Multiplexed PSF Engineering for Three-Dimensional Multicolor Particle Tracking.

Three-dimensional spatiotemporal tracking of microscopic particles in multiple colors is a challenging optical imaging task. Existing approaches require a trade-off between photon efficiency, field of view, mechanical complexity, spectral specificity, and speed. Here, we introduce multiplexed point-spread-function engineering that achieves photon-efficient, 3D multicolor particle tracking over a large field of view. This is accomplished by first chromatically splitting the emission path of a microscope to different channels, engineering the point-spread function of each, and then recombining them onto the same region of the camera. We demonstrate our technique for simultaneously tracking five types of emitters in vitro as well as colocalization of DNA loci in live yeast cells.

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 sub-cellular 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.