Tangent modulus and structural stiffness of the sclera were positively correlated with the inherent birefringence, a result consistent with our previous study 
. Collagen is the most important factor at sclera in relating birefringence to mechanical properties, because the highly organized collagen would result in high mechanical stiffness and resulting high birefringence.
The unpaired t-test and ANCOVA indicated the strain direction dependency of the alteration of the birefringence, i.e., the elevation of birefringence was higher with the meridional strain. A previous study of human skin elasticity, which has more well organized collagen fibers than sclera, showed that skin dermal birefringence is altered by stretching or shrinking 
. The study showed that stretching along the direction of relaxed skin tension lines (RSTL), to which the dermal collagen bundles are aligned, enhanced birefringence. However, stretching in a perpendicular direction to RSTL, and hence perpendicular to the collagen fiber bundle alignment, decreased the dermal birefringence. The authors suggested that the strain along the dermal collagen enhanced the alignment of the collagen fibers, and hence enhanced the dermal birefringence. In a similar manner, a strain perpendicular to collagen fibers destroyed alignment of collagen fibers, and reduced dermal birefringence 
. In the case of sclera, it is known that collagen fibers are highly organized and aligned along a circumferential ring around the scleral canal in the peripapillary region 
. Collagen fibers in the peripheral sclera are more irregularly arranged to form interwoven lamellae, and they exhibit a wide range of diameters, which vary depending on thickness 
. Most importantly, in contrast to the skin dermis, the region we measured (5-mm apart from the ONH) might have relatively randomly oriented collagen fibers. The second-harmonic-generation (SHG) microscopic images of porcine sclera (), which were obtained by a custom-made SHG microscope with an excitation wavelength of 800 nm and pulse duration of 100 fs 
, exemplified the variation of collagen fibers. A similar characteristic has also been known with rat sclera 
. With this random collagen structure, the strain in both directions would promote alignment of collagen fibers and increase the birefringence. This would explain our results of birefringence elevation by the strains in both directions.
Second-harmonic-generation microscopy images of porcine sclera.
It is said that collagen fibers become uncrimped by stretch
. This uncrimping of collagen provides flexibility to the sclera and is believed to provide protection against IOP elevation
. At the same time this uncrimping of the collagen enhances the alignment of collagen fibers, and increased the birefringence.
The strain-directional dependency of the elevation of the birefringence would suggest that collagen was meridionally aligned at the region we examined, although further detailed study is necessary to confirm this hypothesis.
The negative correlation between inherent birefringence and birefringence alteration indicates that higher inherent birefringence tends to have smaller birefringence alteration by strain. This negative correlation is explained as follows. The high inherent birefringence is indicative of highly aligned collagen fibers before applying strain. If collagen has already been well aligned before applying strain, an additional increase of alignment by the strain is limited, and hence the birefringence alteration is only moderate.
It should be noted that, in the birefringence analysis, we have assumed the birefringence was uniformed throughout the sample. However, in practice, the birefringence would have spatial variations. This failure of assumption would results in degradation of reliability of the birefringence quantification. Further development of a data analysis method which accounts for this variation of tissue birefringence will provide us more accurate scleral birefringence analysis.
Our eventual goal is to use the birefringence measurement to assess the biomechanics of human sclera in vivo. In this study, the birefringence was found to be altered by applying stain. Furthermore, the birefringence alteration depended on the strain direction and inherent birefringence. This implies that birefringence of a sclera in vivo would be expected to be affected not only by its mechanical property, but also by intraocular pressure and the shape of the eye globe. This study and future systematic studies will hopefully further characterize the relationship between scleral biomechanics and birefringence, with the eventual goal of making PS-OCT a valuable tool for the clinical diagnosis of glaucoma.