Ariane Deniset-Besseau

Multimodal Nonlinear Imaging of the Human Cornea

PURPOSE To evaluate the potential of third-harmonic generation (THG) microscopy combined with second-harmonic generation (SHG) and two-photon excited fluorescence (2PEF) microscopies for visualizing the microstructure of the human cornea and trabecular meshwork based on their intrinsic nonlinear properties. METHODS Fresh human corneal buttons and corneoscleral discs from an eye bank were observed under a multiphoton microscope incorporating a titanium-sapphire laser and an optical parametric oscillator for the excitation, and equipped with detection channels in the forward and backward directions. RESULTS Original contrast mechanisms of THG signals in cornea with physiological relevance were elucidated. THG microscopy with circular incident polarization detected microscopic anisotropy and revealed the stacking and distribution of stromal collagen lamellae. THG imaging with linear incident polarization also revealed cellular and anchoring structures with micrometer resolution. In edematous tissue, a strong THG signal around cells indicated the local presence of water. Additionally, SHG signals reflected the distribution of fibrillar collagen, and 2PEF imaging revealed the elastic component of the trabecular meshwork and the fluorescence of metabolically active cells. CONCLUSIONS The combined imaging modalities of THG, SHG, and 2PEF provide key information about the physiological state and microstructure of the anterior segment over its entire thickness with remarkable contrast and specificity. This imaging method should prove particularly useful for assessing glaucoma and corneal physiopathologies.

Measurement of the Second-Order Hyperpolarizability of the Collagen Triple Helix and Determination of Its Physical Origin

We performed Hyper-Rayleigh Scattering (HRS) experiments to measure the second-order nonlinear optical response of the collagen triple helix and determine the physical origin of second harmonic signals observed in collagenous tissues. HRS experiments yielded a second-order hyperpolarizability of 1.25 x 10(-27) esu for rat-tail type I collagen, a surprisingly large value considering that collagen presents no strong harmonophore in its amino acid sequence. Polarization-resolved experiments showed intramolecular coherent contributions to the HRS signal along with incoherent contributions that are the only contributions for molecules with dimensions much smaller than the excitation wavelength. We therefore modeled the effective second-order hyperpolarizability of the 290 nm long collagen triple helix by summing coherently the nonlinear response of well-aligned moieties along the triple helix axis. This model was confirmed by HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)(10)](3). We concluded that the large collagen nonlinear response originates in the tight alignment of a large number of small and weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal.

Nonlinear optical imaging of lyotropic cholesteric liquid crystals.

We use nonlinear optical microscopy combining Second Harmonic Generation (SHG) microscopy and Two-Photon Excited Fluorescence (2PEF) signals to characterize collagen lyotropic liquid crystals. We show that SHG signals provide highly contrasted images of the three-dimensional texture of cholesteric patterns with submicrometer lateral resolution. Moreover, simultaneous recording of the 2PEF signal enables in situ quantitative mapping of the molecular concentration and its correlation with the observed textures. We apply this technique to the characterization of biomimetic textures obtained in concentrated collagen liquid solutions. We successfully image biologically relevant organizations that are similar to the collagen organization found as a stabilized state in compact bones.

Imaging and 3D morphological analysis of collagen fibrils

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two‐dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three‐dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.

A bottom-up approach to build the hyperpolarizability of peptides and proteins from their amino acids.

We experimentally demonstrate that some peptides and proteins lend themselves to an elementary analysis where their first hyperpolarizability can be decomposed into the coherent superposition of the first hyperpolarizability of their elementary units. We then show that those elementary units can be associated with the amino acids themselves in the case of nonaromatic amino acids and nonresonant second harmonic generation. As a case study, this work investigates the experimentally determined first hyperpolarizability of rat tail Type I collagen and compares it to that of the shorter peptide [(PPG)10]3, where P and G are the one-letter code for Proline and Glycine, respectively, and that of the triamino acid peptides PPG and GGG. An absolute value of (0.16 ± 0.01) × 10(-30) esu for the first hyperpolarizability of nonaromatic amino acids is then obtained by using the newly defined 0.087 × 10(-30) esu reference value for water. By using a collagen like model, the microscopic hyperpolarizability along the peptide bond can be evaluated at (0.7 ± 0.1) × 10(-30) esu.

Quantitative assessment of collagen I liquid crystal organizations: role of ionic force and acidic solvent, and evidence of new phases

Collagen I is the major structural protein in mammals where it exhibits highly organized fibrillar distributions in connective tissues. In vitro, acidic solutions of collagen I display lyotropic liquid crystal organization. These concentrated organized liquid phases can be stabilized by a pH increase to generate in vitro fibrillar matrices with specific organization. The aim of this work is to understand the mechanisms responsible for liquid crystal chirality at acidic pH in order to guide the synthesis of collagen matrices reproducing the great diversity of organizations found in biological tissues. For this purpose, we quantitatively analyze collagen liquid crystal organization by use of multiphoton microscopy, combining fluorescence and second harmonic generation contrasts. The concentration of the isotropic to liquid crystal phase transition and the evolution of the half pitch of the helical phase with collagen concentration are reported in five physico-chemical conditions using hydrochloric and acetic acids at different pHs and ionic strengths. A new phase transition is observed in highly concentrated solutions ranging from 90 mg ml−1 to 300 mg ml−1 depending on the solvent. Our results bring new quantitative information on collagen chemical physics and further substantiate the on-going analysis of the driving parameters generating twists in liquid crystals. These findings could be advantageously exploited to develop new strategies and protocols for tissue engineering. This is crucial for fundamental studies of cell behavior in biomimetic three-dimensional environments and for medical and pharmaceutical applications.

Achievement of cornea-like organizations in dense collagen I solutions: clues to the physico-chemistry of cornea morphogenesis

Multiphoton and electron microscopic analyses show that acido-soluble collagen I prepared in 5 mM acetic acid (pH 3.5) at concentration above 45 mg mL−1 spontaneously generates liquid crystal phases mimicking plywood organization found in cornea tissues. Those organizations extend for several hundred micrometers. Transmission electron microscopy reveals the presence of small nanofibrils organized in a complex phase, coupling overall smectic and cholesteric organizations together with local order. Those nanofibrils could be the mesogen elements giving rise to this plywood organization. These data provide clues to physico-chemical events that may take place in cornea morphogenesis in vivo. This result is invaluable for bioengineering fields, as this liquid crystal organization paves the way for the generation of collagen based bio-mimetic cornea matrices.

Nonlinear optical response of the collagen triple helix and second harmonic microscopy of collagen liquid crystals

Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to score fibrotic pathologies. However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen triple helices.

Second order hyperpolarizability of the collagen triple helix: Measurement and determination of its physical origin

Collagen is the major protein of the extracellular matrix and plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. As a fundamental brick of the architecture of tissues, it guarantees organs functioning and is crucial in the adaptative response to various tissue injuries. This protein is characterized by triple helical domains and possesses remarkable non linear optical properties. Indeed, collagen fibers exhibit efficient Second Harmonic Generation (SHG) in tissues and SHG microscopy has proved to be a valuable technique to probe the three-dimensional architecture of fibrillar collagen in native and biomimetic tissues and to assess the progression of fibrotic pathologies [1,2].

Concepts to Build Nonlinear Optical Biomaterials in a Bottom-Up Approach

We have performed Hyper-Rayleigh Scattering (HRS) experiments to measure the quadratic hyperpolarizability of several natural amino acids, in particular tryptophan and tyrosine. Values of (29.6+/-0.4)x10-30 esu for tryptophan and (25.7+/-0.03)x10-30 esu for tyrosine have been found. We have then investigated the dependence of the quadratic hyperpolarizability of tryptophan-rich short peptides as a function of the number of tryptophans in the sequence. The experimental findings indicate that the resulting quadratic hyperpolarizability in these peptides cannot be assumed as the mere coherent superposition of the hyperpolarizabilities of the tryptophans contained in the peptide. Our results unambiguously demonstrate that there must be strong interactions between the tryptophans contained in these short peptides. We have also investigated the case of the collagen triple helix. A second order hyperpolarizability of (1.25+/- 0.05)x10-27 esu for rat-tail type I collagen has been measured. In this case, we have been able to model this effective quadratic hyperpolarizability by summing coherently the nonlinear response of elementary moieties forming the triple helix, as opposed to the previous case of the tryptophan-rich peptides.