3d Imaging and Scoring of Collagenous Fibrosis Using Second Harmonic Microscopy

We showed that second harmonic generation (SHG) microscopy presents unique advantages compared to conventional optical techniques to investigate the 3D heterogeneous accumulation of fibrillar collagen during fibrotic pathologies. We compared the SHG signal distribution to anti-collagen I and anti-collagen IV immunohistochemical labeling [1] and we measured the SHG response from collagen I and IV molecular films [2]. These experiments proved that SHG is obtained specifically from fibrillar collagen in tissues. It results in an intrinsically small background noise in the images and enables sensitive measurements. Furthermore, the ability of SHG microscopy to discriminate between fibrillar and non fibrillar collagens is of invaluable interest for pathologists because measurements of fibrillar collagen accumulation provide better scores of severity than scores related to non fibrillar collagen. We therefore proposed phenomenological scores based on the volume density of voxels exhibiting significant SHG signal and on the SHG averaged intensity. We showed that the SHG density score is sensitive to the accumulation of small collagen fibers, whereas the SHG intensity score probes the formation of larger fibers [1]. We applied this methodology to lung fibrosis. We found that combined 2-photon excited fluorescence (2PEF) and SHG imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in the bleomycin murine model, and underlining all characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. We measured density scores in various lung regions, and successfully sorted out bleomycin treated mice and control mice, with a greater sensitivity in the subpleural region [3]. We then measured SHG density and intensity scores in a murine model of hypertensive renal fibrosis, using an image segmentation based on the tissue morphology visualized by endogenous 2PEF signals. We successfully sorted out control and treated mice, and evidenced accumulation of fibrillar collagen in the renal interstitium, which proved that other renal compartments than the glomerulus are affected during hypertensive nephropathies. We also compared the respective distributions of collagen assembly sites, revealed by SHG signals, and of collagen synthesis sites, visualized by 2PEF signals from GFP constructs in transgenic animal models. This approach should help elucidating the role of various enzymes related to the collagen assembly into fibers and proposing new therapeutic approaches [1].