Multiplexed PSF engineering for 3D 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 co-localization of DNA loci in live yeast cells.