Abstract
Purpose :
Adaptive optics scanning light ophthalmoscopy (AOSLO) allows us to view the retina at cellular resolution. However, little is known about the origins of scattered light in methods like confocal and split detection AOSLO. We have developed a scattered light contrast (SLC) microscope to examine scattering properties of retinal structures that can inform strategies for improving cell contrast and ultimately assess function using AOSLO imaging.
Methods :
A custom scattered light contrast (SLC) system was constructed by placing a CMOS camera at the de-scanned confocal plane of a scanning microscope. Retinal vessels and cones were brought into focus with a back-illuminated 780nm LED source using fresh or 4% PFA fixed 13-lined ground squirrel (13-LGS) retina flat-mounted between #1.5 coverslips. Scattered light was then measured at every pixel with the SLC microscope using a 790nm SLD to create 2D image arrays. Digital masks were then applied based on known imaging contrast methods, including confocal and non-confocal split detection. We also applied Fourier transforms to the SLC measurements to determine the magnitude of the phase gradient.
Results :
The figure shows results collected from a blood vessel at the nerve fiber layer and cones using freshly dissected 13-LGS retina. These retinal structures resembled the same structures imaged in vivo with AOSLO (Sajdak et al. 2015; IOVS, Vol.56, 4099). Image quality was reduced when examining fixed retina, and the distribution of scattered light is altered compared to measurements in fresh tissue. Optimal detection geometries vary depending on the retinal structure being examined. For example, our data suggest smaller non-confocal aperture masks may be used to improve contrast for cone photoreceptors compared to larger off-set apertures commonly used for vascular imaging.
Conclusions :
We present a platform for examining contrast mechanisms and developing novel methods for visualizing retinal structure. Cones and blood vessels appear similar between AOSLO and the SLC system when digital masks are applied to fresh tissue data. By measuring the distribution of scattered light at every pixel ex vivo, optimization of known and novel methods of cellular imaging can be directly applied and compared to in vivo AOSLO imaging.
This is a 2020 ARVO Annual Meeting abstract.