Sp5 2 photon microscope

Flexor digitorum brevis fibers were transfected with an N-terminally tagged mCherryGRAF1 complementary DNA (cDNA) using methodology similar to the in vivo electroporation methods previously described [32 ]. Briefly, the footpads of wild-type (WT)¹²⁹ mice were injected with 10 μl of hyaluronidase (8 units). Two hours post injection, mCherryGRAF1 cDNA (2 μg/μl) was injected into the footpad and the muscle was stimulated. The muscle was allowed to recover for 7 days, and then fibers were isolated. The flexor digitorum brevis muscle was dissected and placed in DMEM containing BSA plus collagenase type 2 solution. Dissociated fibers were imaged on Matek confocal microscopy plates (P35G-1.5-14-C; Matek). Utilizing LAS AF Leica Imaging Software, membrane lesions were induced using FRAP Bleach point in the fluorescence recovery after photobleaching (FRAP) wizard protocol. The 405-nm laser was set at 80 % power for 3 s, and images were acquired on a Leica SP5 2 photon microscope. A single image was acquired before damage, upon laser damage, and every 2 s after damage, and then one image every 10 s.

Second‐harmonic imaging microscopy was performed on a Leica SP5 2‐photon microscope with a chameleon ultra laser with pulse width: < 140 fs peak, equipped with two NDD detectors. The collagen channel was excited with a wavelength of 800 nm and detected by a non‐descanned detector (NDD1) with a 400/15 nm cutoff filter. The DAPI channel was excited with a wavelength of 800 nm and detected by a non‐descanned detector (NDD2) with a 460/50 nm cutoff filter. All images were 12‐bit and were acquired with a 20×/1.00 HC PL APO water lens (WD 2.00 mm). Image analysis was performed using Leica application suite X and ImageJ.

Embryos were dechorinated using bleach and stage 10 embryos were manually picked from an agar plate using a Leica fluorescent dissecting microscope. The selected embryos were dorsally or laterally orientated and mounted on a coverslip coated with heptane glue to prevent drift during imaging. A drop of oil was placed on the embryos to maintain their survival. Embryos were imaged using an inverted Leica SP5 2-Photon microscope at 890-nm wavelength, using a 60 × oil immersion lens. Multi-position time-lapse stacks of 20–25 μm, and z-depth of 1.5 was acquired at 2-min intervals over a period of 60 min. Six movies per condition were selected for analysis, and the starting point was defined as the initiation of germband retraction, which is unaffected in the different conditions.