High-resolution adaptive optics (AO) imaging of retinal neurons in living eyes holds promise for improved diagnosis and better assessment of treatment outcomes for retinal diseases. By integrating different imaging modalities, such as scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT), AO has enabled microscopic views of different retinal neurons, including recently reported retinal ganglion cells. In this study, we present a novel design of a multimodal adaptive optics imaging system to investigate the microscopic structure of living human retina. The optical system was designed using Zemax ray tracing software. The system performance was evaluated in terms of image quality and beam displacement. Optical performance was predicted to achieve diffraction limited image quality at the retinal plane and beam displacement was predicted to be >7× smaller than the pitch of Shack-Hartmann lenslet at the pupil planes for scan angles over 3.6°×3.6° field of view. The initial human subject images are presented. High quality photoreceptor images were acquired in both AO-SLO channel and AO-OCT channel simultaneously at 3° temporal from the fovea. Individual cones are delineated in AO-SLO image, the corresponding AO-OCT image showed four main reflections from outer retina, namely external limiting membrane, cone inner segment/outer segment junction, cone outer segment tip, and retinal pigment epithelium. The system allows flexibly alternating between AO-SLO and AO-OCT modes, which provides complementary views of retinal cells, and the potential to improve disease diagnosis and treatment.
Adaptive optics-enabled optical coherence tomography (AO-OCT) and scanning laser ophthalmoscopy (AO-SLO) devices can resolve retinal cones and rods in three dimensions. To evaluate the improved resolution of AO-OCT and AO-SLO, a phantom that mimics retinal anatomy at the cellular level is required. We used a two-photon polymerization approach to fabricate three-dimensional (3D) photoreceptor phantoms modeled on the central foveal cones. By using a femtosecond laser to selectively photocure precise locations within a liquid-based photoresist via two-photon absorption, we produced high-resolution phantoms with μm-level dimensions similar to true anatomy. In this work, we present two phantoms to evaluate the resolution limits of an AO imaging system: one that models only the outer segments of the photoreceptor cells at varying retinal eccentricities and another that contains anatomically relevant features of the full-length photoreceptor. With these phantoms we are able to quantitatively estimate transverse resolution of an AO system and produce images that are comparable to those found in the human retina.
The inner retina is critical for visual processing, but much remains unknown about its neural circuitry and vulnerability to disease. A major bottleneck has been our inability to observe the structure and function of the cells composing these retinal layers in the living human eye. Here, we present a noninvasive method to observe both structural and functional information. Adaptive optics optical coherence tomography (AO-OCT) is used to resolve the inner retinal cells in all three dimensions and novel post processing algorithms are applied to extract structure and physiology down to the cellular level. AO-OCT captured the 3D mosaic of individual ganglion cell somas, retinal nerve fiber bundles of micron caliber, and microglial cells, all in exquisite detail. Time correlation analysis of the AO-OCT videos revealed notable temporal differences between the principal layers of the inner retina. The GC layer was more dynamic than the nerve fiber and inner plexiform layers. At the cellular level, we applied a customized correlation method to individual GCL somas, and found a mean time constant of activity of 0.57 s and spread of ±0.1 s suggesting a range of physiological dynamics even in the same cell type. Extending our method to slower dynamics (from minutes to one year), time-lapse imaging and temporal speckle contrast revealed appendage and soma motion of resting microglial cells at the retinal surface.
Absorption of light by photoreceptors initiates vision, but also leads to accumulation of toxic photo-oxidative
compounds in the photoreceptor outer segment (OS). To prevent this buildup, small packets of OS discs are
periodically pruned from the distal end of the OS, a process called disc shedding. Unfortunately dysfunction in
any part of the shedding event can lead to photoreceptor and RPE dystrophy, and has been implicated in
numerous retinal diseases, including age related macular degeneration and retinitis pigmentosa. While much is
known about the complex molecular and signaling pathways that underpin shedding, all of these advancements
have occurred in animal models using postmortem eyes. How these translate to the living retina and to humans
remain major obstacles. To that end, we have recently discovered the optical signature of cone OS disc shedding
in the living human retina, measured noninvasively using optical coherence tomography equipped with adaptive
optics in conjunction with post processing methods to track and monitor individual cones in 4D. In this study,
we improve on this method in several key areas: increasing image acquisition up to MHz A-scan rates,
improving reliability to detect disc shedding events, establishing system precision, and developing cone
tracking for use across the entire awake cycle. Thousands of cones were successfully imaged and tracked over
the 17 hour period in two healthy subjects. Shedding events were detected in 79.5% and 77.4% of the tracked
cones. Similar to previous animal studies, shedding prevalence exhibited a diurnal rhythm. But we were
surprised to find that for these two subjects shedding occurred across the entire day with broad, elevated
frequency in the morning and decreasing frequency as the day progressed. Consistent with this, traces of the
average cone OS length revealed shedding dominated in the morning and afternoon and renewal in the evening.
Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, however, are often
compromised in ageing and ocular diseases that lead to blindness. Early manifestation of RPE disruption
occurs at the cellular level, but while in vivo biomarkers at this scale hold considerable promise, RPE
cells have proven extremely challenging to image in the living human eye. Recently we addressed this
problem by using organelle motility as a novel contrast agent to enhance the RPE cell in conjunction with
3D resolution of adaptive optics-optical coherence tomography (AO-OCT) to section the RPE layer. In
this study, we expand on the central novelty of our method – organelle motility – by characterizing the
dynamics of the motility in individual RPE cells, important because of its direct link to RPE physiology.
To do this, AO-OCT videos of the same retinal patch were acquired at approximately 1 min intervals or
less, time stamped, and registered in 3D with sub-cellular accuracy. Motility was quantified by an
exponential decay time constant, the time for motility to decorrelate the speckle field across an RPE cell.
In two normal subjects, we found the decay time constant to be just 3 seconds, thus indicating rapid
motility in normal RPE cells.
Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, but are often compromised in ageing and
major ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, and while
biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living
human eye. We present a novel method based on optical coherence tomography (OCT) equipped with adaptive optics (AO)
that overcomes the associated technical obstacles. The method takes advantage of the 3D resolution of AO-OCT, but more
critically sub-cellular segmentation and registration that permit organelle motility to be used as a novel contrast mechanism.
With this method, we successfully visualized RPE cells and characterized their 3D reflectance profile in every subject and
retinal location (3° and 7° temporal to the fovea) imaged to date. We have quantified RPE packing geometry in terms of
cell density, cone-to-RPE ratio, and number of nearest neighbors using Voronoi and power spectra analyses. RPE cell
density (cells/mm<sup>2</sup>) showed no significant difference between 3° (4,892±691) and 7° (4,780±354). In contrast, cone-to-
RPE ratio was significantly higher at 3° (3.88±0.52:1) than 7° (2.31± 0.23:1). Voronoi analysis also showed most RPE
cells have six nearest neighbors, which was significantly larger than the next two most prevalent associations: five and
seven. Averaged across the five subjects, prevalence of cells with six neighbors was 51.4±3.58% at 3°, and 54.58±3.01%
at 7°. These results are consistent with histology and <i>in vivo</i> studies using other imaging modalities.
It has been long established that photoreceptors capture light based on the principles of optical waveguiding. Yet after
decades of experimental and theoretical investigations considerable uncertainty remains, even for the most basic
prediction as to whether photoreceptors support more than a single waveguide mode. To test for modal behavior in
human cone photoreceptors, we took advantage of adaptive-optics optical coherence tomography (AO-OCT, λc=785
nm) to noninvasively image in three dimensions the reflectance profiles generated in the inner and outer segments (IS,
OS) of cones. Mode content was examined over a range of cone diameters by imaging cones from 0.6° to 10° retinal
eccentricity (n = 1802). Fundamental to the method was extraction of reflections at the cone IS/OS junction and cone
outer segment tip (COST). Modal content properties of size, circularity and orientation were quantified using second
moment analysis. Analysis of the cone reflections indicates waveguide properties of cone IS and OS depend on
segment diameter. Cone IS was found to support a single mode near the fovea (≤3°) and multiple modes further away
(<4°). In contrast, no evidence of multiple modes was found in the cone OSs. The IS/OS and COST reflections share a
common optical aperture, are most circular near the fovea, and show no orientation preference.
Optical coherence tomography with adaptive optics (AO-OCT) is a noninvasive method for imaging the living retina
at the microscopic level. We used AO-OCT technology to follow changes in cone photoreceptor outer segment (OS)
length and reflectance. To substantially increase sensitivity of the length measurements, a novel phase retrieval
technique was demonstrated, capable of detecting changes on a nanometer scale. We acquired volume videos of
0.65°x0.65° retinal patches at 1.5° temporal to the fovea over 75 and 105 minutes in two subjects. Volumes were
dewarped and registered, after which the cone intensity, OS length, and referenced phase difference were tracked over
time. The reflections from inner segment/OS junction (IS/OS) and posterior tips of OS (PT) showed significant
intensity variations over time. In contrast, the OS length as measured from the intensity images did not change,
indicative of a highly stable OS length at least down to the level of the system's axial resolution (3μm). Smaller axial
changes, however, were detected with our phase retrieval technique. Specifically, the PT-IS/OS phase difference for
the same cones showed significant variation, suggesting real sub-wavelength changes in OS length of 125±46 nm/hr
for the 22 cones followed. We believe these length changes are due to the normal renewal process of the cone OS that
elongate the OS at a rate of about 100 nm/hr. The phase difference measurements were strongly correlated among Alines
within the same cone (0.65 radians standard deviation) corresponding to a length sensitivity of 31 nm, or ~100
times smaller than the axial resolution of our system.