Dermal Organization in Scleroderma: The Fast Fourier Transform and the Laser Scatter Method Objectify Fibrosis in Nonlesional as well as Lesional Skin

Abstract

SUMMARY:Scleroderma, a chronic, progressive disorder, is characterized by dermal fibrosis with collagen bundles orientated parallel to the epidermis. Simple objective parameters to evaluate disease progression and therapies are needed. We describe two methods, the laser scatter method and the fast Fourier transform (FFT), to measure collagen bundle orientation and spacing. Lesional sclerodermic skin (LS), nonlesional sclerodermic skin (nonLS), and control skin (CS) sections were evaluated for orientation ratio using the laser scatter method. The FFT was used to calculate orientation ratio, variation, and spacing of collagen bundles. Parameters were correlated with local and mean skin score measurements, on a scale of 0 (normal) to 3 (severely sclerotic). With both the laser scatter method and the FFT, orientation ratios of LS (respectively, 2.16 Ϯ 0.33 and 1.83 Ϯ 0.62) were significantly higher than CS (respectively, 1.70 Ϯ 0.35 and 1.38 Ϯ 0.15). NonLS orientation ratios (respectively, 1.92 Ϯ 0.15 and 1.48 Ϯ 0.44) were between LS and CS ratios. Orientation variation and bundle spacing of LS (respectively, 57.3 Ϯ 19.4 and 15.7 Ϯ 5.6 m) were significantly reduced compared to CS (respectively, 73.8 Ϯ 15.0 and 18.9 Ϯ 1.9 m). NonLS orientation ratios (respectively, 57.2 Ϯ 29.0 and 15.6 Ϯ 6.1 m) were similar to LS. Bundles in LS are more parallel, show less variation in orientation, and are more densely packed than in CS. There was a linear correlation between mean skin score and orientation ratio. Local skin score was not linearly correlated to orientation ratio. Our findings suggest that nonLS dermis without clinical sclerosis already shows fibrotic characteristics. Both techniques were easy to use and suitable for objectifying dermal fibrosis in scleroderma lesions. FFT is more accurate and reproducible than the laser scatter method and allows simultaneous pathological evaluation of the location of the analyzed tissue sections. Future studies will need to focus on the correlation between clinical disease severity and collagen bundle characteristics. (Lab Invest 2000, 80:1281-1289.

A Quantitative Approach to Scar Analysis

“…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.…”

“…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.…”

“…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. However, Fourier transform analysis has limitations when applied to analysis of complex biological structures at high magnification.…”

Quantitative analysis on collagen morphology in aging skin based on multiphoton microscopy

“…[15][16][17][18] In this study, the ratio of short axe to long axe of the generated power plot of images was used to estimate the collagen orientation index (N), which was calculated by N = [1-(short/long)]. 16,17 The N of perfectly random tissue is "0," meaning isotropic behavior, and its FFT plot is a circular.…”

Correlation between polarization sensitive optical coherence tomography and second harmonic generation microscopy in skin

“…Collagen organization is important because it is related to mechanical properties and function of the skin [23,24]. Collagen organization was characterized by collagen orientation index which could be obtained from SHG images by either Fast Fourier Transform (FFT) or laser scattering method [17,22,25]. Recently, polarization sensitive SHG (PS-SHG) microscopy, which utilizes polarization sensitivity of SHG intensity from collagen fibers and other SHG source, has been developed to provide more detailed information [21,[26][27][28][29][30].…”