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1From the Center for Biomedical Engineering and Physics, Medical University of Vienna, Austria; and the 2Department of Ophthalmology, General Hospital and Medical University of Vienna, Austria.
| Abstract |
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METHODS. Ten healthy human subjects (mean age, 31 ± 8 years) and two patients with RPE diseases participated in the study. The macular region of one eye of each subject was investigated with a phase-resolved PS-OCT system. The instrument measured backscattered intensity (standard OCT), phase retardation, and (cumulative) birefringent axis orientation, simultaneously. For a quantification of the depolarizing layer, plots of the distributions of retardation and axis orientation within and above this layer were analyzed.
RESULTS. A polarization-scrambling layer (PSL) was observed at the posterior boundary of the retina in PS-OCT images of all volunteers. It was identified in PS-OCT images by determining random retardation and axis orientation in a transverse direction. Measurements in patients with neurosensory retinal detachment, retinal pigment epithelium (RPE) detachment, and RPE atrophy suggest that the PSL is the RPE. The statistical analysis provided objective discrimination of the RPE from the other retinal structures.
CONCLUSIONS. PS-OCT represents a powerful tool for increasing image contrast in ocular tissues. The observed polarization-scrambling nature of the RPE may be used in diseased eyes to locate the RPE or remains of the RPE definitively in OCT images.
Optical coherence tomography (OCT)4 5 6 represents a well-established imaging tool in ophthalmology. The technique offers the possibility of performing a noninvasive optical "biopsy" of tissue (e.g., the human retina) in vivo. Several studies have shown the diagnostic value of the method for investigating retinal diseases.7 8 9 10 11 Recently, new developments12 13 in the field have led to an increased imaging speed with the use of Fourier- or spectral-domain OCT. High-speed imaging of the retina has been achieved with this novel technology,14 15 16 which was subsequently followed by in vivo three dimensional (3D) imaging of normal17 18 and diseased retina.19
In contrast, as known from microscopy, images that represent only the backscattered light intensity (as is the case in standard OCT images) often yield poor image contrast. The possibility of using polarized light to increase image contrast and to gather additional information about the sample is well known and has been used to investigate the polarization properties of the human retina.20 21 22 Probably the most common and commercially available (e.g., GDx; Carl Zeiss Meditec GmbH, Oberkochen, Germany) imaging tool that uses polarized light for retinal imaging is scanning laser polarimetry (SLP). This technique measures the amount of retardation introduced by the retinal nerve fiber layer (RNFL) to obtain information on thickness changes associated with glaucoma.23
However, SLP cannot provide depth-resolved information on the polarization properties of the retina. Polarization sensitive (PS)-OCT24 25 26 combines the ability of OCT to retrieve depth-resolved information with the ability of SLP to retrieve birefringent information. A prototype instrument used this technique to measure birefringence introduced by the RNFL.27 Recently, retardation introduced by Henles fibers in the foveal region and the discovery of a polarization-scrambling layer (PSL) within the retina have been presented.28
In this study, we used PS-OCT to investigate the healthy human macula in vivo. A depolarizing layer was visible in PS-OCT images of all volunteers who participated in the study. For a better identification of the anatomic layer responsible for the depolarization, we performed measurements on patients with macular diseases. Furthermore, we sought to develop an analysis method for quantitative description of the depolarizing layer.
| Methods |
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0 = 841 nm and 
= 51 nm) and was measured with
6.1 µm in air which corresponds to
4.5 µm within the retina (assuming a group refractive index of the retina of 1.38). The system records a B-scan image (OCT tomogram) consisting of 3400 x 500 pixels corresponding to
3.4 x 1 mm (xz image plane) in 0.5 second.
An additional detection channel was implemented from the previous setup, which allowed the recording of scanning laser ophthalmoscope (SLO) images (with similar detection optics as those used for the OCT images) at a frame rate of 5 frames per second (fps) for alignment purposes. The SLO image, which was recorded immediately before (delay of
100 ms) the OCT measurement, was stored to determine the position of the B-scan on the retina.
Control Subject and Patient Selection and Imaging Procedure
All investigations were performed in a protocol that adhered to the tenets of the Declaration of Helsinki and was approved by the ethics committee of the Medical University of Vienna. For this study, 10 healthy, white volunteers (mean age, 31 ± 8 years) without any detectable ocular disease or history of an ocular disease were selected. Apart from a routine eye examination including visual acuity testing, color fundus photography and red-free fundus photography were performed before the measurement with the PS-OCT system. All eyes were rated as normal, with a refractive error of 2 ± 2 D. One eye of each volunteer was investigated with the PS-OCT instrument. Cross-sectional (B-scan) images of the foveal region were recorded and used for this study. No pupil dilation was necessary for this measurement.
To test our hypothesis that the PSL is the RPE, images of two patients (one patient with RPE atrophy, one patient with RPE detachment) were included in the study.
Data Analysis
A combination of anterior segment birefringence and birefringence of Henles fiber layer results in an asymmetric retardation pattern in PS-OCT images of the foveal region.28 This effect is similar to that giving rise to the well-known "bow-tie" pattern in SLP.32 33 To minimize the influence of this effect on the statistical analysis of the PSL, we divided all PS-OCT images at the position of the foveola into two parts: nasal and temporal. The analysis was performed for the nasal part.
In a first step, we extracted the PSL from the OCT images by image segmentation. The PSL represents the last (posterior) strongly reflecting layer in retinal OCT images. Therefore, we used a peak detection algorithm operating on the intensity images to locate the position of the PSL. After this peak detection, we fitted a second-order polynomial through the detected peaks, to exclude erroneous peak locations caused by noise. The fitted polynomial was regarded as the location of the PSL. From the two dimensional (2D) B-scan images, depth lines (corresponding to A-scans) of intensity, retardation, and axis orientation were derived and aligned with respect to the fitted polynomial. The result was a corrected B-scan image in which the PSL appeared as a horizontal line.
A horizontal evaluation line (single pixel width corresponding to a depth extension of 2 µm) parallel to the PSL was interactively shifted in depth by the operator through each image. Distributions of retardation and axis orientation along this evaluation line were plotted at the location of the PSL and at two locations above the PSL. Note that data points with a corresponding intensity below a certain intensity threshold were excluded from this analysis to avoid erroneous data caused by noise.26 The peak of each distribution was regarded as the corresponding retardation and axis orientation of each line. To evaluate the degree of polarization-scrambling, we determined the number of data points (n) with retardations (axis) outside an interval of ±10 ° (20°) from the peak of the distribution. This number was divided by the total number of data points, to normalize the data, and thus obtain a comparable depolarization value.
| Results |
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More information can be obtained from the polarization-sensitive images. The (single-pass) retardation image (Fig. 1B) shows rather constant and low retardation throughout the retina (with the exception of layer 4).28 The observed retardation was induced by birefringence of the anterior segment of the eye (mainly corneal birefringence). The fact that retardation remained constant with depth indicates that these areas of retinal tissue were nonbirefringent. A slight asymmetry of retardation between the left and right sides of the image can be observed at layers 1 to 3. This can be explained by a combined birefringence of the anterior segment and Henles fiber layer (Fig. 1D) .28 Within layer 4, rather random retardation is observed. This randomness is clearer in the enlarged image of a subsection of the foveola region (Fig 1F) . Figure 1C shows the (cumulative) fast birefringent axis orientation of this region. Similar to the retardation image, a fairly constant axis orientation is observed throughout the retina, with the exception of layer 4. (There is more noise in the axes of the inner retinal layers due to the lower intensity obtained from these areas; the range of axes observed in the inner layers was restricted, however, to yellow-orange colors [left] and green-yellow colors [right], whereas the entire range from blue to red is observed in layer 4.) The difference in axis orientations within layers 1 to 3 between foveola and surrounding fovea (which was more pronounced than the difference in the retardation in this region) can be explained by the birefringence of Henles fiber layer. This layer changes the cumulative axis orientation in the region around the foveola; however, because this layer is not present in the foveola, the cumulative axis does not change in this region and remains the same as in the interior layers. Within layer 4, rather random axis orientations are observed.
For a quantitative analysis, the procedure described in the Data Analysis section was implemented. Figure 2 shows an example of the distribution of retardation (obtained from Fig. 1 ) in the different layers. The peak (obtained by a Gaussian fit) of each distribution was regarded as the corresponding retardation in each layer. Note that the retardation measured in layer 4 was higher than that measured in layer 3, probably because of the rather random retardations obtained in layer 4 (Fig. 2C) which broadens the distribution of retardation. Because the retardations provided by the algorithm used are always positive, the distribution is distorted and the center of gravity, which is determined by the fit, is shifted to larger values.
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To test the reproducibility of our system, we evaluated retardations and axis orientations and corresponding DPRs and DPAs in one subject from 10 different measurements at different days spread over a period of 3 months. At layer 3, we measured a retardation
= 17 ± 3° with a corresponding DPR = 17% ± 7% and an axis orientation
= 25 ± 11° and a corresponding DPA = 7% ± 3%. At layer 4, we measured a retardation
= 26 ± 5° with a corresponding DPR = 56% ± 4% and an axis orientation
= 28 ± 11° and a corresponding DPA = 32% ± 5%. Layer 2 showed results essentially similar to those of layer 3. The small errors (SD) of the different values indicate the good reproducibility of the system.
Tables 1 and 2 summarize results from different volunteers. From the data shown in Table 1 , a mean DPR of 14% ± 5% was obtained at layer 3 and a mean DPR of 54% ± 5% at layer 4. From the data in Table 2 , a mean DPA was obtained of 5% ± 2% at layer 3 and a mean DPA of 32% ± 8% at layer 4. Layer 2 showed results essentially similar to those in layer 3.
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| Discussion |
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Because the degree of polarization cannot be directly measured by OCT (as a coherent imaging technique, it always provides a measured degree of polarization equal to 1),38 we introduced a measure based on the local variability of the measured polarization state. Depolarization leads to a random polarization state in PS-OCT images (i.e., the polarization state varies between neighboring speckles in a random manner; therefore, the term polarization-scrambling is sometimes preferred instead of depolarization in the context of PS-OCT). To quantify this effect, we introduced the depolarization values DPR and DPA, which indicate the fraction of pixels in a certain area (in our study, a certain retinal layer) that exhibit a polarization state (quantified by retardation and an axis orientation) outside the range observed normally in the investigated area (layer). The DPR and DPA enable an objective differentiation between polarization-preserving or birefringent structures from depolarizing structures. Our system shows a good reproducibility of the polarization parameters
and
and the depolarization parameters DPR and DPA, tested over an extended period of 3 months. The summarized depolarizations in all volunteers (Tables 1 and 2) showed a clear difference between depolarizations obtained from the depolarizing layer and other retinal layers, indicating the usefulness of these parameters.
Several results of this study can lead to the conclusion that the depolarizing layer is the RPE. First of all, as can be seen in images of a healthy human fovea (Fig. 1) , the depolarizing layer represents the last strongly reflecting layer. Only a little light reaches deeper layers in healthy eyes, due to light absorption and scattering by the pigments of the RPE. Second, images of a patient with retinal and RPE detachment (Fig. 4) show that the depolarizing layer was detached at the location of RPE detachment (a similar observation was made in three other patients). This detachment excludes a possible association of the depolarizing layer with the choriocapillaris. Furthermore, the images indicate that the depolarizing layer cannot be associated with the photoreceptor layer, because this layer is detached from the depolarizing layer in areas of neurosensory retinal detachment (Fig. 4) . Third, images of a patient with RPE atrophy secondary to AMD (Fig. 5) show an enhanced penetration depth at locations where the RPE is missing. These locations coincide with locations of a missing depolarizing layer, which further confirms our associations. A similar effect was observed in another patient with RPE atrophy. Based on our findings, we suggest revising the associations in OCT images of the outer retinal layers35 36 37 with histologic structures. Although further investigations are needed, we think that the following association of the posterior layers is the most plausible: 1 is the ELM, 2 is the IPRL, 3 is the outer segment-RPE junction (OS/RPE), and 4 is the RPE. This description is supported by recently reported high transverse resolution B-scan images which were obtained with adaptive optics.39 These images show clearly a constant spacing between reflections within layer 2, which were attributed to retinal cones. The same spacing can be observed within layer 3 which suggests that layer 3 is part of the cones.
At the current state of investigation we can only speculate about the origin of the depolarization of light within the RPE. A possible explanation that needs further investigation, could be multiple scattering on large nonspherical particles40 (e.g., melanosomes) of the RPE. An observation that could support this hypothesis is the slightly more diffuse and thickened appearance of the RPE in the intensity image (Fig. 1D) compared with the appearance of layers 2 and 3. Because the thickness of the RPE is on the order of the resolution of our instrument, it should appear as a very sharp boundary in the case of single backscattering events. The other interior layers of the retina are rather transparent which can be observed by a weak backscattering signal of these layers (Fig. 1) . Therefore, multiple scattering should not occur within these layers. Note that the strong signal observed from layers 2 and 3 results from a specular reflection within the photoreceptors (which can only be seen if the light is coupled into the photoreceptors) and therefore yields a sharp boundary.
Our results could have impact on other polarization-sensitive imaging methods (such as SLP). SLP maps the retardation of the retina in a depth-integrated manner, assuming that light is backscattered from a posterior retinal layer that maintains the polarization state of reflected light.23 As we have clearly shown, a nonnegligible part of the total backscattered light intensity is backscattered from the RPE and depolarized. Although in eyes with intact posterior retina the fraction of depolarized light may be constant across the image (although this remains to be investigated) and a variability across the population is probably included in the normative database used in a diagnostic glaucoma scanning system (e.g., GDx; Carl Zeiss Meditec, GmbH), care has to be taken in patients with any RPE changes. In this case the fraction of depolarized light will vary across the image, possibly distorting the measured retardance and the derived nerve fiber layer thickness.
The capability of PS-OCT to identify the RPE in OCT images opens a new application for PS-OCT imaging. With this method, it is now possible to locate the RPE or its remains in macular diseases (e.g., AMD, CSC) in a depth-resolved manner, or to identify different kinds of detachments (inner retinal detachment, RPE detachment). This can be of importance in cases with distorted retinal structures, particularly in cases where an identification on the basis of conventional intensity based OCT (including UHR-OCT) images is not possible. Although further investigations are needed, we think that PS-OCT may be capable of distinguishing between the sclera and choroid (Fig. 5) and may therefore be an important tool in measuring the thickness of the choroid.
Furthermore, PS-OCT combined with fast 3D sampling, as made possible by newly developed Fourier-domain OCT,41 could become an essential tool in monitoring RPE diseases and in evaluating new treatment strategies.
| Acknowledgements |
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| Footnotes |
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Submitted for publication December 14, 2005; revised March 20 and June 16, 2006; accepted September 29, 2006.
Disclosure: M. Pircher, None; E. Götzinger, None; O. Findl, None; S. Michels, None; W. Geitzenauer, None; C. Leydolt, None; U. Schmidt-Erfurth, None; C.K. Hitzenberger, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C.
1734 solely to indicate this fact.
Corresponding author: Michael Pircher, Center for Biomedical Engineering and Physics, Medical University of Vienna, Waehringerstrasse 13 1090 Vienna, Austria; michael.pircher{at}meduniwien.ac.at.
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