Second harmonic imaging of collagen organization in connective tissues

Abstract

Type I collagen is a major structural protein in mammals. This biopolymer is synthesized as a triple helix, which self‐assembles into fibrils (diameter: 10‐300 nm) and further forms various 3D organizations specific to each tissue. In recent years Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique for the in situ investigation of the fibrillar collagen structures in matrices or tissues [1]. However, as an optical technique with typically 300 nm lateral resolution, SHG microscopy cannot resolve most of the collagen fibrils. Moreover, in contrast to incoherent fluorescence signals that scale linearly with the chromophore concentration, SHG is a coherent multiphoton signal that scales quadratically with the density of collagen triple helices aligned with the same polarity in the focal volume. Consequently, quantitative SHG measurements have been limited so far to averaged phenomenological parameters [1]. In this study, we correlated SHG and transmission electron microscopies to determine the sensitivity of SHG microscopy and calibrate SHG signals as a function of the diameter of the collagen fibril [2]. To that end, we synthesized in vitro isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter (see figure 1). We observed that SHG signals scale as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. It validated our quantitative bottom‐up approach used to calculate the non‐linear response at the fibrillar scale and demonstrated that the high sensitivity of SHG microscopy originates from the parallel alignment of triple helices within the fibrils and the subsequent constructive interference of SHG radiations. This calibration was then applied to intact rat corneas, where we successfully recovered the diameter of hyperglycemia‐induced fibrils in the Descemet's membrane without having to resolve them [2,3]. Importantly, this calibration only applies to isolated fibrils. Nevertheless, complementary techniques can probe the sub‐micrometer structure of dense distributions of collagen fibrils. In particular, we have shown that polarization‐resolved SHG microscopy can probe the main orientation of collagen fibrils and their orientation disorder within the focal volume [4]. Combination of this modality with traction assays then provides a new method to measure the reorganization of the collagen network upon stretching and to correlate this microscopic response to the biomechanical response at macroscopic scale [4, 5]. In conclusion, our data represent a major step towards quantitative SHG imaging of collagen organization in biomaterials or connective tissues.