The important biological role of collagen-based tissues and the changes produced in the fiber distribution under particular situations (surgery, pathology, external damage, etc.) require tools for the analysis of the collagen organization that might potentially help in early diagnoses. Since collagen structures provide efficient second harmonic generation (SHG) signals, SHG microscopy has emerged as a powerful technique to visualize collagen fibers and qualitatively discriminate normal from abnormal tissues. Here we propose a quantitative method based on the structure tensor to quantify the different organization of collagen patterns in SHG images of ocular tissues. Results show that well-organized collagen distributions present a high degree of isotropy (DoI), a dominant orientation (PO), and a low structural dispersion (SD). On the other hand, the PO vanishes when the collagen tissue is not organized as a consequence of an increase in the SD and a decrease in the DoI. The proposed method is also able to discriminate partially organized samples. The combination of SHG microscopy and the structure tensor is a useful method to objectively classify collagen distributions. Clinical applications of this technique could help in the diagnosis and tracking of pathologies related to collagen disorders in connective tissue.

Two-photon (2P) microscopy is a powerful tool for imaging and exploring label-free biological tissues at high resolution. Although this type of microscopy has been demonstrated in ex vivo ocular tissues of both humans and animal models, imaging the human eye in vivo has always been challenging. This work presents a novel compact 2P microscope for non-contact imaging of the anterior part of the living human eye. The performance of the instrument was tested and the maximum permissible exposure to protect ocular tissues established. To the best of our knowledge, 2P images of the in vivo human cornea, the sclera and the trabecular meshwork are shown for the very first time. Acquired images are of enough quality to visualize collagen arrangement and morphological features of clinical interest. Future implementations of this technique may constitute a potential tool for early diagnosis of ocular diseases at submicron scale.

A polarimetric second harmonic generation (SHG) microscope was used to analyze the dependence between polarization and SHG signal from collagen-based samples. A theoretical model was also developed to investigate the SHG intensity as a function of different polarization states for a set of quasiparallel fibers. Numerical simulations were compared to experimental SHG intensity values and a fairly good agreement was found. Linear polarized light produced periodical changes in the emitted SHG signal with a maximum of intensity corresponding to polarization parallel to the main orientation of the fibers, regardless the ratio of hyperpolarizabilities, $ρ$. A similar behavior was found for elliptical states located along a vertical meridian on the Poincaré sphere (i.e., null azimuth) although the modulation of the SHG signal was different. Our numerical calculations described a dramatic change in this regular trend when $ρ$ changed from positive to negative values. Moreover, we provide an experimental method (based on the analysis of the modulation of the SHG signal) to determine the value of the ratio $ρ$ and, consequently, to obtain information about the internal organization of the collagen fibers.

Corneal cross-linking (CXL) is a surgical procedure able to modify corneal biomechanics and stabilize keratoconus progression. Although it is known that CXL produces changes in corneal collagen distribution, these are still a topic of discussion. Here we quantitatively compare the corneal stroma architecture between two animal models four weeks after in vivo conventional CXL treatment, with second harmonic generation (SHG) imaging microscopy and the structure tensor (ST). The healing stage and the stroma recovery were also analyzed by means of histological sections. Results show that the CXL effects depend on the initial arrangement of the corneal collagen. While the treatment increases the order in corneas with a low level of initial organization, corneas presenting a fairly regular pattern are hardly affected. Histological samples showed active keratocytes in anterior and middle stroma, what means that the recovery is still in progress. The combination of SHG imaging and the ST is able to objectively discriminate the changes suffered by the collagen arrangement after the CXL treatment, whose effectiveness depends on the initial organization of the collagen fibers within the corneal stroma.

Second Harmonic Generation Microscopy: A Tool for Quantitative Analysis of Tissues | InTechOpen, Published on: 2016-09-21. Authors: Juan M. Bueno, Francisco J. Ávila, and Pablo Artal

Collagen organization has been analyzed at both external and internal scales by combining Stokes-vector polarimetry and second harmonic generation microscopy. A significant linear relationship between the diattenuation and the external collagen organization was found. The dominant orientation of the collagen fibers was found to run parallel to the axis of diattenuation. Information on the collagen chirality was obtained from the circular dichroism, which showed also a strong dependence with the internal collagen organization. The results show that certain polarimetric parameters might be useful to extract quantitative information and characterize collagen arrangement.