confocal microscope (Carl Zeiss Microscopy) with a mode-locked Ti:Sapphire
Chameleon Ultra laser (Coherent Inc.) in combination with non-descanned
detection (NDD) to visualize the collagen fibers and quantify their orientation
and distribution as previously described31 (link). The laser was set to 800 nm and emission was filtered
from 380 - 430 nm. Second harmonic generation (SHG) images were collected using
a Plan-Apochromat 20x objective and Zeiss ZEN software. The fiber direction was
estimated using OrientationJ distribution, an ImageJ plug-in (NIH) developed for
directional analysis. A distribution of local angles was generated for each
optical slice, where 0° aligned to the horizontal axis (length-wise along
the tendon) and ± 90° to the vertical axis. The mean and standard
deviation of collagen fiber orientation was calculated from the distribution of
each image. For analysis, eight sections of supraspinatus tendon (each section
was imaged at both proximal and distal ends) were used.