September 2004
Volume 45, Issue 9
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Glaucoma  |   September 2004
Variation of Peripapillary Retinal Nerve Fiber Layer Birefringence in Normal Human Subjects
Author Affiliations
  • Xiang-Run Huang
    From the Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.
  • Harmohina Bagga
    From the Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.
  • David S. Greenfield
    From the Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.
  • Robert W. Knighton
    From the Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.
Investigative Ophthalmology & Visual Science September 2004, Vol.45, 3073-3080. doi:10.1167/iovs.04-0110
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      Xiang-Run Huang, Harmohina Bagga, David S. Greenfield, Robert W. Knighton; Variation of Peripapillary Retinal Nerve Fiber Layer Birefringence in Normal Human Subjects. Invest. Ophthalmol. Vis. Sci. 2004;45(9):3073-3080. doi: 10.1167/iovs.04-0110.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. The retinal nerve fiber layer (RNFL) exhibits linear birefringence due to the oriented cylindrical structure of ganglion cell axons. The birefringence (Δn) depends on the density and composition of axonal organelles. The purpose of this study was to evaluate the distribution of birefringence around the optic nerve head (ONH) in normal subjects.

methods. Birefringence was calculated along circular scan paths around the ONH as Δn = R/T, where R is RNFL retardance measured by scanning laser polarimetry (SLP) and T is RNFL thickness measured by optical coherence tomography (OCT). OCT scans on a 3.4 mm diameter circle were obtained from 26 normal subjects aged 18 to 53 years. Scans on circles with various diameters were obtained from 17 of these subjects.

results. The average reproducibility of Δn measured on three separate days in four subjects was ±0.05 nm/μm. In most subjects Δn varied significantly along a circular path around the ONH, with maxima in superior and inferior bundles, minima temporally and nasally, and a mean of 0.32 ± 0.03 nm/μm. Δn profiles on circles of different diameter were similar, suggesting that Δn did not vary along nerve fiber bundles.

conclusions. RNFL birefringence varies with position around the ONH. This variation may result from known structural differences among nerve fiber bundles that serve different retinal regions. Constant Δn along bundles is consistent with this hypothesis. Measurements of RNFL birefringence may provide a means to detect early subcellular changes in glaucoma.

The retinal nerve fiber layer (RNFL) is formed by the unmyelinated axons of retinal ganglion cells gathered into bundles lying just under the retinal surface. 1 The RNFL is damaged in glaucoma and other optic nerve diseases, and various optical methods have been developed to assess RNFL loss. Among them, scanning laser polarimetry (SLP) incorporates ellipsometry into a confocal scanning laser ophthalmoscope to detect linear birefringence of the peripapillary RNFL, 2 3 and optical coherence tomography (OCT) uses interferometry of low-coherence light to generate cross-sectional images of the retina on a circular path around the optic nerve head (ONH), with the RNFL identified as a brightly reflecting layer lying just beneath the vitreoretinal interface. 4 5  
Linear birefringence is an optical property of many materials, in which light polarized in a direction with higher refractive index travels more slowly than light polarized in the perpendicular direction. The difference in refractive index, Δn, gives the birefringence. For a homogenous material, retardance (R), the delay experienced by the slower component, is proportional to thickness (T), and birefringence can be calculated as retardance per unit thickness: Δn = R/T. (Birefringence is a dimensionless number; we, however, use units of nanometers per micrometer to acknowledge explicitly the relation between retardance and thickness.) An array of parallel cylinders in a medium of different refractive index exhibits a type of birefringence called form birefringence. 6 Such an array behaves as a uniaxial crystal with the direction of highest refractive index parallel to the cylinders. The RNFL is generally assumed to exhibit form birefringence due to oriented cylinders at a spatial scale comparable to the wavelength of light (microtubules or axonal membranes, for example) and theoretical models of RNFL birefringence show that the volume fraction and relative refractive index of the relevant cylindrical organelles and the refractive index of the medium surrounding these organelles combine to determine Δn. 7 8 On this assumption, therefore, differences in Δn must necessarily reflect differences in the density or composition of the axons that make up the RNFL. 
Birefringence of the RNFL has been measured experimentally in several studies. Weinreb et al. 9 used scanning Fourier ellipsometry at 514 nm to measure the retardance at many retinal locations in two fixed macaque eyes and histology to determine RNFL thickness at the same locations. Although individual points showed considerable variation in the ratio of thickness to retardance, data were fit with a straight line passing through zero to obtain a slope of 7.4 μm of thickness per degree of retardance. Recognizing that the retardance represents a double pass through the RNFL and converting retardance expressed as a phase difference (degrees at 514 nm) to retardance expressed as a path length (in nanometers) yields Δn = 0.19 nm/μm. Huang and Knighton 10 measured the retardance of rat RNFL in vitro at multiple wavelengths (400–830 nm), and then measured the thickness of the same nerve fiber bundles in histologic sections. The calculated birefringence was similar across wavelength, averaging 0.23 nm/μm before and 0.19 nm/μm after glutaraldehyde tissue fixation. The wavelength invariance of birefringence justifies comparisons of studies performed at different wavelengths. Cense et al. 11 used polarization-sensitive OCT (PS-OCT) at 830 nm to measure the birefringence of human RNFL in vivo. A study at one location in one subject found Δn = 0.45 nm/μm, 11 and another study of several retinal locations in one subject found Δn ranging from 0.21 to 0.43 nm/μm. 12 Measurements on a circular scan around the ONH in two subjects ranged from 0.12 to 0.4 nm/μm. 13  
The large difference in reported birefringence may mean that RNFL tissue properties differ between species and even between different areas of the RNFL in the same eye. Structurally, in fact, the RNFL does differ between retinal areas. In primate retina the proportion of small axons is lower in nerve fiber bundles nasal to the ONH, higher in arcuate bundles, and highest in papillomacular bundles. 14 Furthermore, the glial content in the bundles varies across the retina. 15 This leads immediately to the question: Does RNFL birefringence reveal these known variations in RNFL structure? If it does, measurement of Δn may provide a means of “optical biopsy”—the ability to detect changes in internal structure that may precede cell death in the early stages of glaucoma. 
The preceding question motivated this study, which was based on the following two hypotheses: (1) birefringence varies across nerve fiber bundles—that is, Δn varies on circular paths around the ONH; and (2) birefringence does not vary along nerve fiber bundles—that is, axonal structure, and thus Δn, does not change rapidly with distance from the ONH. To test these hypotheses, we determined the variation of RNFL birefringence in the peripapillary retina by using SLP to measure RNFL retardance and OCT to measure RNFL thickness and then calculating Δn. A preliminary report of data obtained with a variety of OCT and SLP instruments (Huang et al., IOVS 2003;44:ARVO E-Abstract 3363) supported our hypotheses, as has a recent direct test with PS-OCT in two subjects. 13 For this study we used two commercially available instruments, StratusOCT (Carl Zeiss Meditec, Dublin, CA) and GDx-VCC (Laser Diagnostic Technologies, San Diego, CA), to determine the variation of RNFL birefringence in the peripapillary retina of 26 normal human subjects. 
Materials and Methods
RNFL Thickness Measurement
OCT (StratusOCT; Carl Zeiss Meditec) was used to measure T at 512 points along circular scans around the ONH (Fig. 1) . OCT provides a cross-sectional retinal image, in which the RNFL appears as a layer of high reflectivity (Fig. 1A) . The RNFL thickness along the scan path (Fig. 1B) was extracted from the image by the system (Stratus software, ver. 3.0; Carl Zeiss Meditec). The instrument also provides a video fundus image with superimposed scan path taken after each scan is completed (Fig. 1C) . We exercised care to acquire good-quality video images that showed major blood vessels around the ONH. 
RNFL Retardance Measurement
SLP (GDx VCC; Laser Diagnostic Technologies, Inc.) was used to measure R over a 20° × 40° area of the posterior pole, which included the macula and ONH (Fig. 2) . This instrument incorporates a variable corneal compensator (VCC) to minimize the effect of anterior segment birefringence (mostly from the cornea). The principles underlying the measurement procedure, which includes determining and compensating the anterior segment retardance and then measuring the RNFL retardance, have been described elsewhere. 3 Briefly, the anterior segment retardance is determined from an image acquired with the retardance of VCC set to zero (i.e., no compensation). The pattern of macular retardance depends on the interaction between anterior segment birefringence and the radially oriented birefringence of the Henle fiber layer. Anterior segment birefringence is derived from a retardance profile around the fovea. The VCC is then set to neutralize anterior segment birefringence, and subsequent images measure the retinal retardance. 
The GDx VCC displays a fundus image (Fig. 2A) and retardance image (Fig. 2B) that are stored as digital files. These files were exported for further analysis. The pixels in the retardance image were multiplied by a suitable calibration factor (provided by Laser Diagnostic Technologies, Inc.) to give single-pass retardance in nanometers. 
Extraction of Retardance along the OCT Scan Path
Image processing and data analysis were implemented in commercial software (MatLab, ver. 6.5; The MathWorks, Inc., Natick, MA). 
SLP provides retardance values for a large area around the ONH, whereas OCT gives the RNFL thickness only along a circular scan path. To calculate birefringence, retardance along the corresponding scan path had to be extracted. To locate the OCT scan path in the SLP retardance image, the SLP fundus image (Fig. 2A) was registered to the OCT video image (Fig. 1C) by adjusting image scaling, rotation, and translation in custom software that used a combination of automatic algorithms with manual fine tuning. 16 The size and position of the OCT scan circle were then transferred to the registered SLP fundus and retardance images. 
We used blood vessel locations to check and adjust the image registration. A binary image of blood vessels in the SLP fundus image was obtained by applying a two-dimensional filter and threshold algorithm to the registered SLP fundus image (Fig. 3A) . 17 In the OCT scan, major blood vessels in the retinal cross-sectional image (Fig. 3B , arrows) were identified by their shadows in the outer retina. Blood vessel positions along the scan path in the binary SLP image were plotted as a profile (Fig. 3B , solid line). With well-registered images, the vessel positions in the profile corresponded to the shadow positions, as demonstrated in Figure 3B . Otherwise, SLP image registration was adjusted until the blood vessel positions and shadows were well matched. The retardance profile was derived from the scan circle in the registered retardance image. 
Figure 3C shows a retardance profile around a scan circle. Retardances measured on blood vessels did not represent the full thickness of the RNFL and were excluded from the profile (Fig. 3C , dots). 
Removal of the Residual Birefringence of the Compensated Anterior Segment
Although the variable compensator in SLP provided individualized compensation of anterior segment birefringence, residual birefringence existed in all subjects and typically appeared in a compensated image as a weak bow-tie pattern in the macula (Fig. 2B) . In a manner similar to the determination of anterior segment birefringence, 3 residual birefringence was estimated from the macular pattern by fitting a smooth curve to the retardance profile on a circle around the fovea. The smooth curve varied above and below the average macular retardance by an amount equal to the residual retardance. Residual anterior retardance could change the values of measured RNFL retardance (either up or down) and affect the calculated Δn. We therefore used the macular retardance pattern to perform a correction on the RNFL profile. We assumed that the same variation of retardance seen in the macular profile also occurred in the RNFL profile—that is, we assumed that nerve fiber bundles were radially distributed around the ONH and simply subtracted from the RNFL profile the variation of the smooth curve fit to the macular profile. The assumption that nerve fiber bundles are radially oriented around the ONH is not strictly true, especially for the arcuate bundles, but calculations showed that a variation of ±10° had negligible effect for the small corrections used. This study used corrected RNFL retardance profiles for all birefringence calculations. 
Calculation of Δn
In this study, the RNFL was assumed to be homogenous with depth, and birefringence was calculated as Δn = R/T, where R was the single-pass RNFL retardance measured by SLP and T was the RNFL thickness measured by OCT. Evidence for homogeneity comes from electron microscopy, which shows that although axon diameters range from 0.1 to 3 μm there is no obvious stratification of axons by size, 14 15 and from PS-OCT, which shows linear variation of retardance with depth. 11 12 13  
Subjects
This research adhered to the tenets of the Declaration of Helsinki and was approved by the University of Miami Institutional Review Board. All subjects gave informed consent after the nature and possible consequences of the study were explained. Subjects were judged normal if they had no history of ocular disease, intraocular pressure less than or equal to 21 mm Hg by Goldmann applanation tonometry, normal optic disc appearance, normal perimetry, and refractive errors within ±6 D. 
Subjects were recruited from two study sites, both having the same models and software versions of StratusOCT and GDx VCC. Thirteen subjects were studied at each site (Table 1) . There was no significant difference in age between the two groups, but the residual retardance of anterior chamber birefringence compensation was significantly larger in group 1 (P < 0.001, t-test). At study site 1, one eye of each subject was randomly selected for imaging. Nine eyes were imaged with OCT at a scan radius of 1.73 mm, and four eyes were imaged at scan radii of 1.44, 1.69, 1.90, and 2.25 mm. To determine the reproducibility of the measurements, imaging was repeated on two additional days for these latter four eyes. At study site 2, both eyes of each subject were measured, with five subjects imaged with OCT at scan radii of 1.44, 1.60, 1.70, and 1.85 mm and eight subjects imaged with an additional radius of 1.95 mm. Data from OCT scan circles with 1.69-, 1.70-, and 1.73-mm radii were considered equivalent (3.4 mm diameter). For all subjects, SLP and OCT were performed on the same day. For repeated SLP, the anterior segment birefringence was determined once and used for setting the VCC for all sessions. All images were obtained by experienced operators. 
Results
Calculation and Reproducibility of Δn
Figure 4 shows examples of T and R profiles obtained from one subject on a 3.4-mm diameter circle around the ONH and the birefringence profile calculated from Δn = R/T. To estimate measurement reproducibility, this subject was imaged on three different days over a 3-month period. The three T profiles, obtained by an experienced operator from a subject with good fixation, showed excellent reproducibility (Fig. 4A) . To show the variation in the SLP measurement alone, one OCT scan path was used to extract an R profile from each of the three SLP images. The three R profiles obtained also showed excellent reproducibility (Fig. 4B) . Finally, all nine combinations of OCT and SLP images were registered separately and used to calculate nine profiles of Δn. The mean ± SD of the nine profiles is shown in Figure 4C . Reproducibility measured in three additional subjects was similar (Figs. 4D 4E 4F) . To produce a summary measure of the reproducibility of Δn, the squares of the standard deviation (the variances) at each point were averaged across the Δn profiles from the four subjects. The square root of this average—that is, the average standard deviation for measurements of Δn—was 0.052 nm/μm. 
Variation in RNFL Birefringence around the ONH
A remarkable feature of the calculated Δn shown in Figure 4C is that Δn was not constant, but rather varied significantly along a scan path that crossed nerve fiber bundles. That is, the variation of the Δn profile around the ONH was much larger than the variation of individual points. To explore further this variation across nerve fiber bundles, Δn profiles on a 3.4-mm diameter scan path were measured in 13 eyes of 13 subjects at one study site and 26 eyes of 13 subjects at the other site (Table 1) . For the subjects with both eyes measured, one eye of each subject was randomly selected for comparison. 
Overall appearance of the Δn profiles was similar in most eyes (23/26), with peaks in superior and inferior RNFL and valleys in temporal and nasal RNFL. Figure 5 shows profiles for 12 of 13 eyes in group 1 (left column) and 11 of 13 eyes in group 2 (right column). For these 23 eyes the averages of T, R and Δn over the scan path (profile means) as well as the average Δn in four 60° (2-clock-hour) sectors are summarized in Table 2 . The average birefringence—that is, the profile mean of Δn—was similar within a group (low standard deviation); the sector means within a group, however, were significantly different from each other (P < 0.001, F-test), reflecting the peaks and valleys in Figure 5 . The average T in group 1 was significantly higher than in group 2, and the average R was somewhat lower. As a consequence, Δn in group 1 was significantly higher than in group 2, especially in the superior and inferior sectors. 
One of the 13 eyes in each group was found to have less Δn variation than usual (Fig. 6 , left column). Δn did not show the superior and inferior maxima and the nasal and temporal minima typical of other eyes. Another eye in group 2 had higher than usual nasal values (Fig. 6 , right column). 
The two eyes of the 13 subjects in group 2 were compared. In general, birefringence profiles were mirror symmetric between eyes, and the intrasubject variance was similar in magnitude to the intersubject variance. 
Radial Variation in RNFL Birefringence
To test the hypothesis that birefringence does not vary along nerve fiber bundles, in four eyes of four subjects in group 1 and all 26 eyes of 13 subjects in group 2 we measured Δn profiles on peripapillary circles of different diameters. Figure 7A displays T profiles for one subject on four circles around the ONH. As expected, the RNFL thinned with increasing scan radius. The four OCT scan circles were transferred to the SLP retardance image to yield R profiles along the corresponding scan paths (Fig. 7B) . Retardance also decreased with increasing scan radius. The calculated Δn profiles, however, did not show systematic variation with circle radius (Fig. 7C) . Rather, with the exception of the superior and inferior peaks of the largest circle (thinnest RNFL), the Δn profiles were nearly the same. The average SD of the four Δn profiles in Figure 7C , computed as for Figure 4C , was 0.059. Similar behavior was seen in the Δn profiles of the other three subjects of Figure 4 (Figs. 7D 7E 7F) as well as the 26 eyes in group 2. The average standard deviation across all subjects was 0.046 nm/μm, which is comparable to the reproducibility (Fig. 4) and much less than the magnitude of Δn variation among bundles (Δn ranged from approximately 0.24 to 0.40 nm/μm around the ONH). This result suggests that, at least for the distance covered by the scan circles, Δn does not change significantly along nerve fiber bundles. 
Discussion
RNFL birefringence is an intrinsic property of the tissue and its Δn should depend critically on details of tissue ultrastructure. 7 8 This study combined SLP measurements of RNFL retardance (R) with OCT measurements of RNFL thickness (T) to test whether Δn = R/T would reveal the structural variation of the normal peripapillary RNFL. Profiles of Δn on circular paths around the ONH showed significant variation in most subjects (Figs. 4 5) , which quite plausibly may reflect the different distributions of axonal diameter and glial content seen histologically. 14 15 Profiles on paths of different radii were similar (Fig. 7) , consistent with the concepts that axons, once bundled together, tend to stay together as they converge on the ONH and that the internal structure of axons varies slowly over the distances examined. 
Because both T and R were measured by commercially available devices, the method used in the current study for determining RNFL birefringence can have widespread application. The SLP and OCT images are easily acquired in a clinical setting. Calculation of Δn, however, required postprocessing with custom software. The precision of T and R measurements affect the birefringence calculated, and in this study, although the pattern of Δn variation was similar, a bias existed between the two groups measured with different instruments (Table 2) . Calibration between devices, therefore, is necessary, to compare precisely data from different studies. 
A legitimate concern in the derivation of Δn from measurements made with two different instruments is whether systematic inaccuracies in either could produce apparent variation in Δn when none actually exists. The similar peripapillary pattern in Δn across individuals (Fig. 5) means that any error would have to have a similar pattern, with T overestimated and/or R underestimated in nasal and temporal retina and T underestimated and/or R overestimated in superior and inferior retina. On the assumption that Δn was constant, we calculated that the systematic error in T would have to have been 40 μm to produce the observed variation in Δn, an error not consistent with the RNFL boundaries seen on visual inspection of the OCT images. (It is worth noting, however, that in some sectors of some subjects the RNFL intensity fell to values that made the posterior boundary of the RNFL difficult to distinguish from underlying tissue. This intensity decrease, which was probably caused by the directional reflectance of the RNFL, 18 could have caused errors in T due to failure of the OCT analysis algorithm to detect the true RNFL border.) Similarly, the systematic error in R would have to have been 20 nm, well above the SLP measurement error. The residual retardance of an incompletely compensated anterior chamber produces a patterned retardance bias, but across all subjects the residual retardance was never more than 10.5 nm, and we used a procedure to correct for most of this bias. It seems likely, therefore, that we have measured a true property of the RNFL. Confidence in our conclusion that Δn varies around the ONH also comes from a few subjects measured with PS-OCT, 11 12 13 a fundamentally different method in which Δn measured directly shows similar values and a similar magnitude of variation. Finally, the early in vitro data of Weinreb et al. 9 showed significant scatter around the regression used to characterize RNFL birefringence. Although not suggested by those investigators, spatial variation in Δn of the magnitude found in the current study could explain the scatter that they observed. 
The profiles of Δn around the ONH were similar in most subjects (Fig. 5) . The observed differences in profile shapes (e.g., Fig. 6 ) probably indicate anatomic variation in the spatial arrangement of the RNFL among individuals. Conversely, similar mean values reflected similar average tissue composition across subjects. 
Our finding that Δn varies around the ONH has important consequences for the interpretation of SLP; although the commercial instrument (GDx-VCC; Laser Diagnostic Technologies, Inc.) measures retardance, it expresses these measurements as RNFL thickness—that is, it incorporates implicitly the assumption that Δn is constant. The reported thicknesses cannot be correct everywhere, and changes in SLP measurements could result from changes in either anatomic thickness or birefringence of RNFL. The clinical utility of SLP, however, does not depend on its reported units, and comparisons of SLP parameters with normal values and with individual baseline values remain valid. 
RNFL birefringence may provide a means of “optical biopsy”—the ability to measure a property of the RNFL that characterizes its internal structure. The sensitivity of Δn to axonal ultrastructure suggests that an accurate measurement of Δn may be useful for detecting morphologic abnormalities caused by glaucoma. Such abnormalities may include gliosis or a partial loss of organelles, such as microtubules, that might cause Δn to decrease or shrinkage of ganglion cells, 19 20 21 which might cause Δn to increase. If morphologic change precedes irreversible loss of axons, birefringence measurements may provide an early indicator of glaucoma that may open a therapeutic window during which damage might be prevented. 
In summary, two commercially available technologies, OCT and SLP, have been used in combination to measure RNFL birefringence, an optical property that depends on the structure of nerve fiber bundles. We found that RNFL birefringence in normal subjects varies with position around the ONH, a variation that may result from known structural differences among nerve fiber bundles that serve different regions. Constant Δn along bundles is consistent with this idea. The dependence of Δn on structural variation offers hope that measurement of RNFL birefringence may be able to detect early subcellular changes in glaucoma. To explore this possibility, future studies must measure RNFL birefringence in patients with ocular hypertension and various degrees of glaucomatous damage. 
 
Figure 1.
 
RNFL thickness measurement by OCT. (A) OCT image of peripapillary retinal cross section. (B) RNFL thickness profile derived from the OCT image in (A). (C) Video fundus image of the peripapillary OCT scan path, which in all eyes proceeded from temporal retina through superior, nasal, inferior, and back to temporal retina (TSNIT in B).
Figure 1.
 
RNFL thickness measurement by OCT. (A) OCT image of peripapillary retinal cross section. (B) RNFL thickness profile derived from the OCT image in (A). (C) Video fundus image of the peripapillary OCT scan path, which in all eyes proceeded from temporal retina through superior, nasal, inferior, and back to temporal retina (TSNIT in B).
Figure 2.
 
RNFL retardance measurement from SLP images of a 20° × 40° portion of the posterior fundus. (A) Reflectance image. (B) Retardance image obtained with compensation for anterior segment birefringence. The nonuniform macular pattern reveals weak residual retardance of 7 nm. White horizontal bars: the slow axis of the residual birefringence oriented 0.6° counterclockwise from horizontal.
Figure 2.
 
RNFL retardance measurement from SLP images of a 20° × 40° portion of the posterior fundus. (A) Reflectance image. (B) Retardance image obtained with compensation for anterior segment birefringence. The nonuniform macular pattern reveals weak residual retardance of 7 nm. White horizontal bars: the slow axis of the residual birefringence oriented 0.6° counterclockwise from horizontal.
Figure 3.
 
A retardance profile was obtained from an OCT scan path transferred to the registered SLP image. (A) Binary image of the peripapillary blood vessel pattern derived from the fundus image in Figure 2A . The circle shows the location and the arrow the direction of the OCT scan path. (B) OCT image. Arrowheads: major blood vessels. Solid line: binary profile of the intersections of the scan path and blood vessels in (A). (C) Profile along the transferred OCT scan path of the single-pass retardance in the SLP image. Retardances on retinal vessels (dots) were excluded from further analysis.
Figure 3.
 
A retardance profile was obtained from an OCT scan path transferred to the registered SLP image. (A) Binary image of the peripapillary blood vessel pattern derived from the fundus image in Figure 2A . The circle shows the location and the arrow the direction of the OCT scan path. (B) OCT image. Arrowheads: major blood vessels. Solid line: binary profile of the intersections of the scan path and blood vessels in (A). (C) Profile along the transferred OCT scan path of the single-pass retardance in the SLP image. Retardances on retinal vessels (dots) were excluded from further analysis.
Table 1.
 
Subject Demographics
Table 1.
 
Subject Demographics
Group 1 (Study Site 1) (13 subjects; 13 eyes) Group 2 (Study Site 2) (13 subjects; 26 eyes)
Age (y)* 37 ± 9.5 (22–53) 32.5 ± 8.1 (18–49)
OCT scan radius (mm) 9 eyes: 1.73 10 eyes: 1.44, 1.60, 1.70, 1.85
4 eyes: 1.44, 1.69, 1.90, 2.25 16 eyes: 1.44, 1.60, 1.70, 1.85, 1.95
Residual retardance (nm)* 7.1 ± 1.9 (4–10.5) 4.7 ± 2.1 (1–9)
Figure 4.
 
Reproducibility of RNFL thickness, retardance, and birefringence on a 3.4-mm diameter circle around the ONH in one subject. Data measured on blood vessels were excluded. Data were acquired on three separate occasions over a 3-month period. (A) T profiles from three OCT scans; (B) R profiles from one OCT scan path registered to three separate SLP images; (C) mean (solid line) ± SD (dotted lines) of Δn calculated from the nine pairs of R and T profiles. The average SD (square root of the average variance) for this subject was 0.037 nm/μm. (D–F) Reproducibility of Δn profiles for three other subjects. Average SDs were (D) 0.075 (E) 0.043, and (F) 0.053 nm/μm.
Figure 4.
 
Reproducibility of RNFL thickness, retardance, and birefringence on a 3.4-mm diameter circle around the ONH in one subject. Data measured on blood vessels were excluded. Data were acquired on three separate occasions over a 3-month period. (A) T profiles from three OCT scans; (B) R profiles from one OCT scan path registered to three separate SLP images; (C) mean (solid line) ± SD (dotted lines) of Δn calculated from the nine pairs of R and T profiles. The average SD (square root of the average variance) for this subject was 0.037 nm/μm. (D–F) Reproducibility of Δn profiles for three other subjects. Average SDs were (D) 0.075 (E) 0.043, and (F) 0.053 nm/μm.
Figure 5.
 
Most subjects showed variation in Δn on a path across nerve fiber bundles. (A, D) T profiles; (B, E) R profiles; and (C, F) Δn profiles on a 3.4-mm diameter circle around the ONH in 12 eyes in group 1 (A–C) and 11 eyes in group 2 (D–E). The Δn averages for these two groups were 0.35 ± 0.04 and 0.29 ± 0.02 nm/μm, respectively.
Figure 5.
 
Most subjects showed variation in Δn on a path across nerve fiber bundles. (A, D) T profiles; (B, E) R profiles; and (C, F) Δn profiles on a 3.4-mm diameter circle around the ONH in 12 eyes in group 1 (A–C) and 11 eyes in group 2 (D–E). The Δn averages for these two groups were 0.35 ± 0.04 and 0.29 ± 0.02 nm/μm, respectively.
Table 2.
 
Profile Means of T, R, and Δn and Sector Means of Δn
Table 2.
 
Profile Means of T, R, and Δn and Sector Means of Δn
Group 1 (12 eyes) Group 2 (11 eyes) P
Profile means*
T (μm) 97.80 ± 7.80 105.90 ± 11.60 <0.001
R (nm) 32.50 ± 3.40 30.10 ± 2.90 0.016
 Δn (nm/μm) 0.35 ± 0.04 0.29 ± 0.02 <0.001
Sector means of Δn (nm/μm)*
 Temporal 0.25 ± 0.07 0.21 ± 0.05 0.130
 Superior 0.42 ± 0.05 0.33 ± 0.04 <0.001
 Nasal 0.29 ± 0.08 0.23 ± 0.04 0.050
 Inferior 0.44 ± 0.07 0.37 ± 0.04 0.004
Figure 6.
 
Three subjects with different patterns of Δn. (A–C) Two subjects with less Δn variations than usual. The average Δn in these subjects was 0.31 (solid line) and 0.32 (dashed line) nm/μm, respectively. (D–F) One subject with higher than usual nasal values. The average Δn was 0.34 nm/μm.
Figure 6.
 
Three subjects with different patterns of Δn. (A–C) Two subjects with less Δn variations than usual. The average Δn in these subjects was 0.31 (solid line) and 0.32 (dashed line) nm/μm, respectively. (D–F) One subject with higher than usual nasal values. The average Δn was 0.34 nm/μm.
Figure 7.
 
Radial variation of RNFL birefringence in four subjects of group 1. (A) T profiles obtained on OCT scan circles with radii of 1.44, 1.69, 1.90, and 2.25 mm, as indicated, were used to determine the variation of Δn along nerve fiber bundles in the subject in Figures 4A 4B 4C . (B) Corresponding R profiles from a single SLP image. (C) Calculated Δn profiles. (D–F) Calculated Δn profiles of the three subjects in Figures 4D 4E 4F . Average SDs were (C) 0.059, (D) 0.072, (E) 0.058, and (F) 0.066 nm/μm.
Figure 7.
 
Radial variation of RNFL birefringence in four subjects of group 1. (A) T profiles obtained on OCT scan circles with radii of 1.44, 1.69, 1.90, and 2.25 mm, as indicated, were used to determine the variation of Δn along nerve fiber bundles in the subject in Figures 4A 4B 4C . (B) Corresponding R profiles from a single SLP image. (C) Calculated Δn profiles. (D–F) Calculated Δn profiles of the three subjects in Figures 4D 4E 4F . Average SDs were (C) 0.059, (D) 0.072, (E) 0.058, and (F) 0.066 nm/μm.
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Figure 1.
 
RNFL thickness measurement by OCT. (A) OCT image of peripapillary retinal cross section. (B) RNFL thickness profile derived from the OCT image in (A). (C) Video fundus image of the peripapillary OCT scan path, which in all eyes proceeded from temporal retina through superior, nasal, inferior, and back to temporal retina (TSNIT in B).
Figure 1.
 
RNFL thickness measurement by OCT. (A) OCT image of peripapillary retinal cross section. (B) RNFL thickness profile derived from the OCT image in (A). (C) Video fundus image of the peripapillary OCT scan path, which in all eyes proceeded from temporal retina through superior, nasal, inferior, and back to temporal retina (TSNIT in B).
Figure 2.
 
RNFL retardance measurement from SLP images of a 20° × 40° portion of the posterior fundus. (A) Reflectance image. (B) Retardance image obtained with compensation for anterior segment birefringence. The nonuniform macular pattern reveals weak residual retardance of 7 nm. White horizontal bars: the slow axis of the residual birefringence oriented 0.6° counterclockwise from horizontal.
Figure 2.
 
RNFL retardance measurement from SLP images of a 20° × 40° portion of the posterior fundus. (A) Reflectance image. (B) Retardance image obtained with compensation for anterior segment birefringence. The nonuniform macular pattern reveals weak residual retardance of 7 nm. White horizontal bars: the slow axis of the residual birefringence oriented 0.6° counterclockwise from horizontal.
Figure 3.
 
A retardance profile was obtained from an OCT scan path transferred to the registered SLP image. (A) Binary image of the peripapillary blood vessel pattern derived from the fundus image in Figure 2A . The circle shows the location and the arrow the direction of the OCT scan path. (B) OCT image. Arrowheads: major blood vessels. Solid line: binary profile of the intersections of the scan path and blood vessels in (A). (C) Profile along the transferred OCT scan path of the single-pass retardance in the SLP image. Retardances on retinal vessels (dots) were excluded from further analysis.
Figure 3.
 
A retardance profile was obtained from an OCT scan path transferred to the registered SLP image. (A) Binary image of the peripapillary blood vessel pattern derived from the fundus image in Figure 2A . The circle shows the location and the arrow the direction of the OCT scan path. (B) OCT image. Arrowheads: major blood vessels. Solid line: binary profile of the intersections of the scan path and blood vessels in (A). (C) Profile along the transferred OCT scan path of the single-pass retardance in the SLP image. Retardances on retinal vessels (dots) were excluded from further analysis.
Figure 4.
 
Reproducibility of RNFL thickness, retardance, and birefringence on a 3.4-mm diameter circle around the ONH in one subject. Data measured on blood vessels were excluded. Data were acquired on three separate occasions over a 3-month period. (A) T profiles from three OCT scans; (B) R profiles from one OCT scan path registered to three separate SLP images; (C) mean (solid line) ± SD (dotted lines) of Δn calculated from the nine pairs of R and T profiles. The average SD (square root of the average variance) for this subject was 0.037 nm/μm. (D–F) Reproducibility of Δn profiles for three other subjects. Average SDs were (D) 0.075 (E) 0.043, and (F) 0.053 nm/μm.
Figure 4.
 
Reproducibility of RNFL thickness, retardance, and birefringence on a 3.4-mm diameter circle around the ONH in one subject. Data measured on blood vessels were excluded. Data were acquired on three separate occasions over a 3-month period. (A) T profiles from three OCT scans; (B) R profiles from one OCT scan path registered to three separate SLP images; (C) mean (solid line) ± SD (dotted lines) of Δn calculated from the nine pairs of R and T profiles. The average SD (square root of the average variance) for this subject was 0.037 nm/μm. (D–F) Reproducibility of Δn profiles for three other subjects. Average SDs were (D) 0.075 (E) 0.043, and (F) 0.053 nm/μm.
Figure 5.
 
Most subjects showed variation in Δn on a path across nerve fiber bundles. (A, D) T profiles; (B, E) R profiles; and (C, F) Δn profiles on a 3.4-mm diameter circle around the ONH in 12 eyes in group 1 (A–C) and 11 eyes in group 2 (D–E). The Δn averages for these two groups were 0.35 ± 0.04 and 0.29 ± 0.02 nm/μm, respectively.
Figure 5.
 
Most subjects showed variation in Δn on a path across nerve fiber bundles. (A, D) T profiles; (B, E) R profiles; and (C, F) Δn profiles on a 3.4-mm diameter circle around the ONH in 12 eyes in group 1 (A–C) and 11 eyes in group 2 (D–E). The Δn averages for these two groups were 0.35 ± 0.04 and 0.29 ± 0.02 nm/μm, respectively.
Figure 6.
 
Three subjects with different patterns of Δn. (A–C) Two subjects with less Δn variations than usual. The average Δn in these subjects was 0.31 (solid line) and 0.32 (dashed line) nm/μm, respectively. (D–F) One subject with higher than usual nasal values. The average Δn was 0.34 nm/μm.
Figure 6.
 
Three subjects with different patterns of Δn. (A–C) Two subjects with less Δn variations than usual. The average Δn in these subjects was 0.31 (solid line) and 0.32 (dashed line) nm/μm, respectively. (D–F) One subject with higher than usual nasal values. The average Δn was 0.34 nm/μm.
Figure 7.
 
Radial variation of RNFL birefringence in four subjects of group 1. (A) T profiles obtained on OCT scan circles with radii of 1.44, 1.69, 1.90, and 2.25 mm, as indicated, were used to determine the variation of Δn along nerve fiber bundles in the subject in Figures 4A 4B 4C . (B) Corresponding R profiles from a single SLP image. (C) Calculated Δn profiles. (D–F) Calculated Δn profiles of the three subjects in Figures 4D 4E 4F . Average SDs were (C) 0.059, (D) 0.072, (E) 0.058, and (F) 0.066 nm/μm.
Figure 7.
 
Radial variation of RNFL birefringence in four subjects of group 1. (A) T profiles obtained on OCT scan circles with radii of 1.44, 1.69, 1.90, and 2.25 mm, as indicated, were used to determine the variation of Δn along nerve fiber bundles in the subject in Figures 4A 4B 4C . (B) Corresponding R profiles from a single SLP image. (C) Calculated Δn profiles. (D–F) Calculated Δn profiles of the three subjects in Figures 4D 4E 4F . Average SDs were (C) 0.059, (D) 0.072, (E) 0.058, and (F) 0.066 nm/μm.
Table 1.
 
Subject Demographics
Table 1.
 
Subject Demographics
Group 1 (Study Site 1) (13 subjects; 13 eyes) Group 2 (Study Site 2) (13 subjects; 26 eyes)
Age (y)* 37 ± 9.5 (22–53) 32.5 ± 8.1 (18–49)
OCT scan radius (mm) 9 eyes: 1.73 10 eyes: 1.44, 1.60, 1.70, 1.85
4 eyes: 1.44, 1.69, 1.90, 2.25 16 eyes: 1.44, 1.60, 1.70, 1.85, 1.95
Residual retardance (nm)* 7.1 ± 1.9 (4–10.5) 4.7 ± 2.1 (1–9)
Table 2.
 
Profile Means of T, R, and Δn and Sector Means of Δn
Table 2.
 
Profile Means of T, R, and Δn and Sector Means of Δn
Group 1 (12 eyes) Group 2 (11 eyes) P
Profile means*
T (μm) 97.80 ± 7.80 105.90 ± 11.60 <0.001
R (nm) 32.50 ± 3.40 30.10 ± 2.90 0.016
 Δn (nm/μm) 0.35 ± 0.04 0.29 ± 0.02 <0.001
Sector means of Δn (nm/μm)*
 Temporal 0.25 ± 0.07 0.21 ± 0.05 0.130
 Superior 0.42 ± 0.05 0.33 ± 0.04 <0.001
 Nasal 0.29 ± 0.08 0.23 ± 0.04 0.050
 Inferior 0.44 ± 0.07 0.37 ± 0.04 0.004
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