Ld c apochromat lens 40x 1 1 uv vis ir wd 0

An inverted multiphoton system (TriM-Scope II; LaVision BioTec GmbH; Bielefeld, Germany) with a femtosecond pulsed Ti:Sa laser was used to image muscle samples (water immersion objective, LD C-Apochromat lens 40x/1.1/UV-VIS-IR/WD 0.62, Carl Zeiss, Jena, Germany) for myosin-II and collagen structures. The Ti:Sa laser was tuned to a wavelength of 810 nm. The signal in forward-scattered direction was collected through a band-pass filter at 405 nm ±10 nm (Chroma, Rockingham, VT, USA). The backscattered signal was collected for collagen area measurements. It was separated with a 460 nm beamsplitter, and both beams were collected. The autofluorescence (>460 nm) signal was used to separate myosin and collagen via thresholding from the forward scattered signal. The <460 nm channel images were collected for binary mask denoising, where applicable. Images were acquired as stacked images with a resolution of 1072 × 1072 pixels. Images were processed in Image J (National Institute of Health, Bethesda, MD, USA) to apply gamma correction and adjusted for brightness for enhanced visualization only.

a. Label‐free SHG imaging. Label‐free SHG imaging was performed on an inverse multiphoton microscope (TriMScope, LaVision BioTec, Bielefeld, Germany) with a mode‐locked fs‐pulsed Ti:Sa laser (Chameleon Vision II, Coherent, Santa Clara, CA, USA). The laser was tuned to a wavelength of 810 nm, generating the second harmonic generation signal at 405 nm. The laser was focused into the sample by a water immersion objective (LD C‐Apochromat lens – 40x/1.1/UV–vis‐IR/WD 0.62, Carl Zeiss, Jena, Germany), and the generated SHG signal was detected by an ultra‐sensitive photo multiplier tube (PMT) (H 7422–40 LV 5 M, Hamamatsu Photonics) in transmission mode to target the SHG of myosin‐II.
b. Functional force measurements via the MyoRobot system.^[37, ^38] The MyoRobot was a biomechatronics system for automated assessment of biomechanical active and passive properties as previously described.
c. Functional force measurements via the MechaMorph system.^[31] The MechaMorph was a custom‐engineered device for combined structure–force measurements. A small measurement chamber could be inserted onto the microscope stage below the objective. Single muscle fiber segments could be mounted between a force transducer and a software‐controlled voice coil actuator (VCA) that allows the MechaMorph to perform subsequent isometric force measurements and structural imaging via SHG microscopy.