Quantifying Collagen Fibre Bundles in Mandibular Muscle

Second harmonic generation (SHG) images were acquired using a multiphoton confocal microscope system (LSM880 NLO, Carl Zeiss, Jena, Germany) with an excitation laser (Chameleon Vision II; wavelengths: 680–1080 nm, repetition rate: 80 MHz, pulse width: 140 fs; Coherent Inc., Santa Clara, CA, USA) and an objective lens (Plan-Apochromat 20×/0.8 M27, Carl Zeiss). Following image acquisition, the collagen fibre bundles were quantitatively evaluated using Imaris 8.4 software (Bitplane AG, Zurich, Switzerland). Collagen fibres with a thickness greater than 375 nm, which can be imaged with multiphoton excitation phase-contrast microscopy, were extracted as collagen fibre bundles. Evaluation was carried out by tracing the collagen fibre bundles within the bone in approximately 200 µm² of the mandibular masseter muscle prominence and calculating the mean diameter [27 (link)].