Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.

Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-μm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4',6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastincollagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease;  < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease;  < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy. Impact statement This work demonstrated that second harmonic generation microscopy in conjunction with elastin immunofluorescence provided a comprehensive assessment of multiscale structural re-organization in healing tendon than when restricted to longitudinal collagen fiber alignment alone. Utilizing this approach for tendon histomorphometry is ideal not only to improve our understanding of hierarchical structural changes that occur after tendon injury and during remodeling but also to monitor the efficacy of therapeutic approaches.