June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Author Affiliations & Notes
  • Andres Guevara-Torres
    The Institute of Optics, University of Rochester, Rochester, NY
    Center of Visual Science, University of Rochester, Rochester, NY
  • David R Williams
    The Institute of Optics, University of Rochester, Rochester, NY
    Center of Visual Science, University of Rochester, Rochester, NY
  • Jesse B Schallek
    Center of Visual Science, University of Rochester, Rochester, NY
  • Footnotes
    Commercial Relationships Andres Guevara-Torres, Canon, Inc. (F), University of Rochester (P); David Williams, Canon, Inc. (F), Canon, Inc. (R), Polgenix, Inc. (F), University of Rochester (P); Jesse Schallek, University of Rochester (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4371. doi:https://doi.org/
Abstract
 
Purpose
 

Despite the micron-level resolution of adaptive optics scanning light ophthalmoscopy (AOSLO), many cell classes in the mammalian retina are challenging to image because they provide weak endogenous optical contrast. Here we use split-detector imaging (Scoles et al. , 2014) to enhance the contrast of cell boundaries, rendering them visible in high-resolution imaging.

 
Methods
 

Anesthetized C57BL/6 mice were imaged with an AOSLO using near infrared light. The detection arm of the AOSLO was modified by placing a knife edge at the retinal image plane to direct the left and right half of the point spread function into two photomultiplier tubes (PMTs). Simultaneously captured signals from each PMT were subtracted to generate a differential contrast image. AO was used for defocus control and the vascular layers were used to confirm axial position.

 
Results
 

We resolved a variety of retinal cells that are invisible with conventional confocal AOSLO without contrast agents. We observed a monolayer of photoreceptor distal processes that showed a Yellot’s ring peak at 23 cycles/degree (figure 1a). This corresponds to a photoreceptor density of 477,000 cells/mm2, consistent with previous reports of photoreceptor density in the mouse. When focusing slightly more vitread, we could distinguish a multilayer arrangement of photoreceptor somata within the outer nuclear layer (figure 1b). Above this, at the boundary of the outer plexiform layer, we resolved a sparse mosaic of cells (figure 1c) with a diameter of 11 ± 2 μm (mean ± SD). This mosaic of cells counted by 4 individuals showed a density of 1250 ± 270 cells/mm2 (mean ± SD, 0.25 mm2 analyzed between 10-25 degrees from the optic disc), which is consistent with previous histological reports of horizontal cell density in mouse and more than an order of magnitude different than any other neural cell class in the retina.

 
Conclusions
 

This approach reveals a variety of cell structures that are hidden when imaged by confocal systems. The capability to image horizontal cells and the photoreceptor somata enables quantification of these cell classes over the course of disease. Optimizing this contrast method in the mouse may instruct best strategies for imaging transparent cells in the human retina.  

 
Figure1 Split-detector images of a)mosaic of photoreceptor distal processes b)photoreceptor somas c)horizontal cells
 
Figure1 Split-detector images of a)mosaic of photoreceptor distal processes b)photoreceptor somas c)horizontal cells

 
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