Regions of high mitochondrial density in the ellipsoids give rise to strong intensity signals in OCT. Inner segments of rods and particularly those of cones, i.e.
the ellipsoids, contain a high concentration of mitochondria [59
] which are known to play a role in waveguiding due to their high refractive index. Similarly, the presence of melanosomes in RPE cells [60
] and the tapering of cone outer segments might contribute to an increase in back-scatter in more distal regions.
The difficulty in discerning structure (or signal-carrying speckle) from noise (including signal-degrading speckle) is a very challenging issue for OCT imaging. Several factors may contribute to the wide variation in image quality observed within and across subjects. One possible factor may be the degree of correction of LCA. It is known [26
] that improper subject alignment can result in deviations from optimal correction, and overall subject compliance is one common contributing factor to misalignments. Further work is needed to better understand the factors responsible for the variability in image quality within and across subjects.
Image averaging was used to improve visibility of retinal structures, but averaging alone cannot remove most of the speckle noise present. To better combat this noise, bandpass and a trous filters were utilized. As described in section 2.3, carefully chosen parameters were used to avoid the introduction of artefacts that could confuse artificial structure for real structure. To demonstrate the effect of these two filters, some sample data before and after some postprocessing steps is presented in . A 100 × 100 μm2 patch of RPE mosaic obtained by averaging 15 en face slices is shown in . The 15 en face sections were minimally processed – only image registration was applied to compensate for image motion, followed by rotation of the data by <4° along both orthogonal directions to adjust the tilt of the retina. The power spectrum of is shown in , while in , the power spectrums from each of the 15 en face slices was averaged. Since the power spectrums also appear speckled, the power spectrum in was filtered with BPF4-500 resulting in . A similar sequence of results are shown in and in where the 15 en face sections were filtered using BPF4-500 or ATF, respectively. Although a detailed analysis of the effects of the bandpass and a trous filters is beyond the scope of this work, demonstrates that the use of these filters has not added spurious artefacts to the RPE mosaics (), nor is there any significant change in the radius of Yellott’s rings in the power spectrums (), even after applying bandpass filtering to the power spectrums themselves (). Rather, the ability to discern Yellott’s ring is improved with the use of the filters.
The above analysis has demonstrated that the filters used for speckle noise reduction have not introduced gross artefacts in the resulting RPE cell mosaics or in their power spectrums. To further confirm that the structures visualized were attributed to RPE cell packing and not due to speckle noise or artefacts of the speckle noise filtering process, the en face slice central to the sections piercing through the RPE cell nuclei was cross-correlated to other layers of comparable signal intensity above and below it. A depth dependent plot of the peak cross-correlations is presented in . Since the same filtering was applied to all depth scans considered in this analysis, these results demonstrate that the observed structural details were attributed to real structure and not speckle noise or artefacts due to noise filtering.
Depth-dependent peak cross-correlation between the central most en face section piercing through the RPE cell nuclei and sections at other depths.
Two possible categories of mutations can lead to dichromatic colour vision (colour-blindness) – one which results in photoreceptor loss, while in the other, the substitution of an absent cone class with another [49
]. Although it has been possible to show that these two different genotypes, which have classically been associated with the same dichromat class, can represent different cellular phenotypes, little information was known of the extent of the degeneration distal to the ellipsoids due to a limited depth range. Image quality varied between subjects and within repeated acquisitions at the same or different eccentricities for each subject ().
For the case of photoreceptor imaging in normals and colour-blinds, a significant reduction in signal-producing elements (photoreceptors, in this case) may help to explain why some presentations better contrasted structure from noise than others. Despite this, averaging of several (200) cross-sections () suggested that the majority of signals evident in en face
() and transverse tomograms () were attributed to real structure. Furthermore, this demonstrated that a significant reduction in back-scattered signal was obtained from the photoreceptor layer in two colour-blind individuals, supporting the former model of dichromatic colour vision. Although other imaging techniques [49
] can detect such cone loss, it is only possible to confirm that there has been a complete degeneration of the cones with the high-resolution depth profiling capability of OCT.
A general thickening of the retina was observed in the colour-blind retina relative to that of the normal retina. The thickness of the IS, OS and RPE layers were defined by the centre of the bright bands associated with the ELM and IS/OS junction, and the bottom of the bright band associated with the RPE (). At 5.8° eccentricity (), the thickness of the IS, OS and RPE layers were 25%, 71% and 15% greater in the colour-blind relative to the normal retina. This observed thickening of retinal layers in the colour-blind retina possibly resulted in part from the phagocytosis of non-functional cones but further studies at multiple retinal locations and with larger sample sizes are needed to determine how these differences relate to normal variations with the human population.
The capability of selectively focussing the probing beam onto the nerve fibre layer improved visualization of the nerve fibre bundles and underlying retinal structures. This was previously demonstrated by Zawadzki et al.
] using AO-OCT. A means to track changes in the structure of retinal ganglion cells would offer a useful diagnostic tool for early detection of glaucoma. But due to their dense packing and low-scattering, it is difficult to distinguish ganglion cells without invasive contrast-enhancing agents. Cordeiro et al. [61
] used a prototype Zeiss confocal SLO to image ganglion cells in rats and anesthetized macaque monkeys in vivo
after intravitreal injection of Annexin 5-bound fluorophore. Gray et al.
] also imaged ganglion cells in the macaque monkey in vivo
using SLO and AO-SLO with Rhodamine or Alexa fluorescent dyes.
To improve signal-to-noise, 1D scanning patterns were employed. This technique has a distinct advantage from averaging of tomograms obtained with 2D scanning patterns since for the former, the averaging occurs over the same section of sample (except for translations due to eye motion), thus enhancing the visibility of structures pierced by the sectioning plane. This is in contrast to averaging across a series of adjacent planes, which will blur out details if the average is performed over a layer thickness that exceeds the dimensions of the structures of interest. If eye motions are sufficiently high, this will lead to some spatial averaging of speckle noise (after applying image registration to compensate for any residual motion) while also averaging out detector noise. Although larger sample sizes are needed to verify this result, circular bodies approximately 10–15 μm in size have been observed near the fovea in a normal individual. Averaging across 30 repeated tomograms acquired with only fast-axis scanning significantly reduced speckle noise and improved image contrast. Since no further processing was performed, the structures, with sizes consistent with that of ganglion cells [54
], cannot be attributed to post-processing artefacts.
visualization of RPE cells may help to identify and detect early indicators of diseases associated with their function and to track progression. The RPE nuclei are surrounded by highly scattering organelles () including melanosomes (contained within microvilli that cover phagocytosed OS fragments), mitochondria and lysosomes (). In OCT, these dense, highly scattering structures give rise to bright intensity signals that surround a structured arrangement of dark circular bodies that correspond to the cell nuclei. While it had previously only been possible to image RPE cells in vivo
using autofluorescence imaging [61
], or in a patient with cone loss [63
], RPE cell structure has been probed using OCT for the first time in a subject with a normal and complete photoreceptor mosaic without autofluorescence. The RPE cell spacing was estimated from a manual cell count and this was in agreement with the spacing inferred from the power spectrum of the RPE cell mosaic. And the observed cell density and spacing was consistent with estimates in primates [61
] and humans [62
] using other imaging systems as well as histology in humans [54
]. Moreover, the combination of an ultra-high imaging speed and ultra-high resolution allowed for selectivity in depth sectioning to reveal not only the RPE cell mosaic, but also layers containing structures consistent with the RPE cell soma () and choriocapillaris (), demonstrating the potential of this imaging modality when compared to other technologies with limited depth resolution.
Non-invasive imaging of the lamina cribrosa is of interest since it has been suggested that larger pores and thinning of the connective tissue support might implicate nerve fibre damage due to glaucoma [64
]. In vivo
imaging of the lamina cribrosa was performed by Vilupuru et al.
] using AO-SLO in anesthetized normal and glaucomatous Rhesus monkeys, and in humans by Srinivasan et al.
] and Potsaid et al.
] using OCT, and by Zawadzki et al.
] using AO-OCT. AO-SLO presents a limited depth of field and without AO, uncompensated ocular aberrations severely impairs lateral resolution in OCT. For the first two cases, the axial resolution was restricted (~100 μm and 8 μm, respectively), due to spectral bandwidth limitations. In Zawadzki et al.
], the axial resolution was limited to 6 μm due to uncompensated LCA, and to avoid instability in the AO correction (the AO was applied and fixed to a different retinal location before the subject’s eccentricity was adjusted for imaging of the optic nerve head). We have demonstrated that with a high axial resolution offered by an ultra-broadband light source and LCA compensation, and a high transverse resolution offered by stable AO correction is it possible to visualize collagen fibre bundles in the human lamina cribrosa in vivo
The main limitations inherent in the imaging technique demonstrated here include residual motion artefacts due to eye movements, accuracy of image registration and signal-to-noise, including the deleterious effects of speckle noise. The impact of the first two limitations may be reduced by a further increase in the acquisition speed, but not without posing a further compromise on the third limitation. The signal-to-noise primarily depends on how efficiently the optical system collects and passes the light back-scattered off the retina to the spectrometer (via an optical fibre coupling, in this case), which is primarily affected by optical aberrations. But even a signal free of aberrations will contain OCT signals composed of both signal-carrying and signal-degrading speckle. A great challenge for OCT imaging is in discerning one speckle type from the other.
Although the light incident on the eye is strictly limited to prevent retinal damage [67
], there is possible room for improved image resolution through a better AO correction. Studies [68
] have observed dynamics in ocular aberrations as high as 30 Hz, and thus, enhancements in image resolution could be had by increasing the AO closed-loop speed. Nevertheless, when compared to the system’s predecessor [27
], the implementation of AO in this work was considerably improved since the closed-loop AO correction was performed in real-time, and thus, the geometric aberrations were compensated dynamically throughout the entire duration of image acquisition. Although the subjects’ heads were constrained and accommodation was paralyzed in that earlier work, it would be expected that any microfluctuations in accommodation, tear film changes, or line-of-sight alterations that may have occurred within not only the time elapsed for image acquisition but also between the “freezing” of the AO loop and the commencing of data acquisition would have resulted in aberration changes left uncompensated. When compared to the current system, this effect would have been be exacerbated by the fact that the previous acquisition speed was 6X slower.
This paper demonstrates the successful combination of ultra-high resolution OCT with adaptive optics and longitudinal chromatic aberration compensation and an ultra-high speed image acquisition system to provide detailed morphological structure at the cellular level in normal and pathologic retinas. A high axial resolution offered by an ultra-broadband light source and correction of geometrical and longitudinal chromatic aberrations had been shown [26
] to provide a means of probing cellular structure non-invasively in the living human retina. But only with an ultra-fast image acquisition speed (120,000 depth scans/s) is it possible to overcome deleterious eye motion artefacts to expose weakly-scattering cellular structures in the in vivo
human retina such as ganglion cells and RPE cells. Finally volumetric phenotyping of colour-blinds at the cellular level was accomplished, illustrating the potential of this imaging modality to provide detailed insight of the living retinal micromorphology, which should help to contribute to a better understanding of pathogenesis as well as to improve early diagnosis and therapy monitoring of a variety of retinal diseases. Although it has been shown through preliminary data that the system performance has been markedly improved from its predecessor [27
], further studies are needed to better demonstrate the capabilities of this imaging modality.