Nonlinear optical microscopy is a biocompatible avenue for probing ordered molecular assemblies in biological tissues. As in linear optics, the nonlinear optical response from ordered systems is polarization-sensitive. This dependence can be used to identify and characterize local molecular ordering with micrometer-scale 3D resolution in a nonlinear microscope. In particular, third-harmonic generation (THG) microscopy is a nonlinear optical modality sensitive to the electronic nonlinear susceptibility χ(3) of a material. THG microscopy can be used to map χ(3) spatial variations (i.e. material interfaces), and to probe birefringence. In principle, polarization-resolved THG (P-THG) can therefore be used to probe ordered molecular arrays. However, the orientation, distribution, and nonlinear optical properties of the molecules near the beam focus all affect the detected signal. It is therefore necessary to develop a theoretical method which decouples these effects and permits the extraction of orientational information from P-THG images. In this report, we first present P-THG images of model systems (lipid droplets, multilamellar lipid vesicles) and biological tissues (human skin biopsy) which establish that P-THG is sensitive to lipid ordering and that it is maximized when excitation polarization is parallel to the ordered lipid molecules, giving impetus for the development of a thorough theoretical analysis. We then outline a multiscale model spanning the molecular (nm) and ensemble (μm) scales predicting the PTHG signal, consisting of three main steps: (i) calculation of the molecular electronic hyperpolarizability; (ii) determination of the anisotropic χ(3) for various molecular distribution parameters; and (iii) numerical calculations of the P-THG signal from lipid-water interfaces. This analysis links the measured P-THG response to lipid molecular structure and ordering.
Coherent anti-Stokes Raman scattering (CARS) microscopy is used to determine the distribution and concentration of selected compounds in intact human hair. By generating images based on ratiometric CARS contrast, quantitative concentration maps of both water and externally applied d-glycine are produced in the cortex of human hair fibers. Both water and d-glycine are found to homogeneously distribute throughout the cortical regions of the hair. The ability to selectively detect molecular agents in hair fibers is of direct relevance to understanding the chemical and physical mechanisms that underlie the performance of hair-care products.
Two-photon polymerization (TPP) is an enabling technology that allows fast prototyping of parts with sub-100 nm
resolution. Due to its ability to fabricate microstructures with arbitrary three-dimensional geometries, TPP has been
employed in diverse fields such as photonics, microelectronics, microelectromechanical systems, and microfluidics.
However, no information is available to date that microscopically correlates the experimental conditions used in TPP
with the properties of the ultimate microstructure. We present a study where the distribution of polymer cross-linking in
three-dimensional microstructures fabricated by TPP is visualized by means of nonlinear microscopy. In particular,
coherent anti-Stokes Raman scattering (CARS) microscopy is employed to image polymer microstructures with
chemical specificity. The characterization of the microstructures based on the acquired images permits rational
optimization of the TPP process.
An overview is presented of recent trends in coherent anti-Stokes Raman scattering (CARS) microscopy. We briefly discuss the influence of tissue scattering on the CARS signal, methods for controlling the CARS emission and prospects for surface-enhancement of the CARS radiation.
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