THG imaging experiments were performed with linearly polarized laser light. Indeed, no THG is obtained from isotropic media illuminated with circularly polarized light, even in the presence of heterogeneities.
24 Conversely, TH with circular incident polarization can be used to specifically detect anisotropy with micrometer resolution. For example, media with strong linear anisotropy (birefringence) can efficiently generate TH with circularly polarized excitation.
24 Layered stromal lamellae bear additional subtlety because adjacent lamellae exhibit perpendicular fibril orientations; that is, they may be viewed as stacked slabs with alternate anisotropy directions (noted
x and
y). When the excitation beam (propagating along direction
z) is focused near the interface between two
x-y lamellae, harmonic light from the first lamella emerges with a polarization state different from the one from the second lamella. This creates an effective heterogeneity so that even in the case of weak birefringence, the Gouy-shift–induced destructive interference
17,25 is not complete, and a THG signal is observed at the interface. Excitation with circular polarization is therefore a means to specifically detect anisotropy variations. In
Figure 4 and
Movie S5, a section of the anterior stroma was imaged using SHG, THG with linear incident polarization (lin-THG), and THG with circular incident polarization (circ-THG). Lin-THG reveals optical heterogeneities such as epithelial cell boundaries and stromal keratocytes, whereas circ-THG is obtained specifically from lamellae interfaces. When going from linear to circular excitation, cell signals are decreased by a factor of ≈20, whereas lamellae signals are increased two to three times, providing a convenient means to distinguish the two contributions. We point out that circ-THG and SHG signal maxima are generally not correlated, suggesting that the strongest SHG is obtained inside lamellae, whereas circ-THG is observed at interfaces.