3D Tracking of Continuous Nanoscale Objects in Complex Environments

Understanding 3D behavior of nanoscale biological systems commonly relies on 2D optical imaging combined with computational analyses to extract 3D motion. For example, point-spread function (PSF) engineering approaches infer axial position from the PSF shape but are restricted to sparse objects. To relax the sparsity requirement, rapidly acquired 2D planes can approximate a 3D image as in single-objective light-sheet fluorescence microscopy. Ultimately the brightness and density of emitters limit the speed and accuracy of fluorescence-based 3D tracking. A promising alternative is optical diffraction tomography (ODT), an interferometric technique that infers the 3D refractive index by combining different views of a sample under coherent illumination. Because ODT does not depend on fluorescence it provides high signal at ~1000 volumes per second. Here we describe our recent development of a multi-modal structured illumination (SIM) and ODT microscope based on a digital micromirror device. Using binary patterns to generate SIM and ODT patterns improves speed and reliability compared to previous approaches. We demonstrate our approach by tracking continuous nanoscale objects such as isolated cilia, and study hydrodynamic interactions of diffusing colloidal particles with a boundary.