Abstract
The measurement of three-dimensional (3D) images and the analysis of subcellular organelles are crucial for the study of the pathophysiology of cells and tissues. Optical diffraction tomography (ODT) facilitates label-free and quantitative imaging of live cells by reconstructing 3D refractive index (RI) distributions. In many cases, however, the contrast in RI distributions is not strong enough to effectively distinguish subcellular organelles in live cells. To realize label-free and quantitative imaging of subcellular organelles in unlabeled live cells with enhanced contrasts, we present a computational approach using ODT. We demonstrate that the contrast of ODT can be enhanced via spatial high-pass filtering in a 3D spatial frequency domain, and that it yields theoretically equivalent results to physical dark-field illumination. Without changing the optical instruments used in ODT, subcellular organelles in live cells are clearly distinguished by applying a simple but effective computational approach that is validated by comparison with 3D epifluorescence images. We expect that the proposed method will satisfy the demand for label-free organelle observations and will be extended to fully utilize complex information in 3D RI distributions.
Highlights
The shapes and dynamics of subcellular structures provide important information about biological cells and tissues
Without changing the optical instruments used in Optical diffraction tomography (ODT), subcellular organelles in live cells are clearly distinguished by applying a simple but effective computational approach that is validated by comparison with 3D epifluorescence images
Note that after applying the dark-field ODT algorithm, the tomogram does not have physical refractive index (RI) values anymore, and it is converted into the filtered RI because the low spatial frequency information is reduced or removed
Summary
The shapes and dynamics of subcellular structures provide important information about biological cells and tissues. Fluorescent microscopic techniques have been widely used to image subcellular structures because of their high molecular specificity.. Various molecular markers or fluorescent proteins have been developed to label specific subcellular organelles, including MitoTrackerTM for mitochondria and Hoechst stains for nucleus.. Images produced by ODT suffer from low molecular specificity and are difficult to use in the study of subcellular structures. Organelles such as nuclei and lipid droplets can be identified in RI tomograms.. The presented method, named dark-field ODT, performs numerical high-pass filtering in the 3D spatial frequency domain and generates contrast-enhanced 3D images of subcellular organelles. Without modifying the optical instrument, subcellular organelles in live cells are clearly distinguished, which is validated by comparison with epifluorescence images
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