Measuring Sarcomere Dynamics and Calcium Transients in Skeletal Muscle Fibers

We used the IonOptix Calcium and Contractility System connected with MyoCam-S (IonOptix) in an inverted microscope with a 40× objective to measure sarcomere length kinetics and fluorescence photometry. IonOptix calculates the average frequency of the sinusoidal appearance by altering striation patterns of dark (A-band) and light (I-band) from isolated single myofibers. We applied 250 Hz sampling frequency for sarcomere length kinetics and 1000 Hz for fluorescence photometry with 25 V, 8 ms pulse duration (supplemental information Figure S3), 0.2 Hz field stimulation frequency at 25 °C. This stimulation achieved peak twitch contractions in isolated FDBs which is known to be sensitive to different electrical field configurations and pulse parameters [42 (link)]. For myoplasmic Ca²⁺ transients, the isolated FDB fibers were loaded with 1 μM Fura-2AM (F1221, Invitrogen) in M199 for 10 min at room temperature, and 15 min in M199 for deesterification in the dark. Sarcomere length kinetics and Fura-2AM signals were measured simultaneously. Our data with Fura-2 as an intracellular Ca²⁺ indicator compares well with a recent report using Fura-2 in skeletal muscle [43 (link)].