Daaf Sandkuijl

Nonlinear Optical Properties of Type I Collagen Fibers Studied by Polarization Dependent Second Harmonic Generation Microscopy

Collagen (type I) fibers are readily visualized with second harmonic generation (SHG) microscopy though the molecular origin of the signal has not yet been elucidated. In this study, the molecular origin of SHG from type I collagen is investigated using the time-dependent coupled perturbed Hartree-Fock calculations of the hyperpolarizibilities of glycine, proline, and hydroxyproline. Two effective nonlinear dipoles are found to orient in-the-plane of the amino acids, with one of the dipoles aligning close to the pitch orientation in the triple-helix, which provides the dominant contribution to the SHG polarization properties. The calculated hyperpolarizability tensor element ratios for the collagen triple-helix models: [(Gly3)n]3, [(Gly-Pro2)n]3, and [(Gly-Pro-Hyp)n]3, are used to predict the second-order nonlinear susceptibility ratios, χ(zzz)(2)/χ(iiz)(2) and χ(zii)(2)/χ(iiz)(2) of collagen fibers. From SHG microscopy polarization in, polarization out (PIPO) measurements of type I collagen in human lung tissue, a theoretical method is used to extract the triple-helix orientation angle with respect to the collagen fiber. The study shows the dominant role of amino acid orientation in the triple-helix for determining the polarization properties of SHG and provides a method for determining the triple-helix orientation angle in the collagen fibers.

Hierarchical Model of Fibrillar Collagen Organization for Interpreting the Second-Order Susceptibility Tensors in Biological Tissue

The second-order nonlinear polarization properties of fibrillar collagen in various rat tissues (vertebrae, tibia, tail tendon, dermis, and cornea) are investigated with polarization-dependent second-harmonic generation (P-SHG) microscopy. Three parameters are extracted: the second-order susceptibility ratio, R = [Formula: see text] ; a measure of the fibril distribution asymmetry, |A|; and the weighted-average fibril orientation, . A hierarchical organizational model of fibrillar collagen is developed to interpret the second-harmonic generation polarization properties. Highlights of the model include: collagen type (e.g., type-I, type-II), fibril internal structure (e.g., straight, constant-tilt), and fibril architecture (e.g., parallel fibers, intertwined, lamellae). Quantifiable differences in internal structure and architecture of the fibrils are observed. Occurrence histograms of R and |A| distinguished parallel from nonparallel fibril distributions. Parallel distributions possessed low parameter values and variability, whereas nonparallel distributions displayed an increase in values and variability. From the P-SHG parameters of vertebrae tissue, a three-dimensional reconstruction of lamellae of intervertebral disk is presented.

Efficient frequency doubling of a femtosecond Yb:KGW laser in a BiB 3 O 6 crystal

Efficient frequency doubling of a high-power femtosecond Yb:KGW laser in a nonlinear BiBO crystal is demonstrated. Green second harmonic generation with more than 1.1 W of average power and 41% conversion efficiency was achieved using a single-pass configuration.

Numerical second- and third-harmonic generation microscopy

A full numerical description of second- and third-harmonic generation (SHG and THG) at the focus of a nonlinear microscope is presented. The numerical implementation takes into account reflections and refraction by an arbitrary number of interfaces perpendicular to the optical axis in the focal region. The calculation of the second- and third-harmonic far-field radiation pattern is based on a Green function approach and is presented for any collection direction. The calculations are sped up by using the chirp-z transform for the focusing fields as well as for the far-field radiation calculation. Numerical evidence is presented for deviations in the measurement of the second-order nonlinear susceptibility ratio ρ≡χyyy(2)/χyxx(2) of collagen fibers in SHG microscopy at high excitation numerical aperture. When interface reflections are taken into account, significant direct backward THG is demonstrated from interfaces and multilayer structures.

Differential microscopy for fluorescence-detected nonlinear absorption linear anisotropy based on a staggered two-beam femtosecond Yb:KGW oscillator.

We present a new laser system and nonlinear microscope, designed for differential nonlinear microscopy. The microscope features time-correlated single photon counting of multiphoton fluorescence generated by an alternating pulse-train of orthogonally polarized pulses. The generated nonlinear signal is separated using home-built electronics. Results are presented on fluorescence-detected nonlinear absorption linear anisotropy (FDNALA) of chloroplasts in Asparagus Sprengerii Regel and of Congo Red-stained cellulose.

Differential Polarization Nonlinear Optical Microscopy with Adaptive Optics Controlled Multiplexed Beams

Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.

Interferometric backward third harmonic generation microscopy for axial imaging with accuracy beyond the diffraction limit.

A new nonlinear microscopy technique based on interference of backward-reflected third harmonic generation (I-THG) from multiple interfaces is presented. The technique is used to measure height variations or changes of a layer thickness with an accuracy of up to 5 nm. Height variations of a patterned glass surface and thickness variations of fibroblasts are visualized with the interferometric epi-THG microscope with an accuracy at least two orders of magnitude better than diffraction limit. The microscopy technique can be broadly applied for measuring distance variations between membranes or multilayer structures inside biological tissue and for surface height variation imaging.

Hierarchical model of fibrillar collagen distribution for polarization-resolved SHG microscopy

A hierarchical model of the organization of fibrillar collagen is developed and its implications on polarization-resolved second harmonic generation (SHG) microscopy are investigated. A “ground-up” approach is employed to develop the theory for understanding of the origin of SHG from fibrillar collagen. The effects of fibril ultrastructure and fibril macroscopic organization on the second-order polarization properties of fibrillar collagen are presented in conjunction with recent ab initio results performed on a collagen triple-helix model (-GLY-PRO-HYP-)n. Various tissues containing fibrillar collagen are quantified using a polarization-resolved SHG technique, termed polarization-in, polarization-out (PIPO) and interpreted in light of the aforementioned theory. The method involves varying the incident laser polarization, while monitoring the SHG intensity through an analyzer. From the SHG polarization data the orientation of the fibers, in biological tissue, can be deduced. Unique PIPO signatures are observed for different rat tissues and interpreted in terms of the collagen composition, fibril ultrastructure, and macroscopic organization. Similarities and discrepancies in the second-order polarization properties of different collagen types and ultrastructures will be presented. PIPO SHG microscopy shows promise in its ability to quantify the organization of collagen in various tissues. The ability to characterize the structure of collagen in various tissue microenvironments will aid in the study of numerous collagen related biological process, including tissue diseases, wound repair, and tumor development and progression.

Nonlinear Multimodal Microscope with Increased Axial Resolution Using Interferometric Backwards Third Harmonic Generation

A new nonlinear optical microscope based on interference of multiple reflected third harmonic waves is presented. The signal can be used to infer surface height variations with up to 5 nm accuracy. We demonstrate the technique by imaging fibroblasts.

Axial Super-resolution Microscopy Based On Backward Third Harmonic Generation

We present a far field imaging modality capable of sub 45 nm axial resolution based on backward third harmonic generation from a few-layer structure. The method is applicable for transparent (including biological) sample imaging.