High-throughput multicolor 3D localization in live cells by depth-encoding imaging flow cytometry

Capturing the dynamics of live-cell populations with nanoscale resolution poses a significant challenge in microscopy, primarily due to the speed-resolution tradeoff of existing approaches. While flow cytometry has sufficient throughput, subsample detail is largely inaccessible. Here we show that imaging flow cytometry, which replaces the point-source detector(s) of flow cytometry with a camera to record 2D images, is compatible with 3D localization microscopy by point-spread-function engineering, which encodes an emitter’s depth into the emission pattern captured by the camera. By exploiting the laminar-flow profile in microfluidics, 3D positions can be extracted from cells or other objects of interest by calibrating the depth-dependent response of the imaging system using fluorescent microspheres mixed with the sample buffer. This approach enables us to image up to tens of thousands of objects each minute, producing high-resolution 4D (x,y,z,t) measurements at the population scale. We apply our technique to measure the dynamics of chromatin compaction in live cells in real-time.