Morphometry of dermal collagen orientation by Fourier analysis is superior to multi‐observer assessment

Abstract

In human dermis, collagen bundle architecture appears randomly organized, whereas in pathological conditions, such as scar tissue and connective tissue disorders, collagen bundle architecture is arranged in a more parallel fashion. Histological examination by one or two observers using polarized light is the most common method to determine collagen orientation. The hypothesis on which this study is based is that an objective image analysis technique, Fourier analysis, would improve the reliability (are the measurements reproducible?) and the accuracy (does the method measure what it is supposed to measure?) of collagen orientation assessment, compared with observer ratings. Fourier analysis was applied to 271 images of scar tissue and normal skin that were acquired by confocal laser-scanning microscopy. Observers rated the same areas using polarized light as well as the confocal microscopy images. Computer images consisting of different types of ellipses were generated with a fixed orientation. Observers and Fourier analysis evaluated the images to evaluate accuracy. The inter-observer reliability was acceptable when at least three observers rated polarized light images (r > 0.69), whereas two observers were sufficient for rating confocal microscopy images (r > 0.71). Fourier analysis correlated better with observer ratings of confocal microscopy images (r = 0.69) than with polarized light microscopy images (r = 0.42). Fourier analysis was more accurate than four observers for the evaluation of the 'true' orientation for almost all types of computer-generated images. For the first time it is shown that Fourier image analysis is suitable for the morphometry of dermal collagen orientation and leads to a superior measurement of collagen orientation compared with subjective histological evaluation by several experts. If an evaluation is performed by conventional light microscopy, at least three observers are required to attain an acceptable inter-observer reliability.

A Quantitative Approach to Scar Analysis

“…3,4 Fourier transform analysis failed to show a statistically significant difference between WT and FMOD Ϫ/Ϫ unwounded dermis and between scar tissue and unwounded tissue of WT animals. A possible explanation for this observation may be the fact that we used higher-magnification images of the collagen bundles compared with previous studies using Fourier analysis (1000ϫ objective versus 5ϫ).…”

“…A possible explanation for this observation may be the fact that we used higher-magnification images of the collagen bundles compared with previous studies using Fourier analysis (1000ϫ objective versus 5ϫ). 3,5,12 Unlike Fourier transform analysis, fractal calculations change as the analyzed pixel size in the image gets smaller. Therefore as the image resolution is increased, more accurate F D and L values are obtained.…”

“…Because 85% of the dermis consists of collagen, 1 dermal elasticity and strength are primarily determined by collagen with lesser contributions from elastin and other extracellular matrix constituents. 2, 3 At present, no consistent methodologies exist for quantitatively assessing dermal collagen architecture. Despite the integral nature of collagen ultrastructure and architecture to wound healing research, few studies have been conducted on evaluation of collagen architecture, with most studies published decades ago.…”

“…5 Currently traditional scar morphology and collagen architectural analyses are performed by one or two observers, using conventional light microscopy in combination with polarized light. 6,7 More objective methods such as X-ray diffraction, 8,9 laser scattering, 10,11 and Fourier transform analysis 3,4,12 also have been recently described. Head-to-head comparisons of these modalities indicate that Fourier transform analysis, which provides information about collagen bundle orientation, randomness, and spacing, 12 appears to be the superior method.…”

Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils

“…These methods exhibit spatial resolutions that enable imaging of the ultrastructure of bone, where the ultrastructural elements (mineralized collagen fibrils or fibril bundles) can be visually identified. Quantification of the orientation and arrangement of the ultrastructure is performed by image post-processing of the acquired images, either through specialized orientationsensitive algorithms [53][54][55] or, most commonly, through two-dimensional (2D) or 3D Fourier transform (FT) [56], which allow the orientation and degree of orientation (DO) to be derived [57][58][59][60]. It should be noted that the indirect assessment of the organization of mineralized collagen fibrils by imaging techniques can lead to artefacts, which are discussed in the introduction of the respective subsection.…”