As shown in the inset of
Figure 2A , the retina was simulated as two linear retarders, one for background tissue underlying the RNFL (R
1) and the other for nerve fiber bundles only (R
2). To take the imperfect extinction of the PC′A system into account, we added a weak depolarizer (D) after P. The transmission intensity of the simulated retina was calculated by Mueller calculus.
28 29 Bundle contrast was defined as
B = (
R b −
R g )/
R g , where
\(R_{b}\) was the intensity of a light beam transmitted through a bundle and its background and
\(R_{g}\) was the intensity of a light beam transmitted through background only. Parameters used in the simulation were based on retardance measurements of bundles and gaps in a subset of experiments. The simulation showed that
B strongly depended on
\({\delta}_{C{^\prime}}\) (Fig. 2A) . To achieve the most sensitive measurement of
B,
\({\delta}_{C{^\prime}}\) for the highest
B was used. For weak tissue birefringence, B is approximately a parabolic function of bundle retardance
\({\delta}_{B}\) . Due to depolarization, variation of B with
\({\delta}_{B}\) lies on a monotonic portion of the curve that is approximately linear for small changes in
\({\delta}_{B}\) (solid curve in
Fig. 2B ). In experiments, test images were taken at different
\({\delta}_{\mathrm{C}{^\prime}}\) near 180° until nerve fiber bundles were displayed as bright stripes with best contrast at each wavelength to be tested. Images at these
\({\delta}_{\mathrm{C}{^\prime}}\) were then collected two to three times per minute at 500 nm and every 10 to 20 minutes at 440, 600, 700, and 780 nm throughout an experiment. To ensure that images with best bundle contrast were recorded, we also obtained images at δ
C′± 5°. To compare measurements of the same bundles over time, an entire set of images was registered by horizontal and vertical translations. Approximately 10 rectangular measurement areas of 4 to 6 pixels each were chosen both on bundles and on nearby gaps between bundles, and the measured intensities of the pixels in each area were averaged. (Examples of bundle and gap areas are shown in
Figs. 3A and 5A .) Only bundles that were oriented near vertical and had approximately uniform surrounding gaps were chosen for analysis. To evaluate changes in birefringence, the contrast of each bundle area was calculated as
B ij = (
R bi −
R gj )/
R gj (
i, j = 1, 2, … ), where
R bi and
R gj were the average intensities of the
ith bundle and
jth gap areas, respectively. Note that bundle intensity
R b includes contributions of nerve fiber bundles and the underlying retina.
16 Average bundle contrast
\({\bar{B}}\) and the SEM were calculated from all
B ij (
i ×
j ≈ 100). Data analysis was implemented in MatLab (The MathWorks, Inc., Natick, MA).