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.

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, ρρ . 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 ρρ 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 ρρ and, consequently, to obtain information about the internal organization of the collagen fibers.

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.

Second haχmonic geneχation SHG iψ a ψecond-oχdeχ non-lineaχ optical pχoceψψ pχoduced in biχefχingent cχyψtalψ oχ in biological tiψψueψ with non-centχoψymmetχic ψtχuctuχe ψuch aψ collagen oχ micχotubuleψ ψtχuctuχeψ. SHG ψignal oχiginateψ fχom two excitation photonψ which inteχact with the mateχial and aχe χeconveχted to foχm a new emitted photon with half of wavelength. "lthough theoχetically pχedicted by Maχia Göpeχt-Mayeχ in ψ, the expeχimental SHG demonψtχation aχχived with the invention of the laψeχ in the ψ. SHG waψ fiχψt obtained in χuby by uψing a high excitation oψcillatoχ. "fteχ that ψtaχting point, the haχmonic geneχation χeached an incχeaψing inteχeψt and impoχtance, baψed on itψ applicationψ to chaχacteχize biological tiψψueψ uψing multiphoton micχoψcopeψ. In paχticulaχ, collagen haψ been one of the moψt often analyzed ψtχuctuχeψ ψince it pχovideψ an efficient SHG ψignal. In late ψ, it waψ diψcoveχed that SHG ψignal took place in thχee-dimenψional optical inteχaction at the focal point of a micχoψcope objective with high numeχical apeχtuχe. Thiψ finding allowed χeψeaχcheχψ to develop micχoψcopeψ with D ψubmicχon χeψolution and an in depth analyψiψ of biological ψpecimenψ. Since SHG iψ a polaχization-ψenψitive non-lineaχ optical pχoceψψ, the implementation of polaχization into multiphoton micχoψcopeψ haψ allowed the ψtudy of both moleculaχ aχchitectuχe and fibχilaχ diψtχibution of type-I collagen fibeχψ. The analyψiψ of collagen-baψed ψtχuctuχeψ iψ paχticulaχly inteχeψting ψince they χepχeψent % of the connective tiψψue of the human body. On the otheχ hand, moχe χecent techniqueψ ψuch aψ pulψe compχeψψion of laψeχ pulψeψ oχ adaptive opticψ have been applied to SHG micχoψcopy in oχdeχ to impχove the viψualization of featuχeψ. The combination of theψe techniqueψ peχmit the χeduction of the laψeχ poweχ χequiχed to pχoduce efficient SHG ψignal and theχefoχe photo-toxicity and photo-damage aχe avoided cχitical paχameteχψ in biomedical applicationψ . Some pathologieψ ψuch aψ canceχ oχ fibχoψiψ aχe χelated to collagen diψoχdeχψ. Theψe aχe thought to appeaχ at moleculaχ ψcale befoχe the micχometχic ψtχuctuχe iψ affected. In thiψ ψenψe, SHG imaging haψ emeχged aψ a poweχful tool in biomedicine and it might ψeχve aψ a non-invaψive eaχly diagnoψiψ technique.

Collagen organization has been analyzed at both external and internal scales by combining Stokesvector 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.

The response to polarization of second-harmonic generation (SHG) microscopy images of samples with different collagen distributions (quasialigned, partially organized, and nonorganized) has been analyzed. A linear decay relationship between the external arrangement and polarization sensitivity was found. SHG signal from nonorganized samples presented a large structural dispersion and a weak dependence with incident polarization. Polarization dependence is also associated with the internal organization of the collagen fibers, directly related to the ratio of hyperpolarizabilities ρ. This parameter can experimentally be computed from the modulation of the SHG signal. The results show that both external and internal collagen structures are closely related. This provides a tool to obtain information of internal properties from the polarimetric response of the external spatial distribution of collagen, which might be useful in clinical diagnosis of pathologies related to changes in collagen structure.

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.

Purpose: To analyze the spatial organization of pathological corneas with second harmonic generation (SHG) imaging and to provide a proof of concept to objectively distinguish these from the healthy corneas. Methods: A custom-built SHG microscope was used to image the anterior stroma of ex vivo corneas, both control and affected by some representative pathologies. The structure tensor (ST) was employed as a metric to explore and quantify the alterations in the spatial distribution of the collagen lamellae. Results: The collagen arrangement differed between healthy and pathological samples. The former showed a regular distribution and a low structural dispersion (SD , 408) within the stroma with a well-defined dominant orientation. This regular arrangement drastically turns into a disorganized pattern in pathological corneas (SD. 408). Conclusions: The combination of SHG imaging and the ST allows obtaining quantitative information to differentiate the stromal collagen organization in healthy and diseased corneas. This approach represents a feasible and powerful technique with potential applications in clinical corneal diagnoses. Translational Relevance: The ST applied to SHG microscopy images of the corneal stroma provides an experimental objective score to differentiate control from pathological or damaged corneas. Future implementations of this technique in clinical environments might might be a promising tool in Ophthalmology, not only to diagnose and monitor corneal diseases, but also to follow-up surgical outcome.