Polarization sensitive optical coherence tomography (PS-OCT) is a functional extension of OCT. In addition to imaging based on tissue reflectivity, PS-OCT also enables depth-resolved mapping of sample polarization properties such as phase-retardation, birefringent axis orientation, Stokes vectors, and degree of polarization uniformity (DOPU). In this study, PS-OCT was used to investigate the polarization properties of melanin. In-vitro measurements in samples with varying melanin concentrations revealed polarization scrambling, i.e. depolarization of backscattered light. Polarization scrambling in the PS-OCT images was more pronounced for higher melanin concentrations and correlated with the concentration of the melanin granules in the phantoms. Moreover, in-vivo PS-OCT was performed in the retinas of normal subjects and individuals with albinism. Unlike in the normal eye, polarization scrambling in the retinal pigment epithelium (RPE) was less pronounced or even not observable in PS-OCT images of albinos. These results indicate that the depolarizing appearance of pigmented structures like, for instance, the RPE is likely to be caused by the melanin granules contained in these cells.
©2012 Optical Society of America
Ever since its first presentation in 1991, optical coherence tomography (OCT) has found applications in manifold fields . Its ability to perform non-invasive, non-contact imaging of weakly scattering structures with micrometer-scale resolution in real time has made OCT particularly attractive for investigating biological tissues in vivo [2–4]. The recent development of OCT techniques using Fourier domain detection has led to an immense increase of detection sensitivity which allowed for recording three-dimensional (3D) OCT data sets within a few seconds [5–8]. Consequently, OCT has been applied for volumetric high-resolution imaging in biomedical fields such as endoscopic investigations of the gastrointestinal tract or intravascular probing. The most prominent application of OCT, however, is in ophthalmology where it has become an essential tool in everyday clinical routine. OCT’s ability to resolve single retinal layers has been used for diagnosis of pathology, monitoring disease progression and follow-up imaging of response to therapy . Current developments of OCT technology pursue further improvement of image resolution, increasing imaging speeds and finding new ways to enhance image contrast.
Polarization sensitive (PS) optical coherence tomography is often referred to as a “functional” extension of OCT and provides depth-resolved imaging of the polarization properties of sample structures . Other than standard, intensity-based OCT, which provides image contrast based on the amount of light backscattered from different sites inside the sample, PS-OCT opens access to an additional contrast channel: By illuminating the sample with light in a well-defined polarization state and by using a polarization sensitive detection unit, not only reflectivity but also the polarization state of light can be assessed three-dimensionally. From the detected polarization state, phase retardation, optic axis orientation as well as Stokes vectors can be calculated [10–13]. PS-OCT has also been demonstrated for 3D assessment of depolarization and diattenuation as well as for imaging of complete Jones matrices and Müller matrices [14–17].
In the human eye, PS-OCT imaging allows one to distinguish structures with birefringent (e.g., corneal stroma, retinal nerve fiber layer), polarization preserving (e.g., stroma of the iris, photoreceptor layer), and depolarizing (e.g., pigment epithelia of iris and retina) properties [12,18–23]. It was shown that the polarization-scrambling, i.e. depolarizing, characteristics of the retinal pigment epithelium (RPE) can be used for segmenting that layer . In diseased retinas, this enables it to assess retinal disorders – such as drusen areas and volumes as well as the areas of atrophic zones in age-related macular degeneration (AMD) – in a quantitative way [24,25].
Recently, we investigated the depolarizing character of the RPE in the macula region of healthy volunteers . An example for macular PS-OCT imaging is shown in Fig. 1 . The RPE’s depolarization appeared most pronounced close to the center of the fovea and decreased in the periphery (Fig. 1(B) and (D)). However, the origin of the polarization scrambling is still unclear and could be caused by the outer morphology of the RPE cells or by their structural composition. In addition to regular cell organelles like mitochondria or cell nuclei, the human RPE cells contain different pigments in granular shape: melanin, lipofuscin, and composites of both . While the lipofuscin concentration has a minimum in the fovea and increases in the periphery of the posterior pole (Fig. 1(G)), the concentration of melanin shows the inverse characteristic with a peak concentration in the fovea (Fig. 1(H)) [27–29]. Since the macular distribution of melanin appears congruent with the depolarization pattern measured with PS-OCT , this implies that melanin could be the origin of the RPE’s polarization scrambling property. In order to verify this hypothesis, measurements in phantoms are necessary.
In this paper, we present a melanin granule model to investigate the origin of polarization scrambling in PS-OCT images (based on ). In addition to results from measurements in these pigment phantoms, we demonstrate 3D PS-OCT imaging of retinas with regular pigmentation and – for the first time – ocular albinism featuring irregular pigmentation.
2.1 Melanin phantoms
Melanin models of different concentrations were generated to simulate the effect of RPE pigmentation. From raw eumelanin (Sigma Aldrich), ellipsoidal melanin particles were produced by dissolving in a sodium carbonate solution and re-precipitating upon addition of calcium chloride . A narrow size distribution of the melanin particles with a maximum at ~530 nm was observed by dynamic light scattering (DLS) measurements which were performed with an ALV/CGS-3 compact goniometer system with an ALV/LSE-5003 light scattering electronics and multiple τ digital correlator at 25 °C (Fig. 2(D) ). This size corresponds well to the dimension of melanin granules in the human RPE cells reported in literature  (Fig. 2(F)). Scanning electron micrographs of the powder, which were recorded on a FEI Quanta 200 Mk2, revealed particles in the respective sizes and shapes (cf. Fig. 2(A)–2(C)).
Using distilled water, dispersions with melanin volume concentrations Cmel = Vmel:Vwater in the range from 1:1 to 1:100 were prepared. The suspensions were treated with ultrasound for 30 seconds in order to break up particle agglomerates and to prevent the formation thereof.
2.2 PS-OCT system
PS-OCT imaging of both melanin phantoms and human retinas was performed using our recently reported clinical PS-OCT instrument . In brief, the system design is based on a free-space Michelson interferometer with additional polarization optics. A broadband superluminescent diode (Superlum Diodes, Inc.) centered at 839 nm with an FWHM bandwidth of 58 nm was employed as a light source. Circularly polarized light was used to illuminate the sample. The sample arm power of ~700 μW was well below the safety limits for retinal imaging given by ANSI and IEC [33,34]. At the interferometer exit, the OCT signal was split into two orthogonal polarization components, which were simultaneously detected by two identical spectrometer units. Every readout of the spectrometer line scan cameras yielded the spectral data necessary to compute not only depth-resolved reflectivity, but also phase retardation, optic axis orientation and Stokes vectors for one depth profile . Operating at an A-line rate of 20 kHz, the system sensitivity was ~98 dB for each polarization channel.
The PS-OCT system includes an additional detection channel, which allows it to record scanning laser ophthalmoscope (SLO) images by detecting the horizontally polarized light portion of light returning from the sample into the source arm. The PS-OCT and SLO subsystems share the same light source and sample arm optics. Controlled by a field programmable gate array (FPGA), the system can record and display OCT and SLO images in an alternate fashion, which allows for real-time monitoring of the retinal position during alignment of the eye under investigation.
Using this PS-OCT system, 3D data sets (1024 × 64 × 1024 voxels, 12 × 12 × 3.3 mm3, orientation: x × y × z) of melanin suspensions were recorded. The liquid suspensions were imaged inside of a cylindrical glass container immediately after ultrasonification (Fig. 2(E)). The samples were imaged in 3D in order to be insensitive to container alignment in x- and y-direction. Evaluation of a PS-OCT data set recorded in the empty container confirmed the polarization preserving characteristics of the container material.
For retinal imaging, 3D data sets of both 1024 × 64 × 1024 and 512 × 128 × 1024 voxels (6.2 × 6.7 × 3.3 mm3) were recorded using the PS-OCT instrument. Healthy subjects as well as albino patients were imaged. The study protocol adhered to the tenets of the declaration of Helsinki and was approved by the ethics committee of the Medical University of Vienna. Written informed consent was obtained from each subject prior to the study.
2.3 Quantification of depolarization – degree of polarization uniformity
In order to assess polarization scrambling in PS-OCT images, a quantity called degree of polarization uniformity (DOPU) was used. For each B-scan, the spectral signals recorded at the two exits of the PS-OCT instrument were processed using regular SD-OCT processing including background subtraction, remapping to equidistant sampling in k-space, numerical dispersion compensation, and Fourier transformation . The Stokes vector elements (I, Q, U, V)T were calculated from the amplitudes and phases of the two polarization channels’ OCT signals for each image pixel. The first Stokes vector element I (corresponding to backscattered intensity) was used to identify pixels with low intensity, which consequently were excluded from further calculation. The Stokes vector elements of the remaining pixels were then normalized and averaged in a sliding evaluation window yielding Qmean(x,y,z), Umean(x,y,z), and Vmean(x,y,z). The degree of polarization uniformity, DOPU, was then computed as 
3.1 Melanin phantoms
PS-OCT imaging was performed in melanin suspensions with 13 different volume concentrations ranging from Cmel = 0.01 to Cmel = 1. Figures 3(A) and (B) show reflectivity B-scan images of the suspensions with Cmel = 1:60 and Cmel = 1:3, respectively. The corresponding DOPU B-scan images are shown in Figs. 3(C) and (D). The DOPU images are color coded from blue (DOPU = 0) to red (DOPU = 1). Pixels with intensities below a threshold set to ~3.5 standard deviations of the noise intensity above the mean noise level are displayed in grey. While the reflectivity images exhibit rather similar appearance, obvious changes can be observed in the DOPU images for different Cmel. In order to evaluate the depolarization characteristics of the melanin suspension, the average DOPU was computed for the different concentrations Cmel in an area of 5.86 (x) × 0.84 (z) mm2 (500 (x) × 260 (z) pixels).
For high melanin concentrations, Cmel = 1:1 to 1:5, DOPU values are rather constant at 0.39 ± 0.01. These low DOPU values indicate strong polarization scrambling, i.e. the highly depolarizing characteristic of the suspension with high melanin concentration. Starting from Cmel = 1:7.5, a monotonic increase of DOPU values can be observed as Cmel decreases. At low melanin concentrations, high DOPU values indicate less depolarization in the phantoms.
3.2 Depolarization imaging in the normal and albino human retina
In the previous section, we demonstrated the dependence of depolarization in PS-OCT images on the pigment concentration in melanin phantoms. Next, we show examples of PS-OCT images in human subjects with different retinal pigmentation.
Retinal PS-OCT images of a normal subject are shown in Fig. 4 . In the reflectivity B-scan image, the retinal layers appear as known from standard OCT (Fig. 4(A)). Figure 4(D) shows details of the outer retinal layers: The layers associated with the photoreceptors – i.e., the external limiting membrane (ELM), the junction of the inner and outer photoreceptor segments (IS/OS), and the end tips of the photoreceptors (ETPR) – can be observed as thin, hyperreflective lines. Posterior to the photoreceptors, the RPE appears as a diffuse, hyperreflective layer. In the corresponding phase retardation images, rather constant values can be observed in the polarization preserving outer retinal layers (Figs. 4(B) and 4(E)). In contrast, the RPE exhibits strongly varying retardation values, which manifest in a dotted appearance with pixel colors ranging from blue over green to orange. This polarization scrambling characteristic of the RPE is also obvious in the DOPU images (Figs. 4(C) and 4(F)). While most retinal layers exhibit DOPU values close to 1, indicating rather uniform polarization, depolarization in the RPE manifests in lower DOPU values.
PS-OCT imaging was also performed in the eyes of two subjects with ocular albinism of different stages of lack of pigmentation. However, obvious differences in the appearance of the RPE can already be observed in albino patient 1 presenting with mild depigmentation. While apart from the obvious foveal hypoplasia, the inner retinal layers look similar to the corresponding layers in normal subjects, obvious differences can be observed in the RPE/BM complex in the reflectivity B-scan images (Figs. 5(A) and 5(D)): The previously fuzzy hyperreflective structure now appears as two tightly spaced thin lines. Further, polarization scrambling is less obvious in the phase retardation images (Figs. 5(B) and 5(E)). This translates into unusual, high DOPU values in the RPE, and thus into a more uniform shading of all retinal layers including the RPE (Figs. 5(C) and 5(F)).
Even more pronounced differences are apparent in another albino patient (Fig. 6 ). While also in this patient the RPE/BM structures can be observed as clearly separated lines rather than one diffuse layer (Figs. 6(A) and 6(D)), their different appearance in the PS-OCT images is evident at first glance: Neither in the phase retardation images (Figs. 6(B) and 6(E)) nor in the DOPU images (Figs. 6(C) and 6(F)), the specific depolarization contrast can be observed in the RPE. Looking at this albino patient’s RPE in the retardation and DOPU images, the RPE appears rather blue and reddish, respectively – just like the polarization preserving photoreceptors.
PS-OCT has emerged as a functional extension of OCT. Imaging the sample’s polarization properties has enabled additional image contrast and quantitative measurements. While tissue birefringence had already been a subject of investigations in early reports on PS-OCT [10,37,38], polarization scrambling in PS-OCT has been discovered and investigated only recently [20,26,39]. In the human eye, depolarization was found in the pigment epithelia of the retina and iris, as well as in the choroid. Depolarization in the RPE was used as a tissue specific contrast and further as a useful tissue property for segmentation of this layer . The distribution of the depolarizing appearance of the RPE was found to be congruent with the distribution of the melanin granules . As a logical consequence to these findings, we performed PS-OCT measurements in melanin phantoms in order to investigate the polarization properties of isolated melanin granules in various concentrations.
The results detailed in section 3.1 showed a strong correlation between melanin granule concentration and polarization scrambling. For high melanin concentrations in the phantoms, depolarization manifested in low DOPU values, saturating with a plateau at melanin concentrations Cmel ≥ 0.2. For lower concentrations of the ellipsoidal melanin particles in the water suspension, an exponential relationship between Cmel and DOPU values can be observed: where A = −0.1408 and B = −0.144 were found by logarithmic fitting of the experimental data (R2 = 0.984). These results suggest that there may be a connection between the polarization scrambling observed in the human RPE and the melanin granules contained in the RPE cells.
The polarization state of light backscattered from tissue depends on the size, shape, refractive index, and concentration of the scattering particles as well on the polarization state of incident light [40–42]. While already single scattering events can modify the polarization state of backscattered light, the effective, accumulated polarization state after multiple such scattering events may be random, i.e. depolarized. In order to illustrate the effect of multiple scattering events on the PS-OCT signal, a standard sample was used [40,42,43]. In exact analogy to the experiments with the melanin phantoms described above, suspensions of polystyrene spheres (Sigma Aldrich) with 1.1 µm mean particle size were investigated. PS-OCT measurements were performed at different latex bead concentrations. The results shown in Fig. 7 reveal a similar dependence of depolarization on the particle concentration: On one hand, severe depolarization manifests as a plateau for higher concentrations of polystyrene spheres. For lower microparticle concentrations, on the other hand, an increase of DOPU values can be observed. Note, however, that the DOPU at the plateau settles at a slightly higher value. Also, the break of slope (between the logarithmic decrease and the plateau) occurs at a lower volume concentration. These differences between the depolarization characteristics of the polystyrene and melanin samples may be attributed to differences in shape and optical properties. However, the overall similarity of the PS-OCT measurements in the suspensions of latex beads to those in melanin granules indicates the similar relationship between scattering and depolarization. Further in-depth investigations of depolarization and its dependence on particle shape, size and concentration are to be exploited in future PS-OCT studies.
Retinal PS-OCT imaging in normal subjects yields a repeatable pattern with high DOPU values (i.e., no depolarization) in the neural retina and low DOPU values (i.e., strong depolarization) in the RPE (cf. e.g [14,26], and Figs. 1 and 4). In subjects with RPE disorders, such as patients suffering from late-stage non-exudative AMD (geographic atrophy), the depolarizing layer vanishes wherever the RPE cells are gone [19,24,25]. In contrast, highly pigmented choroidal naevi appear as polarization scrambling structures . Here, we are filling the missing link between the complete lack of RPE cells and the presence of unusual ocular pigmentation by investigating PS-OCT images of albino patients. The effect of irregular pigmentation of the RPE cells can be observed as a reduction or as a complete lack of polarization scrambling in the RPE in Figs. 5 and 6, respectively.
Albinism is an inherited disorder of melanin biosynthesis . Structural abnormalities resulting from the disrupted normal retinal development, such as foveal hypoplasia as one of the more commonly associated ocular phenotypes, have been investigated using standard spectral domain OCT [46,47]. While the absent or reduced melanin pigment in the fundi was not imaged with polarization contrast in those studies, a similar appearance of the RPE in the fovea centralis – two tightly spaced thin layers rather than one diffuse layer at the level of the RPE (cf. Figs. 4–6) – was observed . It is remarkable that similar features of the RPE/BM complex were observed in the periphery of the macula in normal subjects using ultrahigh resolution OCT [48,49]. The structural differences between normals and albino patients may be connected to a lower level of highly scattering melanin pigment where the RPE/BM complex appears less fuzzy. The fuzzy appearance may be explained by multiple scattering of light by the melanin granules. Multiple scattering effects could also be responsible for the thickened, smeared out appearance of the RPE, which is actually a single cell layer with reported thicknesses of only 10-16 µm in the macular region [28,32,50]. Furthermore, multiple scattering or scattering at particles with dimensions close to the wavelength are possible causes of the light’s polarization changes that can be observed as polarization scrambling. Just like the previously discussed reduction of polarization scrambling in the peripheral RPE, the structural observations are also in agreement with the fact that the normal melanin pigmentation has a maximum in the fovea centralis (cf. Fig. 1(H)).
Our PS-OCT results agree well with earlier reports using other techniques: Ultrastructural investigations of macular pigmentation have shown that there is an inverse relationship between RPE lipofuscin concentration and RPE melanin concentration : While RPE melanin has a maximum in the fovea centralis, the amount of RPE lipofuscin has a consistent dip there . Furthermore, it was found in an animal study that near-infrared autofluorescence associated with RPE melanin was observable in a pigmented rat retina and almost not visible in the albino rat retina . This result was in agreement with the lack of photoacoustic signals linked to RPE melanin in albino rat retinas, while the RPE was well visible in pigmented rats . In contrast, lipofuscin autofluorescence was observed in the RPE of both pigmented and albino rats [52,53]. Similar findings were reported in a histopathological study of human oculocutaneous albinism . All of these results are in excellent agreement with the results from PS-OCT imaging presented in this paper and underscore the thesis that polarization scrambling observed in the RPE is connected to the melanin contained in these cells.
Using ultrastructural morphometry, Feeney-Burns et al. found that on average, 3.5 – 8% of a human RPE cell is occupied by melanin granules . From our phantom measurements (Fig. 3), average DOPU values between 0.49 and 0.61 would be expected for Cmel = 0.035 to 0.08. These numbers are in good agreement with the actual DOPU values observed in the normal retinas (green to blue mapping in Figs. 1(B), 1(D) and 4(C)) as well as with our recently reported investigation of the macular depolarization patterns in the human RPE . There, mean DOPU values between 0.39 and 0.60 were measured in the central and peripheral regions of the macula, respectively. This consistence between the morphometric investigations of the RPE anatomy and the depolarization measurements indicate that PS-OCT could enable in vivo quantification of melanin concentration in the RPE and possibly other pigmented structures.
The results presented in this study are the first PS-OCT imaging results in the human retina in albinism. Only very recently, PS-OCT was performed for the first time in the retina of pigmented and albino rats . In good agreement with the PS-OCT images in humans presented here, the retina in albino rats lacked polarization scrambling structures, whereas strong depolarization was observed in the highly melanin pigmented RPE and choroid. Polarization scrambling in the human choroid has not yet been investigated in detail, and also a more in-depth study of PS-OCT in human albino patients could help to shed some more light on the relationship of (ocular) pigmentation and polarization sensitive imaging. So far, PS-OCT imaging has just been used to segment depolarizing structures like the RPE in a binary fashion. Using quantitative depolarization data such as the DOPU, it may be possible to assess pigmentation irregularities (for instance, mild RPE depigmentation in early stage dry AMD) not only with high sensitivity but also in 3D and in real-time.
Having such a diagnostic tool for 3D quantification of melanin would not only have great impact on retinal diagnostics but could also be of great value for assessment of pathology and tissue health of other pigmented structures. With PS-OCT, polarization scrambling was also detected in the iris pigment epithelium and in the human skin [23,56]. Compared to other techniques used for pigment specific imaging such as (auto)fluorescence imaging, PS-OCT enables depth-resolved imaging rather than integrating signals from all depths for one transverse position. Recently, photoacoustic microscopy (PAM) was demonstrated for depth-resolved imaging of melanin in the human eye [53,57]. PAM is a novel imaging technique based on the spatially resolved detection of optical absorption. However, unlike for OCT, expensive short pulse light sources are required for PAM. In contrast to PAM, PS-OCT could serve as a single modality capable of both micrometer scale resolution structural imaging and tissue-specific mapping in 3D.
In this study, we investigated the polarization properties of melanin with PS-OCT. Melanin phantoms were prepared from ovoid melanin particles suspended in water at different concentrations. PS-OCT images of these suspensions revealed a correlation between the phantoms’ polarization scrambling characteristics and the melanin concentration. Depolarization (manifesting in low DOPU values) was more pronounced for higher concentrations of melanin, and decreased for lower melanin concentrations. The polarization scrambling character of the melanin suspensions was in analogy to that of pigmented ocular structures like the RPE, which contain melanin granules of similar size and shape. Therefore, it is likely that the depolarizing appearance of the pigment epithelia of iris and retina be caused by the melanin granules contained within. Retinal PS-OCT imaging in albino patients and subjects with normal pigmentation revealed pronounced polarization scrambling in the normal RPE, while less or even absent depolarization was observed in the albinos’ RPE. These results suggest that PS-OCT might be a powerful technique for 3D imaging of pigmented structures.
The authors thank Christoph Wölfl at Medical University of Vienna as well as David Stifter and Martin Wurm at Recendt GmbH for their outstanding technical support. Fruitful discussions with Chao Zhou, WooJhon Choi, and James G. Fujimoto at Massachusetts Institute of Technology are gratefully acknowledged. Financial support from the European Union (project FUN OCT, FP7 HEALTH, contract no. 201880) and from the Austrian Science Fund (FWF grant P19624-B02) is gratefully acknowledged.
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