Phase-modulated multi-foci microscopy for rapid 3D imaging

Abstract

3D imaging technology is pivotal in monitoring the functional dynamics and morphological alterations in living cells and tissues. However, conventional volumetric imaging associated with mechanical z-scanning encounters challenges in limited 3D imaging speed with inertial artifact. Here, we present a unique phase-modulated multi-foci microscopy (PM3) technique to achieve snapshot 3D imaging with the advantages of extended imaging depths and adjustable imaging intervals between each focus in a rapid fashion. To accomplish the tasks, we utilize a spatial light modulator (SLM) to encode the phases of the scattered or fluorescence light emanating from a volumetric sample and then project the multiple-depth images of the sample onto a single charge-coupled device camera for rapid 3D imaging. We demonstrate that the PM3 technique provides ∼55-fold improvement in imaging depth in polystyrene beads phantom compared to the depth of field of the objective lens used. PM3 also enables the real-time monitoring of Brownian motion of fluorescent beads in water at a 15 Hz volume rate. By precisely manipulating the phase of scattered light on the SLM, PM3 can pinpoint the specific depth information in living zebrafish and rapidly observe the 3D dynamic processes of blood flow in the zebrafish trunk. This work shows that the PM3 technique developed is robust and versatile for fast 3D dynamic imaging in biological and biomedical systems.