BPS2026 – Direct 3D intracellular diffusion measurement with dual channel phase mask light sheet fluorescence microscopy in S. cerevisiae

Diffusive motion is a primary mechanism of intracellular transport. Tagging mesoscale particles for single-particle tracking has enabled in-vivo measurements of 2D diffusion, but resolving their full 3D motion remains challenging. Conventional volumetric approaches such as confocal or even light sheet fluorescence microscopy often lack the temporal resolution required to follow fast mesoscale dynamics. Point-spread-function (PSF) engineering avoids slow axial scanning by encoding depth-axis position into the image PSF. We present a 3D approach that uses a polarization-preserving, dual-channel phase mask setup to encode axial position into a single image plane while recovering photons from both polarization channels using a spatial light modulator. We reconstruct 3D trajectories of genetically encoded multimeric probes in Saccharomyces cerevisiae with a time resolution of 10’s of milliseconds, opening the possibility of studying 3D diffusion in this organism.