Extended Data Fig. 2: Comparison of image-based tracking and imaging (spinning disk confocal) with active-feedback tracking and complementary 3D imaging (3D-TrIm).

Fluorescently-labeled virus-like particles (eGFP-Vpr VSVG) were introduced to cultures of live HeLa cells stained with SiR650-actin (100 nm). a-e, Data collected on Andor Dragon fly spinning disk confocal microscope. The same area was sampled continuously. Each volume has a depth of 8 μm split into 16 z planes. Virus particles and cells were imaged simultaneously with a camera exposure time of 40 msec. f-j, Data collected on 3D-TrIm microscope. A single virus particle was tracked continuously (1 msec sampling shown) and the surrounding area was imaged 5 μm below the particle and 3 μm above, to give an approximate 8 μm volume. The resulting trajectories over the entire acquisition period are shown and color-coded by time. Gray box in XZ projections shows the size of the spinning disk imaging volume for comparison. Experiments were performed in live-cell image solution with 2% FBS and maintained at 37 °C.