Since many real fibrous tissues have multilayer microstructures with layers aligned in different directions, we considered further samples consisting of two layers of cylindrical scatterers with different orientations and alignments, as shown in Fig. 3(b). It is known from a previous study4 that the birefringence induced by aligned cylindrical scatterers is affected by the diameter, the optical properties, and the alignment of the cylinders, as well as the scattering coefficient and thickness of the layer. In the simulations, we kept the parameters of the first layer constant and changed the scattering coefficient and the other parameters of the second layer to examine in detail how the scattering-induced anisotropy of the two layers affected the total retardance and the equivalent extraordinary axis of the sample. In Figs. 5(a) and 5(b), the cylinders were perfectly aligned without any fluctuations in the orientation. The horizontal axis represents the retardance of the second layer of cylindrical scatterers obtained by decomposing the simulated Mueller matrices. For Figs. 5(c) and 5(d), the orientations of the cylindrical scatterers followed a Gaussian distribution, whose full width at half maximum (FWHM) was . In the simulations, the diameter of the cylinder was , the first layer cylinder scattering coefficient was , the second layer cylinder scattering coefficient changed from to , and the thickness of the two layers was 1 cm, corresponding to a change in retardance from 0 to 1.14 rad. The different shapes show data for different intersection angles between the directions of the two layers of cylindrical scatterers. The solid lines represent the calculated results for BBS. We also investigated a mixed model with two types of cylindrical scatterers and different alignment orientations in the two layers. The simulation results in Fig. 5 were very similar to those shown Figs. 4(a) and 4(b). This good agreement confirmed that the anisotropic optical features from the coupling of the two layers of cylindrical scatterers with different orientations were approximately equivalent to the features observed for the combination of two pieces of birefringent media with different extraordinary axes.