Theoretical, numerical and experimental study of geometrical parameters that affect anisotropy measurements in polarization-resolved SHG microscopy.

International audiencePolarization-resolved second harmonic generation (P-SHG) microscopy is an efficient imaging modality for in situ observation of biopolymers structure in tissues, providing information about their mean in-plane orientation and their molecular structure and 3D distribution. Nevertheless, P-SHG signal build-up in a strongly focused regime is not throroughly understood yet, preventing reliable and reproducible measurements. In this study, theoretical analysis, vectorial numerical simulations and experiments are performed to understand how geometrical parameters, such as excitation and collection numerical apertures and detection direction, affect P-SHG imaging in homogeneous collagen tissues. A good agreement is obtained in tendon and cornea, showing that detection geometry significantly affects the SHG anisotropy measurements, but not the measurements of collagen in-plane orientation

Application of classical models of chirality to surface second harmonic generation

International audienceTwo classical models (Kuhn and Kauzmann) are extended to calculate the second-order nonlinear response of an isotropic layer of chiral molecules. Calculation of the various nonlinear susceptibilities (electric dipolar, magnetic dipolar, and electric quadrupolar) is performed and applied to the derivation of the second harmonic field radiated by the molecules. It is shown that the two models give strikingly different results about the origin of the chiral response in such experiments. Previously published results are analyzed in view of this calculation which allows to understand the different interpretations proposed. This calculation emphasizes the interest of surface second harmonic generation to access information about the microscopic origin of optical activity in chiral molecules. © 2001 American Institute of Physics

In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy

International audienceWe demonstrate multimodal nonlinear optical imaging of historical artifacts by combining Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (2PEF) microscopies. We first identify the nonlinear optical response of materials commonly encountered in coatings of cultural heritage artifacts by analyzing one- and multi-layered model samples. We observe 2PEF signals from cochineal lake and sandarac and show that pigments and varnish films can be discriminated by exploiting their different emission spectral ranges as in luminescence linear spectroscopy. We then demonstrate SHG imaging of a filler, plaster, composed of bassanite particles which exhibit a non centrosymmetric crystal structure. We also show that SHG/2PEF imaging enables the visualization of wood microstructure through typically 60 µm-thick coatings by revealing crystalline cellulose (SHG signal) and lignin (2PEF signal) in the wood cell walls. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin. SHG/2PEF imaging thus appears as a promising non-destructive and contactless tool for in situ 3D investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale. © 2012 OS

In situ three-dimensional monitoring of collagen fibrillogenesis using SHG microscopy.

International audienceWe implemented in situ time-lapse Second Harmonic Generation (SHG) microscopy to monitor the three-dimensional (3D) self-assembly of collagen in solution. As a proof of concept, we tuned the kinetics of fibril formation by varying the pH and measured the subsequent exponential increase of fibril volume density in SHG images. We obtained significantly different time constants at pH = 6.5 ± 0.3 and at pH = 7.5 ± 0.3. Moreover, we showed that we could focus on the growth of a single isolated collagen fibril because SHG microscopy is sensitive to well-organized fibrils with diameter below the optical resolution. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials

Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy.

International audienceWe extend second-harmonic generation (SHG) microscopy to the measurement of sarcomere length in unstained living cardiac myocytes with 20-nm accuracy. We quantify individual sarcomere shortening in the presence of saxitoxin and find that it is in agreement with mechanical measurements of atrial tissue contracture. This functional application of SHG microscopy is generally applicable to quantify the physiological effects of drugs on contractile tissue. Our data also suggest that packed myosin heads in sarcomere thick filaments are responsible for the large second-harmonic endogenous signal in muscle tissue

Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy

International audienceWe recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin. © 2005 Optical Society of Americ

Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics

International audienceThe assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects

Polarization-resolved second-harmonic generation in tendon upon mechanical stretching

International audienceCollagen is a triple-helical protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical and physical properties of most organs. Second-harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. In this article, we use a multiscale nonlinear optical formalism to bring theoretical evidence that anisotropy of polarization-resolved SHG mostly reflects the micrometer-scale disorder in the collagen fibril distribution. Our theoretical expectations are confirmed by experimental results in rat-tail tendon. To that end, we report what to our knowledge is the first experimental implementation of polarization-resolved SHG microscopy combined with mechanical assays, to simultaneously monitor the biomechanical response of rat-tail tendon at macroscopic scale and the rearrangement of collagen fibrils in this tissue at microscopic scale. These experiments bring direct evidence that tendon stretching corresponds to straightening and aligning of collagen fibrils within the fascicle. We observe a decrease in the SHG anisotropy parameter when the tendon is stretched in a physiological range, in agreement with our numerical simulations. Moreover, these experiments provide a unique measurement of the nonlinear optical response of aligned fibrils. Our data show an excellent agreement with recently published theoretical calculations of the collagen triple helix hyperpolarizability. Copyright © 2012 Biophysical Societ

Wavelength dependence of nonlinear circular dichroism in a chiral ruthenium-tris(bipyridyl) solution

International audienceNonlinear circular dichroism is studied in a solution of ruthenium-tris(bipyridyl) salt in one-beam and pump-probe experiments by tuning the laser wavelength across the circular dichroism structure. The dispersion of the nonlinear circular dichroism is measured. This wavelength dependence is well accounted for by a model calculation where nonlocality is included in the optical response of a two-coupled-oscillator system. This calculation also allows us to address the question of the contribution of electric quadrupolarization to the nonlinear optical activity of an isotropic liquid of chiral molecules. © 2002 The American Physical Societ

Second Harmonic Generation imaging of collagen fibrillogenesis

International audienceDevelopment of nonlinear optical microscopy has significantly improved three-dimensional (3D) imaging of biological tissues in recent years. In particular, collagen has been shown to exhibit endogenous Second Harmonic Generation (SHG) signals and SHG microscopy has proved to enable the visualization of collagen architecture in tissues with unequalled contrast and specificity [1, 2]. Type I collagen is a major structural protein in mammals and shows highly structured macromolecular organizations specific to each tissue. It is synthesized by cells as triple helices, which self-assemble outside the cells into fibrils that further form fibers, lamellae or other three-dimensional (3D) networks. This assembly mechanism depends critically on the collagen concentration, as well as on the temperature, pH and ionic strength of the solution in vitro. Thorough characterization of collagen fibrillogenesis is crucial to understand the biological mechanisms of tissue formation and tissue remodeling in response to a variety of pathologies. Booming of tissue engineering furthermore requires advanced in situ quantitative imaging techniques to verify whether the tissue substitutes display appropriate biomimetic 3D organization for cell culture scaffolds or functional implants. In this study, we continuously monitored the formation of collagen fibrils by time-lapse SHG microscopy [3]. Fibrillogenesis was triggered in a controlled way by increasing the pH in a dilute solution of collagen I. The fibril density was measured every 10 to 20 minutes as the number of voxels with significant SHG signal in 3D image stacks [1]. Our results showed reproducible dynamics of fibrillar collagen formation that could be changed by tuning the pH (see figure 1). We also monitored the growth of single fibrils and measured the length increase over time, which had never been reported before using an optical technique. We then correlated these SHG images to TEM images at nanometer-scale resolution by blocking the fibrillogenesis at early stages and drying the samples. It showed that SHG microscopy allows imaging of fibrils with a diameter down to 30-50 nm in our experimental conditions. We finally investigated surface-mediated fibrillogenesis by adding silica nanoparticles to the solution [4]. We used Two-Photon excited fluorescence (2PEF) microscopy to visualize the fluorescently-died nanoparticles and quantify the self-assembly of collagen around these nanoparticles. In conclusion, SHG microscopy enabled sensitive and well contrasted 3D visualization of collagen fibrillogenesis in a non invasive way. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials. 0 200 400 600 800 0.0 0.5 1.0 1.5 2.0 2.5 Pixel fraction (%) Time (min) (d) (a) (c) (b) Fig 1. 3D reconstruction of SHG images of collagen fibrillogenesis (at pH=6.5) after a) 170 b) 410 and c) 730 minutes; d) Experimental kinetics of fibril density in the SHG images (black dots) with exponential fitting (red line). References [1] M