Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J. Biophoton. 3, 34-43

L.E.N.S. and Department of Physics, University of Florence, Sesto Fiorentino, Italy.
Journal of Biophotonics (Impact Factor: 4.45). 09/2009; 3(1-2):34-43. DOI: 10.1002/jbio.200910062
Source: PubMed


We have used nonlinear imaging to evaluate collagen organization in connective tissue ex-vivo samples. Image analysis methods were tested on healthy dermis, normal scars, and keloids. The evaluation of the second harmonic to autofluorescence aging index of dermis (SAAID) has allowed a first characterization of tissues by scoring the collagen/elastin content. Further analyses on collagen morphology in healthy dermis and keloids were performed by image-pattern analysis of SHG images. The gray-level co-occurrence matrix (GLCM) analysis method has allowed classification of different tissues based on the evaluation of geometrical arrangement of collagen fibrillar bundles, whereas a pattern analysis of the FFT images has allowed the discrimination of different tissues based on the anisotropy of collagen fibers distribution. This multiple scoring method represents a promising tool to be extended to other collagen disorders, as well as to be used in in-vivo skin-imaging applications.

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Available from: Dimitrios Kapsokalyvas, Oct 05, 2014
    • "In particular, SHG microscopy can provide morphological and functional images of anisotropic biological structures with high hyperpolarizability, such as collagen. Thanks to its high sensitivity to collagen, SHG microscopy has been used to reveal collagen organization in a variety of tissues, including human dermis, keloid, and tumor microenvironment [7] [8] [9]. Nevertheless applications to human cardiac tissues [5;10], and in particular to the relation between atrial collagen and AF [11;12], remain sparse. "
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    ABSTRACT: The assessment of collagen structure in cardiac pathology, such as atrial fibrillation (AF), is essential for a complete understanding of the disease. This paper introduces a novel methodology for the quantitative description of collagen network properties, based on the combination of nonlinear optical microscopy with a spectral approach of image processing and analysis. Second-harmonic generation (SHG) microscopy was applied to atrial tissue samples from cardiac surgery patients, providing label-free, selective visualization of the collagen structure. The spectral analysis framework, based on 2D-FFT, was applied to the SHG images, yielding a multiparametric description of collagen fiber orientation (angle and anisotropy indexes) and texture scale (dominant wavelength and peak dispersion indexes). The proof-of-concept application of the methodology showed the capability of our approach to detect and quantify differences in the structural properties of the collagen network in AF versus sinus rhythm patients. These results suggest the potential of our approach in the assessment of collagen properties in cardiac pathologies related to a fibrotic structural component.
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    • "Experimental techniques providing volume 3D images of the collagen structure of tissues do already exist in the literature. For example, we note the multiphoton fluorescence microscopy techniques [6] [7] [8], which have the advantage to avoid any preparation of the sample for the observation of collagen due to its autofluorescence. "
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    ABSTRACT: The assessment of the three-dimensional architecture of collagen fibers inside vessel walls constitutes one of the bases for building structural models for the description of the mechanical behavior of these tissues. Multiphoton microscopy allows for such observations, but is limited to volumes of around a thousand of microns. In the present work, we propose to observe the collagenous network of vascular tissues using micro-CT. To get a contrast, three staining solutions (phosphotungstic acid, phosphomolybdic acid and iodine potassium iodide) were tested. Two of these stains were showed to lead to similar results and to a satisfactory contrast within the tissue. A detailed observation of a small porcine iliac vein sample allowed assessing the collagen fibers orientations within the medial and adventitial layers of the vein. The vasa vasorum network, which is present inside the adventitia of the vein, was also observed. Finally, the demonstrated micro-CT staining technique for the three-dimensional observation of thin soft tissues samples, like vein walls, contributes to the assessment of their structure at different scales while keeping a global overview of the tissue. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.
    Full-text · Article · May 2015 · Comptes rendus biologies
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    • "Some collagen classification metrics that are discussed in the skin aging section, also find applications in wound healing. Cicchi et al. also assessed three different approaches, including employing a gray-level co-occurrence matrix (which was studied further by Ferro et al.170), a fast Fourier transform and a measure known as the SAAID, or Second-harmonic Autofluorescence Aging Index of Dermis.142 Rather than determining one superior technique, it was found that each was effective at a particular length scale. "
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    ABSTRACT: This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.
    Full-text · Article · Jan 2014 · Journal of Innovative Optical Health Sciences
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