Caustic wavefront encoded imaging for snapshot three-dimensional fluorescence microscopy

Abstract

Abstract High-resolution, three-dimensional fluorescence microscopy is widely used in biology and neuroscience. The challenges of conventional three-dimensional fluorescence microscopy which relies on scanning the focal spot across the object include limited imaging cycles due to photobleaching of the fluorophores, ambiguous spatiotemporal information in dynamic samples due to long scanning times, and mechanical perturbation during the scanning process. In this paper, we report a snapshot three-dimensional fluorescence microscopy method (CausWEI) where three-dimensional sample information is encoded in a single wide-field image by engineering a high-contrast, laterally invariant point-spread function composed of caustics generated via the interaction of a uniform, thick glass sample holder and a high-numerical aperture objective. The three-dimensional information is computationally reconstructed from the caustic pattern recorded at the camera plane. The method can be implemented with a wide-field fluorescence microscope, without any internal modification in the microscope optics. We qualitatively and quantitatively evaluate CausWEI’s capabilities and limitations with reference fluorescent beads, neural cells on three-dimensional scaffolds, and spinal cord tissue sections. CausWEI microscopy is of importance when fluorescently labelled features are located in a depth range significantly larger than the depth-of-field of the objective lens.