In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

Abstract

Phytoplankton is a minor fraction of the global biomass playing a major role in primary production and climate. Despite improved understanding of phytoplankton diversity and genomics, we lack nanoscale subcellular imaging approaches to understand their physiology and cell biology. Here, we present a complete Focused Ion Beam -Scanning Electron Microscopy (FIB-SEM) workflow (from sample preparation to image processing) to generate nanometric 3D phytoplankton models. Tomograms of entire cells, representatives of six ecologically-successful phytoplankton unicellular eukaryotes, were used for quantitative morphometric analysis. Besides lineage-specific cellular architectures, we observed common features related to cellular energy management: i) conserved cell-volume fractions occupied by the different organelles; ii) consistent plastid-mitochondria interactions, iii) constant volumetric ratios in these energy-producing organelles. We revealed detailed subcellular features related to chromatin organization and to biomineralization. Overall, this approach opens new perspectives to study phytoplankton acclimation responses to abiotic and biotic factors at a relevant biological scale.

In situ3D comparison ofChlorella pyrenoidosawith nuclear-irradiated mutagenic strains by using focused ion beam milling and cryo-electron tomography

“…2E, F, Table S5). This result is similar with the previous observation (25). The chloroplast consists of thylakoid membrane, pyrenoid, starch granules and chloroplast matrix, and is encompassed by the chloroplast envelope (Video 5).…”

“…The cell wall of our high-pressure frozen cell samples were broken (see Fig. 1), and we noticed the similar phenomenon from other works (25). The reason may be that the external cryoprotectant (1-hexadecene) was not spread out evenly to the cell samples, thus the samples were suffered directly from the instant high pressure (over 2000 bars), which may destroy the cell wall of the sample.…”

“…With the development of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and its biological applications, it is now possible to image intact cells in 3D by FIB-SEM (24, 25). Furthermore, cryo-FIB milling coupled with cryo-electron tomography (cryo-ET) provides in situ snapshots of frozen-hydrated cells at sufficient resolution (26) to further view the subcellular architecture within the organelles.…”

Comparative Structural and Functional Analyses of the Fusiform, Oval, and Triradiate Morphotypes of Phaeodactylum tricornutum Pt3 Strain

“…Expression of a mitochondrion marker like a subunit of the glycine decarboxylase complex as an eGFP-fusion protein resulted in a fluorescence pattern near the complex plastid in the fusiform cells (Liu et al, 2016). Moreover, in P. tricornutum Pt1 fusiform cells, a continuous network of mitochondria sitting on the plastid is also clearly described through focused ion beam-scanning electron microscopy (Bailleul et al, 2015;Flori et al, 2017, Uwizeye et al, 2020. Such physical contacts between the two organelles may possibly facilitate exchange of energy.…”

Cytoklepty in the plankton: a host strategy to optimize the bioenergetic machinery of endosymbiotic algae

“…From the aligned FIB-SEM stack, images were binned in Fiji (https://imagej.net/Fiji), and regions of interest representing Phaeocystis cells were cropped. Segmentation and 3D reconstruction were performed using the work-flow developed in (64), and geometry measurements were provided using algorithms provided at (https://gitlab.com/clariaddy/stl_statistics and https://gitlab.com/clariaddy/mindist). Briefly, segmentation of organelles and vacuoles of Phaeocystis (considered as regions of interest) was carried out with 3DSlicer software (65) (www.slicer.org), using a supervised semi-automatic pixel classification mode (3 to 10 slices automatically segmented for each ROI).…”