As the frequency of ultrasound is inversely proportional to the wave length and beam width, GHz range ultrasound realizes the nano-level ultrasonic imaging. In the present study, a prototype of ultrasonic nano-level imaging system using a single pulsed ultrasound is proposed. A 500-ps width single electric pulse was input into an ultrasonic transducer made of ZnO with the central frequency of 500 MHz, which corresponded to 2-μm focal spot. The reflections were captured by 2GSa/s analogue-digital converter. The transducer was driven by two linear servo motors to scan XY-plane above the specimen. The scanning field was available from 2.4 × 2.4 mm to 40 × 40 μm with 300 × 300 pixels. The waveform from the tissue area contains two reflections at the surface and at the interface between the tissue and slide glass. Frequency domain analysis was performed by analyzing the interference between the two reflections in order to calculate the thickness and sound speed of the specimen. Normal and atherosclerotic human coronary arteries were frozen, sliced approximately 5- μm and mounted on a glass slides. Pure water was used for the coupling medium between the transducer and tissue. Three layered structure of coronary artery was clearly visualized by the system. Especially, calcification and lipid core showed a significant difference of sound speed. The values of speed of sound were within same range as our previous measurements. In visualization of cultured nerve cells, saline was used for the coupling medium. The resolution was enough to visualize a single cell and the process of the cell was clearly seen. A prototype of ultrasonic nano-imaging system which was able to visualize a single cell was developed. The system is useful for intraoperative pathological examination, study of lowfrequency ultrasonic images, and assessment of biomechanics of the tissues at a nano-level.