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Capturing high-speed structural dynamics of contracting myocytes is an important requirement for understanding the biophysical processes occurring during contraction. We present our recent technological innovations that allow for real-time three-dimensional (3D) observation of the moving cardiac muscle tissue in beating chick embryo hearts. In vivo dynamics in the beating heart muscle tissue are visualized using the multicontrast nonlinear optical microscope. The setup employs two femtosecond laser beams with staggered pulse trains, which allow for the simultaneous measurement of second harmonic generation at perpendicular laser polarizations. Fast refocusing deep inside the sample is achieved by the deformable mirrors, which allow imaging moving structures in 3D. The microscopy setup revealed intricate interactions between sarcomeres during contracting and quiescent periods of the myocytes. The …