Micro-mechanical damage of needle puncture on bovine annulus fibrosus fibrils studied using polarization-resolved Second Harmonic Generation(P-SHG) microscopy.

Needle injection has been widely used in spinal therapeutic or diagnostic processes, such as discography. The use of needles has been suspected in causing mild disc degeneration which can lead to long-term back pain. However, the localised microscopic damage caused by needles has not been well studied. The local progressive damage on a microscopic level caused by needle punctures on the surface of bovine annulus fibrosus was investigated. Four different sizes of needle were used for the puncture and twenty-nine bovine intervertebral discs were studied. Polarization-resolved second harmonic generation and fluorescent microscopy were used to study the local microscopic structural changes in collagen and cell nuclei due to needle damage. Repeated 70 cyclic loadings at ±5% of axial strain were applied after the needle puncture in order to assess progressive damage caused by the needle. Puncture damage on annulus fibrosus were observed either collagen fibre bundles being pushed aside, being cut through or combination of both with part being lift or pushed in. The progressive damage was found less relevant to the needle size and more progressive damage was only observed using the larger needle. Two distinct populations of collagen, in which one was relatively more organised than the other population, were observed especially after the puncture from skewed distribution of polarization-SHG analysis. Cell shape was found rounder near the puncture site where collagen fibres were damaged.

Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging.

We analyze the influence of the anisotropy of molecular and biological samples on polarization resolved nonlinear microscopy imaging. We show in particular the detrimental influence of birefringence on Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) polarization resolved microscopy imaging, which, if not accounted for, can lead to an erroneous determination of the sample properties and thus to a misinterpretation of the read-out information. We propose a method to measure this birefringence and account for this effect in nonlinear polarization resolved experiments.

Probing molecular order in zeolite L inclusion compounds using two-photon fluorescence polarimetric microscopy.

We investigate the local static molecular orientational behavior in zeolite L inclusion compounds by polarimetric two-photon fluorescence microscopy. This technique, based on the polarized read-out of the signal under a tunable incident polarization state, provides refined information on molecular disorder that is not achievable using traditional fluorescence anisotropy. Moreover, the polarimetric microscopy imaging scheme permits a spatial investigation of possible heterogeneities, with a submicrometric resolution. The study performed on different fluorescent molecules inserted in zeolite L channels evidence a degree of disorder for either small or flexible structures.

Defocused imaging of second harmonic generation from a single nanocrystal.

We demonstrate the direct imaging of the second harmonic generation radiation from a single nonlinear nanocrystal using defocused nonlinear microscopy. This technique allows the retrieval of complete information on the 3D orientation of a nanocrystal as well as possible deviations from its purely crystalline nature, in a simple experimental implementation. The obtained images are modeled by calculation of the radiation diagram from a nonlinear dipole that accounts for the excitation beam, the crystal symmetry and the particle size. Experimental demonstrations are performed on Potassium Titanyl Phosphase (KTP) nanocrystals. The shape and structure of the radiation images show a strong dependence on both crystal orientation and field polarization state, as expected by the specific nonlinear coherent coupling between the induced dipole and the excitation field polarization state.

Local second-harmonic generation enhancement on gold nanostructures probed by two-photon microscopy.

The localization of surface second-harmonic generation (S-SHG) enhancements from granular gold structures that exhibit local plasmon resonance was investigated. A two-photon microscopy technique was used to perform high spatial resolution S-SHG imaging. The magnitude and the spatial density of S-SHG enhancement confined in submicroscopic regions are strongly dependent on the morphology of the gold's surface. Polarization measurements of local S-SHG responses reveal the local field anisotropy in enhancement regions and furthermore prove the incoherent and strongly depolarized nature of the emission, which is attributed to ultrafast fluctuations of the enhancement location in the focal volume.

Polymer-based microcavity with photoencoded quadratic nonlinearity.

Functional electro-optic polymer thin films embedded in microcavity structures have been poled by an all-optical procedure based on the interference of multiphoton absorption processes. The photoinduced X((2)) tensor was then further addressed at modal resonance for the fundamental wavelength, leading to significant enhancement of the second-harmonic-generation efficiency. An order-of-magnitude enhancement, which is due to electric field resonant conditions inside the microcavity, has been probed by an optical parametric oscillator, in comparison with a single-path thin-film configuration. This configuration opens new perspectives in the realm of nonlinear photonic device processing.

Optical methods for exploring dynamics of single copies of green fluorescent protein.

Single copies of four different phenolate ion mutants of the green fluorescent protein (GFP) exhibit a complex blinking and fluctuating behavior, a phenomenon that is hidden in measurements on large ensembles. Both total internal reflection microscopy and scanning confocal microscopy can be used to study the blinking dynamics, and autocorrelation analysis yields histograms of the correlation times for many individual molecules. While the total internal reflection method can follow several single molecules simultaneously, the confocal method offers higher time resolution at the expense of parallelism. We compare and contrast the two methods in terms of the ability to follow the complex dynamics of this system.

Control of the polarization dependence of optically poled nonlinear polymer films.

We demonstrate the possibility of controlling the symmetry properties of photoinduced chi((2)) macroscopic susceptibility in polymer films. Ellipsometric adjustment of the write beams allows one to monitor the macroscopic chi((2)) symmetry from a dipolar to an octupolar configuration. Experimental results are in agreement with an irreducible spherical tensor-based model jointly applied to the molecular beta hyperpolarizability and field tensors. We found a purely octupolar polarization-independent photoinduced second-harmonic-generation response in a Dispersed Red 1-methyl methacrylate thin film. Such a configuration, as generated by an octupolar tensor write field tensor made up of counterclockwise circularly polarized omega and 2omega beams, is not within the reach of the classical electric-field poling technique.