Quantitative nanohistological investigation of scleroderma: an atomic force microscopy-based approach to disease characterization

Abstract

Scleroderma (or systemic sclerosis, SSc) is a disease caused by excess crosslinking of collagen. The skin stiffens and becomes painful, while internally, organ function can be compromised by the less elastic collagen. Diagnosis of SSc is often only possible in advanced cases by which treatment time is limited. A more detailed analysis of SSc may provide better future treatment options and information of disease progression. Recently, the histological stain picrosirius red showing collagen register has been combined with atomic force microscopy (AFM) to study SSc. Skin from healthy individuals and SSc patients was biopsied, stained and studied using AFM. By investigating the crosslinking of collagen at a smaller hierarchical stage, the effects of SSc were more pronounced. Changes in morphology and Young's elastic modulus were observed and quantified; giving rise to a novel technique, we have termed "quantitative nanohistology". An increase in nanoscale stiffness in the collagen for SSc compared with healthy individuals was seen by a significant increase in the Young's modulus profile for the collagen. These markers of stiffer collagen in SSc are similar to the symptoms experienced by patients, giving additional hope that in the future, nanohistology using AFM can be readily applied as a clinical tool, providing detailed information of the state of collagen.

Keywords: adjunct diagnosis; collagen; nanohistology; picrosirius red; rheumatology.

Figure 1

LM images of control (A and B) and SSc (C and D) biopsies taken in unpolarized and polarized light (10× magnification). Abbreviations: LM, light microscopy; SSc, systemic sclerosis.

Figure 2

SEM images of control (A and B) and SSc (C and D). Notes: Scale bars of (A) and (C): 20 µm, 1,000× magnification; scale bars of (B) and (D): 2 µm, 15,000× magnification. Circled area in (D) shows an area of high collagen disorder due to SSc. Abbreviations: SEM, scanning electron microscopy; SSc, systemic sclerosis.

Figure 3

AFM analysis of control skin sample in random locations. (A) Standard LM was performed on a healthy control skin sample, and four areas of interest (A–D) were marked (10× magnification). (B) AFM image of area D from (A), showing interface between three different collagen regions (scale bar 2 µm). (C) Young’s modulus obtained by transverse collagen on fibrils in locations A–D from (A). No areas were significantly different, P<0.05 (Kruskal–Wallis). Abbreviations: AFM, atomic force microscopy; LM, light microscopy.

Figure 4

AFM analysis of control skin sample. (A) Polarized LM showing red, red/yellow, yellow and green areas of interest (10× magnification). (B) AFM error image of red area showing collagen present and highlighting an area of collagen (scale bar 2 µm). (C) AFM error image of green area showing minimal intact collagen (scale bar 2 µm). (D) AFM error image of highlighted red area with inset showing a line profile from the height image (scale bar 800 nm). Abbreviations: AFM, atomic force microscopy; G, green; LM, light microscopy; R, red; R/Y, red/yellow; Y, yellow.

Figure 5

Young’s modulus values for control and SSc skin samples. (A) Young’s modulus obtained by transverse indentation on collagen fibrils in each area. Yellow and green areas are statistically different (indicated with asterisks) compared with red, P>0.05 (Kruskal–Wallis ANOVA). (B) Young’s modulus obtained by transverse indentation on collagen fibrils in each area. Red/yellow, yellow and green areas are statistically different (indicated with asterisks) compared with red, P>0.05 (Kruskal–Wallis ANOVA). Abbreviations: ANOVA, analysis of variance; G, green; R, red; R/Y, red/yellow; SSc, systemic sclerosis; Y, yellow.

Figure 6

AFM analysis of SSc skin sample. (A) Polarized LM showing red, red/yellow, yellow and green areas of interest (10× magnification). (B) AFM error image of red area showing collagen present and highlighting an area of collagen (scale bar 2 µm). (C) AFM error image of green area showing minimal intact collagen (scale bar 2 µm). (D) AFM error image of highlighted red area with inset showing a line profile from the height image (scale bar 800 nm). Abbreviations: AFM, atomic force microscopy; G, green; LM, light microscopy; R, red; R/Y, red/yellow; SSc, systemic sclerosis; Y, yellow.