In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy

Abstract

The transparency and mechanical strength of the cornea are related to the highly organized three-dimensional distribution of collagen fibrils. It is of great interest to develop specific and contrasted in vivo imaging tools to probe these collagenous structures, which is not available yet. Second Harmonic Generation (SHG) microscopy is a unique tool to reveal fibrillar collagen within unstained tissues, but backward SHG images of cornea fail to reveal any spatial features due to the nanometric diameter of stromal collagen fibrils. To overcome this limitation, we performed polarization-resolved SHG imaging, which is highly sensitive to the sub-micrometer distribution of anisotropic structures. Using advanced data processing, we successfully retrieved the orientation of the collagenous fibrils at each depth of human corneas, even in backward SHG homogenous images. Quantitative information was also obtained about the submicrometer heterogeneities of the fibrillar collagen distribution by measuring the SHG anisotropy. All these results were consistent with numerical simulation of the polarization-resolved SHG response of cornea. Finally, we performed in vivo SHG imaging of rat corneas and achieved structural imaging of corneal stroma without any labeling. Epi-detected polarization-resolved SHG imaging should extend to other organs and become a new diagnosis tool for collagen remodeling.

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

“…In addition, deep imaging inside biological tissues can strongly affect the polarization linearity of the excitation field that propagates through the overlying tissue layers before reaching the focal volume. Recently, it has been demonstrated that the birefringency of collagen strongly affects the polarization of the incident field and this effect has to be taken into account for a proper modelling of SHG polarization anisotropy measurements within thick tissues [122,123]. Further, deep tissue imaging inside strongly scattering media requires the positioning of the dichroic mirror as close as possible to the objective lens in order to maximize light collection in the detection path.…”

Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry

“…Incorporation of polarization-dependent analysis into SHG imaging has been used for the discrimination of different biological tissues (15), differentiation of tumor and normal tissues (16), and many other biological applications as well as the study of surfaces and crystals (17)(18)(19)(20)(21)(22)(23). Despite the success of previous polarization-dependent SHG imaging approaches, two major limitations exist.…”

Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains

“…PR-SHG measurements of monolayers at interfaces have previously been shown to enable discrimination between samples with similar nonlinear optical properties (Begue, Everly et al, 2009;Begue & Simpson, 2010;Begue, Moad et al, 2009). Further, polarization-dependent SHG microscopy has a rich history of enabling structural and orientational studies Amat-Roldan et al, 2010;Chang et al, 2011;Latour et al, 2012;Duboisset et al, 2012;Stoller et al, 2002;Mansfield et al, 2007;Nucciotti et al, 2009;Filippidis et al, 2009;Madden et al, 2011;Tuer et al, 2011;Brideau & Stys, 2012). PCA offers the advantages of simplicity, generality and the absence of required training when extracting the core features of high-dimensional data.…”