While quantitative analysis of dynamic biological cell motions in vivo is of great biomedical interest, acquiring 3-D (plus time) information is difficult due to the lack of imaging tools with sufficient spatial and temporal resolution. A novel 3-D high-speed microscopic imaging system is developed to enable 3-D time series data acquisition, based on a defocusing technique (DDPIV). Depth coordinate Z is resolved by the triangular image patterns generated by a mask with three apertures forming an equilateral triangle. Application of this technique to microscale imaging is validated by calibration of targets spread over the image field. 1-µm fluorescent tracer particles are injected into the blood stream of 32 h post-fertilization developing zebrafish embryos to help describe cardiac cell motions. 3-D and velocity fields of cardiovascular blood flow and trajectories of heart-wall motions are obtained.