供有关视网膜功能的客观信息。但是，包括与光色素消耗无直接关系的视网膜反射率变化在内的许多因素，使其解释变得复杂。我们探究了这些因素，并提出了一种将其影响降至最低的方法。
使用自适应光学扫描激光检眼镜（AOSLO）测量猴子在用514纳米光进行光色素漂白前后光感受器反射率的变化。同时记录514纳米和794纳米处的反射率测量值。比较了几种归一化方法以提取光色素的表观光密度。
我们发现794纳米反射率中存在与刺激相关的波动，这些波动与光色素吸收率无关，在视杆细胞和视锥细胞中均会出现。这些变化的幅度接近与色素消耗直接相关的变化幅度，因此无法使用红外反射率进行归一化。我们转而使用一种空间归一化方法，该方法避免了近红外区域的波动，以及一种旨在将来自感受器以外层的光降至最低的共聚焦AOSLO。然而，这些方法得出的视紫红质表观密度估计值出奇地低（动物1：0.073±0.006，动物2：0.032±0.003）。
这些结果证实了早期的观察结果，即在暗适应过程中，光色素吸收的变化不是视网膜反射率变化的唯一来源。看来，在视网膜密度测定中，历史上降低视锥细胞光色素表观密度的杂散光主要来自光感受器本身附近的层。尽管存在这些复杂情况，但该方法仍为视网膜功能提供了有价值的客观测量方法。
