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Digital Holography (DH) is a label-free optical microscopy technique which allows reconstructing the Quantitative Phase Maps (QPMs) of transparent biological specimens. In a QPM, the phase-contrast is endogenous and is due to the Refractive Index (RI) and thickness differences. Although phase-contrast allows a quantitative characterization of the whole biological sample, it is often not enough to ensure an adequate intracellular segmentation, also because of the lack of exogenous markers, e.g., fluorescent dyes. Here we investigate a biological strategy for increasing the intracellular contrast inside epidermal onion cells to recognize their nuclei within the QPMs. Plant cells continuously undergo dehydration-hydration loops during their lifetime since dehydration is reversible when plasmolysis is not reached. Therefore, by setting specific environmental temperature and humidity, we can induce dehydration, thus provoking the water evaporation from the vacuole and therefore increasing the nucleus-cytoplasm contrast. Moreover, the reduction of the turgor pressure causes a rearrangement of the cytoskeleton, thus allowing nuclear roto-translations. We exploit an ad-hoc algorithm to estimate the nucleus rolling angles around the image plane. Then, we perform phase-contrast tomography to reconstruct the three-dimensional (3D) RI distribution of the plant cells’ nuclei by operating in complete reversible conditions, i.e., before plasmolysis when no cell damage has occurred. Finally, we segment the nuclear tomograms to isolate the 3D nucleoli, thus providing quantitative measurements about their volumes, dry masses, and RI statistics. In this way, DH can be further exploited for the label-free and non-invasive analysis of several plant cell lines at the nuclear and sub-nuclear level.
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