Ping-Jung Su

Determination of Collagen Nanostructure from Second-Order Susceptibility Tensor Analysis

A model is proposed to describe the polarization dependence of second harmonic generation (SHG) from type I collagen fibrils. The model is based on sum-frequency vibrational spectrum experiments that attribute the molecular origins of collagen second-order susceptibility to the peptide groups in the backbone of the collagen α-helix and the methylene groups in the pyrrolidine rings. Applying our model to a polarization SHG (P-SHG) experiment leads to a predicted collagen I peptide pitch-angle of 45.82° ± 0.46° and methylene pitch-angle of 94.80° ± 0.97°. Compared to a previous model that accounts for only the peptide contribution, our results are more consistent with the x-ray diffraction determination of collagen-like peptide. Application of our model to type II collagen from rat trachea cartilage leads to similar results. The peptide pitch-angle of 45.72° ± 1.17° is similar to that of type I collagen, but a different methylene pitch-angle of 97.87° ± 1.79° was found. Our work demonstrates that far-field P-SHG measurements can be used to extract molecular structural information of collagen fibers.

The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue

Using excitation polarization-resolved second harmonic generation (SHG) microscopy, we measured SHG intensity as a function of the excitation polarization angle for type I and type II collagens. We determined the second order susceptibility (χ((2))) tensor ratios of type I and II collagens at each pixel, and displayed the results as images. We found that the χ((2)) tensor ratios can be used to distinguish the two types of collagen. In particular, we obtained χ(zzz)/χ(zxx) = 1.40 ± 0.04 and χ(xzx)/χ(zxx) = 0.53 ± 0.10 for type I collagen from rat tail tendon, and χ(zzz)/χ(zxx) = 1.14 ± 0.09 and χ(xzx)/χ(zxx) = 0.29 ± 0.11 for type II collagen from rat trachea cartilage. We also applied this methodology on the label-free imaging of engineered cartilage tissue which produces type I and II collagen simultaneously. By displaying the χ((2)) tensor ratios in the image format, the variation in the χ((2)) tensor ratios can be used as a contrast mechanism for distinguishing type I and II collagens.

Discrimination of collagen in normal and pathological skin dermis through second-order susceptibility microscopy.

Polarization-resolved, second harmonic generation (P-SHG) microscopy at single pixel resolution is utilized for medical diagnosis of pathological skin dermis. In analyzing the large area, pixel by pixel, second-order susceptibility of normal and pathological skin dermis, we found that P-SHG can be used to distinguish normal and dermal pathological conditions of keloid, morphea, and dermal elastolysis. Specifically, we found that the second order susceptibility tensor ratio of d(33)/d(31) for normal skins is 1.27+/-0.20, while the corresponding values for keloid, morphea, and dermal elastolysis are respectively 1.67+/-0.29, 1.79+/-0.30, and 1.75+/-0.31. We also found that the histograms of the d(33)/d(31) ratio for the pathological skins contain two peak values and are 1.5 times wider than that of the normal case, suggesting that the pathological dermal collagen fibers tend to be more structurally heterogeneous. Our work demonstrates that pixel-resolved, second-order susceptibility microscopy is effective for detecting heterogeneity in spatial distribution of collagen fibers and maybe used for future clinical diagnosis and in vivo studies of collagen pathological conditions.

Quantifying thermodynamics of collagen thermal denaturation by second harmonic generation imaging

Time-lapse second harmonic generation (SHG) microscopy was applied for the extraction of thermodynamic parameters of collagen thermal denaturation. We found that at sufficiently high temperatures, temporal dependence of SHG intensity from the isothermal treatment of chicken dermal collagen was single exponential and can be modeled by the Arrhenius equation. Activation energy and the frequency factor of chicken dermal collagen thermal denaturation were determined using temporal decays of SHG intensity at different temperatures. Our results show that time-lapse, high temperature SHG imaging can be used to quantify kinetic properties of collagen thermal denaturation within a microscopic volume of 1 nl.

Image heterogeneity correction in large-area, three-dimensional multiphoton microscopy

Large-area multiphoton laser scanning microscopy (LMLSM) can be applied in biology and medicine for high sensitivity and resolution tissue imaging. However, factors such as refractive index mismatch induced spherical aberration, emission/excitation absorption and scattering can result in axial intensity attenuation and lateral image heterogeneity, affecting both qualitative and quantitative image analysis. In this work, we describe an image correction algorithm to improve three-dimensional images in LMLSM. The method consists of multiplying the measured nonlinear signal by a three-dimensional correction factor, determined by the use of twophoton images of the appropriate specimens and specimen absorption and scattering properties at the excitation and emission wavelengths. The proposed methodology is demonstrated in correcting multiphoton images of objects imbedded in uniform fluorescent background, lung tissue, and Drosophila larva.

Characterization of optical-aberration-induced lateral and axial image inhomogeneity in multiphoton microscopy

The effects of off-axis optical aberration in multiphoton microscopy and the resulting lateral and axial image inhomogeneity are investigated. The lateral inhomogeneity of the scanning field is demonstrated by second harmonic generation (SHG) imaging of fasciae and two-photon fluorescence (TPF) microscopy of thin fluorescent samples. Furthermore, refractive index mismatch-caused intensity attenuation of the TPF signal at central and peripheral regions of the scanning frame is measured using homogeneous 10-microM sulforhodamine B samples with refractive indexes of 1.33 and around 1.465. In addition to characterizing image field convexity, we also found that image resolution degrades away from the optical axis. These effects need to be accounted for in both qualitative and quantitative multiphoton imaging applications.

The Use of Second-Order Susceptibility as Contrast Mechanism for Label-Free Imaging of Biological Tissue

This review paper conveys state-of-the art research on second-order susceptibility microscopy: physical origin, devices and instrumentation, applications in biological system, and prospects of clinical applications. The organization of this paper started with an overview of second harmonic generation (SHG) in biological medium. Illustrating with figures system architecture of second-order susceptibility and manipulation of polarization are introduced, which is the central scheme in this imaging modality. Several applications of SHG susceptibility imaging in biological and biomedical sciences are then discussed. This review paper is finally concluded with future prospects of susceptibility imaging in clinical settings.

Discrimination of collagen in normal and pathological dermis through polarization second harmonic generation

We used polarization-resolved, second harmonic generation (P-SHG) microscopy at single pixel resolution for medical diagnosis of pathological skin dermis, and found that P-SHG can be used to distinguish normal and dermal pathological conditions of keloid, morphea, and dermal elastolysis. We find that the histograms of the d33/d31 ratio for the pathological skins to contain two peak values and to be wider than that of the normal case, suggesting that the pathological dermal collagen fibers tend to be more structurally heterogeneous. Our work demonstrates that pixel-resolved, second-order susceptibility microscopy is effective for detecting heterogeneity in spatial distribution of collagen fibers.

Dynamic multiphoton imaging of reversible and irreversible thermal changes in collagen tissues

Collagen is the major component of skin, tendon, cartilage, cornea, and, as a main structural protein it is the key determinant of thermo-mechanical properties of collagen-rich tissues in mammals. Thermal damage of chicken dermis and tendon, bovine leg tendon, and other collagen contained tissues were investigated with the use of second harmonic generation (SHG) and two-photon excited auto-fluorescence microscopy and spectroscopy. Samples were heating in a temperature-controlled water bath in the temperature range 18-90° C. SHG time-lapse imaging and analysis of intensity decay showed that the collagen thermal destruction depended on both temperature and heating time, and can be modeled by the Arrhenius equation. Temporal decay of SHG signal from the chicken dermis was single exponential during isothermal treatment at temperatures above 60º C that allowed to determine activation energy and frequency factor of skin collagen denaturation. Furthermore, two-exponential decay and partially reversible change in SHG intensity were registered during the tendon thermal treatment. A simple laser system and procedure is proposed for a real-time monitoring of collagen fiber thermal modification within a microscopic volume of 1 nl.

Utilizing Two-Photon Fluorescence and Second Harmonic Generation Microscopy to Study Human Bone Marrow Mesenchymal Stem Cell Morphogenesis in Chitosan Scaffold

A major goal of tissue engineering is to cultivate the cartilage in vitro. One approach is to implant the human bone marrow mesenchymal stem cells into the three dimensional biocompatible and biodegradable material. Through the action of the chondrogenic factor TGF-β3, the stem cells can be induced to secrete collagen. In this study, mesenchymal stem cells are implanted on the chitosan scaffold and TGF-β3 was added to produce the cartilage tissue and TP autofluorescence and SHG microscopy was used to image the process of chondrogenesis. With additional development, multiphoton microscopy can be developed into an effective tool for evaluating the quality of tissue engineering products.