Adam Tuer

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.

Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections

Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.

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.

Three‐dimensional visualization of the first hyperpolarizability tensor

With polarization dependent second harmonic generation (SHG) microscopy becoming a more popular method for investigating the structure of biological materials, there is a need to develop tools with which to understand and interpret the observed SHG properties. Quantum mechanical calculations of the hyperpolarizability tensor have become a popular method for understanding the SHG properties of biomolecules. Visualization of the full hyperpolarizability tensor, termed the unit sphere representation, has been developed to provide insight and intuition on the relationship between SHG properties and molecules. A single vector representation is also presented, which approximates the SHG properties of molecules for certain cases, where the anisotropy is negligible.

Enhancement of third harmonic contrast with harmonophores in multimodal non-linear microscopy of histological sections

Histological investigations of biological tissue benefited tremendously from staining different cellular structures with various organic dyes. With the introduction of new imaging modalities such as second harmonic generation (SHG) and third harmonic generation (THG) microscopy, the demand for novel dyes that enhance the harmonic signals has arisen. The new labels with high molecular hyperpolarizability have recently been termed harmonophores. In this study, we demonstrate that hematoxylin, the standard histological stain used in H&E (hematoxylin and eosin) staining, enhances the microscopic THG signal. Hematoxylin has an affinity for basophilic structures such as the cell nucleus, ribosomes and mitochondria, while eosin stains structures such as the cytoplasm, collagen and red blood cells. The histological sections of H&E stained cancerous prostate tissue found in transgenic adenocarcinoma of the mouse prostate (TRAMP) have been investigated with the multimodal SHG, THG and multiphoton excitation fluorescence (MPF) microscope. Strong THG signal revealed intracellular structures originating where the hematoxylin stain resides, while SHG imaging of the tissue showed the presence of collagen fibrils in the extracellular matrix. The MPF was mostly present in the extracellular matrix. The spectrally and temporally resolved MPF revealed that most of the fluorescence originates from the eosin. The THG image did not correlate with MPF confirming that the harmonic signal originates from hematoxylin. Multimodal nonlinear microscopy adds invaluable information about cellular structures to the widely used bright field investigations of H&E stained histological sections, and can be efficiently used for morphological studies as well as cancer diagnostics.

Ab initio Calculations of the Linear and Nonlinear Optical Properties of Amino Acids

A number of proteins can assemble into chiral structures that display strong nonlinear optical activity. For instance, proteins such as myosin and collagen exhibit intense second harmonic generation (SHG). A large number of experimental studies on the SHG of proteins have been conducted; however few predictive models have been proposed that reliably relate the macroscopic SHG properties to the amino acids present in the peptidic chain. In this study, the linear polarizability (α), first (β) and second hyperpolarizability (γ) of all twenty amino acids was investigated by time-dependent Hartree-Fock calculations under physiological conditions. Ab initio calculations were performed using the GAMESSUS computational chemistry package. We have found that the aromatic amino acids give rise to the largest mean α, β and γ values. With this finding, we hope to apply this method to protein structures in order to understand how second harmonic signal is generated from individual amino acids, as well as, recognize how manipulation of the secondary structure of proteins might enhance SHG and third harmonic generation (THG).

Ultrafast Yb:KGd(WO4)2 laser for multimodal biomedical imaging with reduced photodamage

We report on the development and successful application of a femtosecond Yb:KGd(WO4)2 laser for multimodal imaging of various biological samples. Its operation at longer wavelengths, 1029 nm, provided efficient excitation and greatly reduced sample photobleaching. The laser produced ~300 fs pulses with up to 100 nJ of energy at 14.3 MHz repetition rate. This laser system enabled continuous imaging of various live samples for prolonged periods of time. The details of laser development and fluorescence imaging of isolated chloroplasts are reported.

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.

Second-Order Nonlinear Optical Properties of Fibrillar Proteins

Quantum mechanical calculation of collagen-like protein model’s first hyperpolarizability aided in predicting the second-order nonlinear optical properties of collagen in tissue. Polarization dependent second harmonic generation microscopy experiments confirmed the model’s predictions.