Three-dimensional localization microscopy in live flowing cells

Capturing the dynamics of live cell populations with nanoscale resolution poses a significant challenge, primarily owing to the
speed-resolution trade-off of existing microscopy techniques. Flow cytometry would offer sufficient throughput, but lacks subsample
detail. Here we show that imaging flow cytometry, in which the point detectors of flow cytometry are replaced with a
camera to record 2D images, is compatible with 3D localization microscopy through point-spread-function engineering, which
encodes the depth of the emitter into the emission pattern captured by the camera. The extraction of 3D positions from subcellular
objects of interest is achieved by calibrating the depth-dependent response of the imaging system using fluorescent
beads mixed with the sample buffer. This approach enables 4D imaging of up to tens of thousands of objects per minute and
can be applied to characterize chromatin dynamics and the uptake and spatial distribution of nanoparticles in live cancer cells.