Specific Second Harmonic Generation Research Articles

Characterization of fibrillar collagen isoforms in infarcted mouse hearts using second harmonic generation imaging.

We utilized collagen specific second harmonic generation (SHG) signatures coupled with correlative immunofluorescence imaging techniques to characterize collagen structural isoforms (type I and type III) in a murine model of myocardial infarction (MI). Tissue samples were imaged over a four week period using SHG, transmitted light microscopy and immunofluorescence imaging using fluorescently-labeled collagen antibodies. The post-mortem cardiac tissue imaging using SHG demonstrated a progressive increase in collagen deposition in the left ventricle (LV) post-MI. We were able to monitor structural morphology and LV remodeling parameters in terms of extent of LV dilation, stiffness and fiber dimensions in the infarcted myocardium.

Detection of the Tendon Properties in Posterior Tibial Tendinopathy in Three-Dimensional Space Using High Resolution Multiphoton and Second Harmonic Generation Imaging

Category:HindfootIntroduction/Purpose:Posterior tibial tendon dysfunction is the most common cause of acquired flat foot deformity in adults, and results in significant morbidity. The exact etiology of this condition is still unknown. Posterior tibial tendon is predominately made up of fibrillar collagens and we hypothesize that their structural properties such as the collagen fiber density, orientation properties and cross-linking density essentially control the biomechanical properties of posterior tibial tendons. In this study, our aim is to visualize and quantitate fibrillar collagen distributions, their organization, cross-link densities and the fiber orientation (using Fourier analysis) in three distinct regions of pathologic posterior tibial tendon human samples namely proximal, middle and distal regions.Methods:After an institutional IRB approval we obtained diseased posterior tibial tendon total excision samples from 10 patients that had reconstructive procedures for posterior tibial tendinopathy at our institution and two fresh non-frozen healthy posterior tibial tendon samples from cadavers were used as the control. This research mainly utilized well established multiphoton microscopy and harmonic generation methodologies which compared proximal, middle and distal 1/3 of the tendon samples; multiphoton microscopy provides a powerful imaging method for evaluation of remodeling of fibrillar collagen structures deep within tissues. Ultra-short IR laser pulses served as an excitation source to produce multiphoton excitation fluorescence (MPEF) from endogenously fluorescent macromolecular systems and to induce highly specific second harmonic generation (SHG) signals from fibrillar collagens. Since MPEF and SHG involve different principles and contrast mechanisms, they were captured simultaneously to provide spectrally and spatially resolved, quantitative imaging of complex structures.Results:We systematically examined the nature of fibrillar collagen remodeling in relatively thick posterior tibial tendon tissues. Representative 3D forward SHG (FSHG), backward SHG (BSHG) and auto-fluorescence (MPEF) images originating from fibrillar collagen matrix from relatively thick (˜50 microns) posterior tibial tendon sections of three different regions namely proximal, middle and distal regions are shown in Figure 1. Computed Orientation Index values obtained from Fourier analysis show statistically significant differences particularly between proximal and middle regions (decreased orientation of collagen fibers in the middle region, p<0.0001). The cross-linking densities determined from the ratio of auto-fluorescence (MPEF) to BSHG again show differences particularly between proximal and middle regions (increased cross-linking in the middle region, p<0.01).Conclusion:The multiphoton and harmonic generation microscopy can be a powerful high resolution imaging method requiring minimal sample preparation that can provide structural information about spatially and spectrally resolved fibrillar collagens in three different posterior tibial tendon tendon regions. We found that middle third of the pathologic tendons show increased cross-linking and decreased orientation ratio of the collagen fibers which can provide insight into the regional based collagen remodeling and improved understanding of earliest stages of tendon destruction that could lead to improvements in the treatment of this condition.

Quantitative assessment of forward and backward second harmonic three dimensional images of collagen Type I matrix remodeling in a stimulated cellular environment.

The structural remodeling of collagens is important in several biological processes including wound healing, tendon repair and adaptation, fibrosis and morphogenesis. Multiphoton microscopy is efficient in the induction of highly specific second harmonic generation (SHG) signal from non-centrosymmetric macromolecules such as fibrillar collagens. Although the detectors in the reflection geometry have been normally employed for capturing the backward scattered SHG considering the wide range of engineered thick tissue applications, there are still questions about the generated 3D collagen structures because of the directional pattern of SHG signals. The present study dealt with an in vitro collagen-fibroblast raft or bioartificial tendon model where the stimulation of fibroblast cells induced lateral orientation of collagen Type I fibers. The SHG signals originating from 3D collagen matrix were captured simultaneously in both forward and backward scattering directions. Our structural analysis indicates that collagen fibers formed in such in vitro model systems are predominantly of uniform sizes and are aligned preferentially in the lateral direction. The criss-cross arrangements of laterally oriented fibers are evident in the initial stages of contraction but eventually those laterally oriented collagen fibers are found to be aligned in parallel to each other as well as to the fibroblasts after an extended period of contraction. Our comprehensive quantitative assessment of simultaneously captured forward and backward 3D SHG image datasets, which includes the SHG signal decay, fiber diameter, cell dimensions, colocalization profiles, the 3D voxel volumes and Fourier analysis, indicates strong correlation of structural features identified in forward and backward directions.

Minimally invasive multiphoton and harmonic generation imaging of extracellular matrix structures in lung airway and related diseases

Multiphoton microscopy has become a powerful imaging method for minimally invasive evaluation of extracellular matrix (ECM) and cellular structures deep within tissues in their native environments. This technology, which uses ultra-short femto-second laser pulses as the excitation source, is efficient in multiphoton excitation fluorescence (MPEF) of endogenously fluorescent macromolecular systems and induction of highly specific second harmonic generation (SHG) signals from non-centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of ECM as well as cellular morphologies in lung or airway tissue with spectral specificity and sensitivity. These imaging modalities are minimally invasive since structures deep within tissues can be visualized without the need for tissue fixation and/or sectioning. Much of the traditional histological and chemical procedures associated with conventional microscopy methods, which may alter native structure of lung tissue samples, can be circumvented to generate more accurate 3D morphological and fine structural information. In addition to outlining basic principles associated with MPEF and SHG microscopy methods, this review reports potential uses of these high resolution imaging modalities in lung structural imaging. We place special emphasis on imaging 3D structural features of airways, visualizing and quantifying ECM remodeling associated with mouse asthma model as well as the potential uses for multiphoton microscopy in in vitro airway applications.