Using our lab built two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) hybrid confocal imaging system, we observed, for the first time, the dynamic sarcomeric addition process in a rat cardiomyocyte cell culture system; this culture system expressed in vivo-like myofibril structure and mimicked mechanical overload experienced in a heart muscle tissue. Micro-grooved topographic patterned substrates com- bined with electrical stimulation are used to achieve the in vivo-like myofibril structure. After cardiomyocytes aligned, longitudinal and transverse mechanical stretch was applied to cardiomyocytes in parallel or perpendicu- lar, respectively, to the direction of alignment via stretching the substrates to mimic mechanical overload. Z discs, in which alpha-actinin expressed, have been proposed to involve in the process. TPEF detected alpha-actinin that labeled with enhanced yellow florescent protein via plasmid transfection. SHG is intrinsic to noncentrosym- metric structures, thus was used to detect myosin, a polar molecule expressed in myofibril. Pulse splitter system and synchronized recording system was introduced on TPEF-SHG imaging system to reduce the photodamage during live cell imaging. In our study, TPEF-SHG imaging system was used to study the dynamic process of sarcomeric addition in in vivo-like culture model under mechanical overload. This microscopic technique is ideal for tracking sarcomeric components to successively assemble onto pre-exist myofibrils and for revealing the role of Z discs played in sarcomeric addition. Transition of Z discs from continuous to broadened striation and from broadened to uniform striation under stretch has been observed. We concluded that continuous Z discs is the place of new sarcomeric addition.