Abstract
Single-cell phase-contrast tomography promises to become decisive for studying 3D intracellular structures in biology. It involves probing cells with light at wide angles, which unfortunately requires complex systems. Here we show an intriguing concept based on an inherent natural process for plants biology, i.e., dehydration, allowing us to easily obtain 3D-tomography of onion-epidermal cells’ nuclei. In fact, the loss of water reduces the turgor pressure and we recognize it induces significant rotation of cells’ nuclei. Thanks to the holographic focusing flexibility and an ad-hoc angles’ tracking algorithm, we combine different phase-contrast views of the nuclei to retrieve their 3D refractive index distribution. Nucleolus identification capability and a strategy for measuring morphology, dry mass, biovolume, and refractive index statistics are reported and discussed. This new concept could revolutionize the investigation in plant biology by enabling dynamic 3D quantitative and label-free analysis at sub-nuclear level using a conventional holographic setup.
Highlights
Optical microscopy of transparent biological specimens is mostly governed by two outstanding concepts, namely marker-based and label-free imaging
The latest advances in phase-contrast tomography (PCT) based on quantitative phase imaging (QPI), and the recent harmonic optical tomography (HOT) approach have extended the variety of specimens that can be analyzed, including nonlinear and inhomogeneous objects as well as strongly scattering specimens, paving the way to the tomographic analysis of model organisms based on QPI23–26
We studied the dehydration process through Digital holography (DH) time-lapse experiments and we determined an optimal time window to observe the sample before plasmolysis starts
Summary
Optical microscopy of transparent biological specimens is mostly governed by two outstanding concepts, namely marker-based and label-free imaging. Both are devoted to induce a contrast mechanism to enable the visualization of all the sample structures that are transparent at the wavelength of the light probe. Specific information can be gained by accurately changing the marker, in order to excite the sole sub-cellular structures of interest. Phase-contrast tomography (PCT) probes the sample from different directions to achieve resolving capability along the optical axis and to measure the sample refractive index 3D distribution[16,17,18,19,20,21,22]. The latest advances in PCT based on QPI, and the recent harmonic optical tomography (HOT) approach have extended the variety of specimens that can be analyzed, including nonlinear and inhomogeneous objects as well as strongly scattering specimens, paving the way to the tomographic analysis of model organisms based on QPI23–26
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