Acoustic manipulation of a 3-mm glass sphere in vitro along a 3D path seen from two angles. An ultrasound imaging transducer was placed in the center opening of the array, and its 2D image was aligned to correspond with one camera (A) and then with an orthogonal camera (D). The red “×” marks the acoustic focus of the array which was used as a reference in ultrasound to target the sphere. Images displayed are superimposed from Movies S2, S3, and S4. The ultrasound images of the sphere were identified by different colors at different times in postprocessing (A and D). The measured paths (displayed as a moving average) are shown in each image and the intended paths are also shown in B, E and C, F. The maximum discrepancy occurred farthest from the focus where the effective potential well was the shallowest. (Scale bar, 1 mm.)