Photon-efficient three-dimensional simultaneous multicolor particle tracking by multiplexed PSF engineering

Real-time microscopic tracking of multicolor fluorescent probes in 3D is of key importance for understanding a variety of biological processes. Point-Spread-Function (PSF) engineering is an effective tool for such tasks – by modulating the PSF of the optical system, emitters can be tracked with nm-scale resolution in 3D by means of computational analysis [1] . Existing approaches that make use of PSF engineering for high spatiotemporal-resolution tracking, while aiming to provide spectral information, typically separate the light to multiple channels, thus have a reduced Field of View (FOVs) [2] . Alternatively, multicolor phase masks are used, which suffer from low efficiency due to strong optical constraints and high sensitivity to manufacturing errors [3] .