In vitro model for endogenous optical signatures of collagen

Abstract

Type I collagen is a major component of the extracellular matrix as well as many tissue engineered models. To understand changes in collagen related models over time, it is important to evaluate collagen dynamics with noninvasive techniques. Fluorescence spectroscopy provides a method to noninvasively measure endogenous collagen fluorescence. Additionally, second harmonic generation (SHG) imaging of collagen produces high resolution images of the fibrils. In this study, a novel in vitro collagen measurement chamber was developed for measurement in standard spectroscopic cuvette chambers and microscopic imaging. The fluorescence of polymerized collagen was found to be highly variable, primarily depending on incubation time after polymerization. Changes in fluorescence over time were consistent with increases at UVA excitation wavelengths (lambda ex = 360 nm) and decreases at UVC excitation wavelengths (lambda ex = 270 nm), suggesting changes in nonenzymatic association of the collagen fibrils. SHG imaging of the collagen suggested that a stable network formed during polymerization. Unlike the fluorescence emission, SHG images from the gels varied little with time suggesting that SHG is not as sensitive to cross-linking or fibril-fibril associated changes. The developed measurement system will allow further studies on the effect of enzymatic cleavages and structural alterations on collagen fluorescence and SHG.

Endogenous Optical Biomarkers of Ovarian Cancer Evaluated with Multiphoton Microscopy

“…The GLCM provides texture features based on gray-level statistical patterns between neighboring pixels. In particular, the correlation feature, a measure of intensity correlation as a function of pixel distance, relates to collagen fibril structure by indicating fibril size and separation (29). The correlation was calculated for distances ranging from 1 to 30 pixels (2-60 Am) in the horizontal direction (vertical and diagonal correlations yielded similar results).…”

“…To quantitatively assess these collagen-related changes, we applied GLCM texture analysis to the SHG images. The correlation feature extracted from the GLCM provided an estimate of collagen fibril organization and structure (29). Because this feature is an average from the entire image, images dominated by more defined, structured fibrils result in a substantial reduction in correlation with distance (steeper slope).…”

Fully automated, quantitative, noninvasive assessment of collagen fiber content and organization in thick collagen gels

“…For example, SHG images of 3 mg/ml collagen type I gels reported by Kirkpatrick et al exhibit a very similar morphology to the images acquired from the acellular gels of our study, consisting of a highly disorganized mesh of collagen fibers. 21 Analysis based on the correlation of SHG intensity as a function of pixel distance did not reveal any significant changes in the organization of collagen fibers during the first two days of observation following the onset of polymerization. However, by day 8 the gels exhibited lower correlation distances, consistent with the decreased levels of OI and entropy that we observe in the acellular gels of this study over 14 days of observation.…”

“…17,18 Finally, the degree of organization and alignment within fibrillar bundles has been found to significantly influence the detected SHG signal. 19 The analytical tools that have been presented so far for characterizing collagen organization relied at least to a certain extent either on user input, 8,12,17,[19][20][21][22] or on computationally intense calculations that confine the region of interest to a limited region. 9 Here, we present the use of an adaptive thresholding approach in combination with Fourier-and a Hough-transform based analysis as a relatively simple and efficient means of acquiring quantitative information on the content, orientation, and local organization of collagen fibrils.…”

Development of a highly sensitive spectral camera for cartilage monitoring using fluorescence spectroscopy

“…The different collagen types and elastin are among the most important fluorophores in the extracellular matrix (Tuan and Brian, 2003). The collagen fluorescence is caused by pyridinoline, a crosslinking compound of collagen (Uchiyama et al, 1981;Kirkpatrick, 2006). Because of differences in the fluorescence properties of the different collagen types, it is possible to distinguish collagen I and II non-invasively by recording the fluorescence spectra after UV excitation.…”