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A. Dubra, Y. Sulai, D. R. Williams; Microscopic in vivo Imaging of Human Inner Retina With a Phase Adaptive Optics Scanning Laser Ophthalmoscope. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1200.
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Though adaptive optics (AO) has allowed high resolution imaging of photoreceptors with reflected light in the living human eye, in vivo non-invasive cellular imaging of the inner retina has proven more difficult due to its transparency. In order to address this issue, we have developed a phase AO scanning laser ophthalmoscope for imaging the inner retina in vivo.
There are numerous phase imaging methods, and most of them require the ability to control the light as it passes through the pupil planes of the imaging system. We have designed a confocal laser scanning ophthalmoscope that allows independent manipulation of the entrance and exit pupils, so that various phase imaging techniques can be implemented. For example, we have recorded pairs of images at 796 nm, by diverting the light passing through the left and right halves of the exit pupil to different detectors. This technique is called split-detector. The signal-to-noise ratio of these images is increased by registering and averaging multiple frames. The phase gradient image is calculated by dividing the difference of the averaged image pairs by their sum. Reflectance images are also produced by averaging the image pairs. The instrument was designed with a 7.75 mm diameter pupil at the eye, and uses a confocal pinhole (2.1 Airy disks in diameter) to provide increased axial sectioning. The transverse resolution of the instrument is limited by optical blur to better than 2 µm. The low temporal coherence of the light source (<20 µm) and the image averaging eliminate speckle. The combined optical power of the imaging and wavefront sensing sources at the subject’s cornea was below 250 µW at all times.
The reflectance images of the inner retina show reproducible fine cellular scale structure at multiple depths. The contrast of these features is enhanced by the use of phase imaging techniques. The observed structure is consistent with variations in refractive index at a cellular spatial scale, and resembles that seen in histological examination of post-mortem retinas with phase microscopy.
We have demonstrated that structure corresponding to cells in the inner retina can be resolved reproducibly in the living human eye at light levels that are well within safety limits. Moreover, the images are speckle-free. These non-interferometric phase imaging methods have potential for revealing changes in inner retinal mosaics, such as the ganglion cell mosaic, which is affected by glaucoma.
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