Towards 3-D snapshot volumetric imaging: Novel methods of microscopy and image reconstruction to achieve 3-D volumes with single snapshot exposures

Authors

Matthew K. Daddysman, Alan Selewa, Xiang Huang, Toan Huynh, Justin Jureller, Nicola J. Ferrier, Mark Hereld, Norbert F. Scherer

Abstract

Limited imaging bandwidth is an important consideration in microscopy where hardware limitations of frame rate (25 Hz for an EMCCD) and sensitivity set a limit on the number of frames that can be acquired per second. The limited imaging resources can be allocated into either z-spatial (slices) or temporal resolution images. However, biological processes such as rapid diffusion or active transport inside cells often require the maximal temporal resolution, thus sacrificing 3D z-slices to properly track objects. To overcome this limitation, our approach is to collect multiple focal planes simultaneously with each image. We use a diffractive optical element to project 9 or 25 z-slices with precise focal shifts of the same image onto a single EMCCD. The collection of multifocal stacks gives a “snapshot” of carefully selected planes in the 3D volume of the imaging area of interest. We then use these select planes to reconstruct the full 3D volume to gain 3D positional information about the object(s) of interest (Figure, 4 μm bead). With this approach, the microscopy bandwidth can be fully committed to temporal resolution while trading x,y field of view for z-spatial resolution. We use this system to measure for the first time the 3D rotational diffusion of an oblong extended object (immobile bacterium) to confirm predictions about rotation diffusion. We also track vesicles undergoing active transport along the cytoskeleton in live cultured cells. Furthermore, this reconstruction will be coupled with interferometric and structured illumination microscopy techniques for super-resolution imaging.