AP(s) and (2) to track CaV1.1 individual VSD rearrangement in muscle fibers. We first demonstrate the physiological relevance of AP(s) optical measurement for ECC study. AP-like depolarization applied to muscle fibers reveals differences in the amount and kinetics of VSD recruitment when compared to long depolarization; and also shows a decrease in RyR1 Ṙ and occurrence of CaV1.1 ICa within AP train, both in a frequency dependent way. Next, to characterize individual VSD activation, we developed a variation of functional site-directed fluorometry (FSDF) allowing individual VSD tracking in dissociated muscle cells. This gave us the ability to detect optically individual VSD motion in response to AP. Through this technique, we demonstrated individual VSD activation heterogeneity. We then combined this approach with voltage clamp to perform voltage clamp fluorometry (VCF), allowing characterization of individual VSD, IQ, Ṙ, and ICa in response to imposed depolarization. This is a first in the implementation of VCF in native cells. We showed that VSDs have independent voltage dependence, and that VSD-II is implicated in RyR1 Ṙ while VSD-I and -IV are involved in ICa gating. Overall, this work provides valuable insights into ECC that could be the basics for CaV1.1 therapeutic regulating ICa and Ṙ independently.