T-tubules are invaginations of the plasma membrane that conduct the electrical depolarization to the cardiomyocyte core, allowing a fast and synchronous Ca2+ release. We have recently developed an ultrafast random access multi-photon (RAMP) microscope that, in combination with a customly synthesized voltage-sensitive dye (VSD), is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We found that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, we found that AP propagation into the pathologically remodeled tubular system frequently fails and may be followed by local spontaneous electrical activity (Sacconi et al. PNAS 2012). To clarify the link between tubular abnormalities and Ca2+-dependent arrhythmias, we combine the advantage of RAMP microscope with a double staining approach to optically record tubular AP and, simultaneously, the corresponding local Ca2+ transient. Isolated rat cardiomyocytes were co-loaded with the VSD and a green calcium indicator. Although the calcium and voltage probes can be excited at the same wavelength, the large Stokes shift of the VSD emission allows us to use spectral unmixing to resolve the voltage and calcium responses. The capability of our technique in probing spatiotemporal relationship between Ca2+ and electrical activity was explored in a model of acute detubulation in which failure to conduct AP in disconnected t-tubules causes local delay of Ca2+ transient rise.