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

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.