Cell complexity in prokaryotic cells, whether Bacteria or Archaea, is generally considered low, with a single compartment. A few examples, however, have been recently reported showing evidence for compartmentalization [e.g. 1,2,3]. These studies gained in particular from improved sample preparation methods (plunge freezing or high-pressure freezing), adequate follow-up methods, and 3D imaging of the samples, either by FIB-SEM or by electron tomography. Here we report on our analyses of the ultrastructure of exceptional Archaea of the genera Ignicoccus and Pyrococcus, which show remarkable physiological characteristics (e.g. optimal growth at T~85 up to 100°C, under anoxic conditions), and unusual ultrastructural features. Although prokaryotes, these cells have a diameter of > 1 µm, which does not allow for reasonable cryoTEM observation without sectioning. Thus, samples were harvested by gentle concentration on ultrafiltration membranes, cryo-immobilized by high-pressure freezing, freeze-substituted, and finally resin-embedded; this enabled us to perform 3D ultrastructural analysis by (S)TEM tomography and immuno-localization studies on sections of the same resin block.
So far, we are about to routinely investigate single or serial sections (ranging from 200 up to 800 nm in thickness), on a transmission electron microscope operated as TEM or STEM at 200 keV. For STEM, the illumination is adjusted to have an almost parallel beam, i.e. small convergence angle, resulting in an extended depth of focus . Under these conditions, if the sample is imaged at high tilt angle (e.g. up to 70 deg), all visible image details are seen ‘in focus’, which greatly facilitates image alignment during 3D image reconstruction using IMOD (Boulder, Co, USA). In addition, the system in use allows parallel acquisition of BF and DF signal. Reconstruction and visualization is done using either WBP or SIRT in IMOD, and final manual segmentation using AMIRA®.
Our 3D data show novel and remarkable ultrastructural features: (A) Ignicoccus cells possess two membranes; in the intermembrane compartment, there is a complex endogenous membrane system. Attached to the outer cellular membrane, we have localized enzyme complexes for proton gradient generation, ATP and acetyl-CoA synthesis, and CO2 fixation . (B) Pyrococccus cells possess flagella of the archaeal type , i.e. extracellular filaments involved in motility and cell-cell interaction, and a cytoplasmic membrane and an S-layer, and a so far unknown layer inside the cytoplasm presumbably involved in anchoring or organizing the flagellar bundle.
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To cite this abstract:Reinhard Rachel, Veronika Heinz, Jennifer Flechsler, Ralph Witzgall, Thomas Heimerl; Serial section (S)TEM tomography of prokaryotic cells. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/serial-section-stem-tomography-of-prokaryotic-cells/. Accessed: December 2, 2023
EMC Abstracts - https://emc-proceedings.com/abstract/serial-section-stem-tomography-of-prokaryotic-cells/