Multiscale investigation of collagen structure in human skin and gel matrices using polarization resolved second harmonic generation microscopy

Abstract

Collagen is critical to the structure and function of skin tissues, with the collagen I/III ratios influencing fibrillogenesis, fiber organization, and skin mechanics. Abnormal collagen organization, such as in fibrosis or scar tissue, compromises both skin functionality and aesthetics. In this study, we employed label-free polarization resolved second harmonic generation (PSHG) microscopy to investigate collagen structure in artificial collagen matrices with various Col I/III ratios at the fibril scale ( $\sim$ 1 to $3\mu \text{m}$ ) and in ex vivo human healthy and scarred skin at the fiber scale ( $\sim 10$ to $20\mu \text{m}$ ). Complementary third harmonic generation (THG) microscopy provided additional structural information. Our results indicate that an increasing Col I/III ratio is associated with longer fibril length, higher PSHG intensity, and a reduced effective $\alpha$ -helix pitch angle of fibrils. In pure Col I, the effective $\alpha$ -helix pitch angle is determined to be $47.{72}^{\circ }$ . These observations indicate alterations in fibril assembly. Furthermore, although the $\alpha$ -helix pitch angle of fibers in both healthy and scarred skin was approximately $46.7^{\circ }$ , healthy skin exhibited $24\%$ greater variability in fiber orientation, suggesting a more randomized organization compared to scar tissue. THG imaging further revealed a higher cellular density in scar tissue, consistent with the inflammatory activity associated with wound healing. Immunohistochemical (IHC) staining using dermatansulphate and Col III-specific antibodies confirmed that the Col I/III ratio is higher in healthy skin (2.2) than in scarred skin (1.6). These findings underscore the potential of PSHG microscopy for label-free, quantitative assessment of collagen structure across multiple scales, with THG offering complementary cellular insights. This integrated approach represents a promising strategy for real-time, in vivo monitoring and automated quantification of collagen organization in clinical applications, including dermatology, burn treatment, and fibrosis monitoring.

Keywords: Collagen gel; Collagen type I and type III; Polarization second harmonic generation; Scar skin.

Fig. 1

(A) Schematic diagram of the experimental setup. SL, scan lens; TL, tube lens; M, mirror; L1, L2, L3 focusing lens. (B) Starch granule images with 18 polarizations (from $0^{\circ }-{170}^{\circ }$ in step size of ${10}^{\circ }$). White arrows indicate the polarization angles of the laser.

Fig. 2

Polarization resolved SHG measurements on different structures. (A) PSHG images (red) recorded at polarization angle $\varphi =0^{\circ }$ of starch granule, human and mouse ECM, mouse muscle, alongside THG signals (green). (B) PSHG intensity profiles of a randomly selected pixel of interest. Color maps (C-F), frequency distributions (G-I), and mode values (J) of structure angle, ${\chi }_{15}/{\chi }_{31},{\chi }_{33}/{\chi }_{31}$, and $\alpha$-helix pitch angle for amylopectin, collagen fibers, and myofibrils. Same color represents same structure, one curve/dot represent one sample. * stands for p<0.05, ** for p<0.01, *** for p<0.001 and ns for no significant difference.

Fig. 3

(A) Western blot (WB) images show bands around $\sim$115 kDa in collagen gel samples with decreasing Col I proportions, detected using a Col I antibody. Lane numbers correspond to the Col I proportions. A representative cropped blot is shown, with all original blots provided in Supplement 1, Figure S1. (B) Quantitative analysis showing the Col I incorporation (exposure time: 100 s). Data from two individual blots are represented in the graph. (C) PSHG images of collagen gel matrices recorded at polarization angle $\varphi =0^{\circ }$. (D) Mean SHG intensity profiles with different polarization angles. Collagen fibrils (E) length and (F) thickness in matrices with different Col I/III ratios. (G) The mode values of ${\chi }_{15}/{\chi }_{31},{\chi }_{33}/{\chi }_{31}$ and $\alpha$-helix pitch angle of different collagen gel matrices. Triangles represent the results of trial 1, and stars represent the results of trial 2. For each trial 5 ROIs were measured.

Fig. 4

(A) SHG/THG images of cross section of a fresh hypertrophic scar skin sample, measured with the cross-section face to the objective. Color maps (B - D) and histogram distributions (E - G) of ${\chi }_{33}/{\chi }_{31},{\chi }_{15}/{\chi }_{31}$ and $\alpha$-helix pitch angle for two boxed regions.

Fig. 5

(A) SHG/THG, Hematoxylin and eosin (H&E) staining images, and fiber orientation maps of frozen sections from healthy skin and three types of scar tissue. Scale bar 100 $\mathrm{\mu }$m. Total area is $1\times 0.8{\text{mm}}^2$. (B) Fiber orientation distribution and (C) fiber mask. (D) Quantitative analysis of the standard deviation of fiber orientation distribution and fiber density of healthy skin (n=5) and scar skin (n=7).

Fig. 6

PSHG and IHC analysis of healthy and scar skin tissue. (A) Color maps of ${\chi }_{15}/{\chi }_{31}$, ${\chi }_{33}/{\chi }_{31}$ and $\alpha$-helix pitch angle, providing collagen structure information. (B) IHC staining for dermatansulphate and Col III. Scale bar 100 $\mathrm{\mu }$m. (C) Mean mode values of ${\chi }_{15}/{\chi }_{31}$, ${\chi }_{33}/{\chi }_{31}$ and $\alpha$-helix pitch angle of healthy (n = 5) and scarred (n = 7) skin samples. (D) The ratio of Col I/III analyzed from IHC images. Dermatansulphate was used as proxy for Col I. Shown in mean, error bars represent SD.