DOI: 10.6084/m9.figshare.24033777
title: FIB-SEM data from <i>Anaeramobea flamelloides</i> BUSSELTON2.

description: <b><i>Focused-ion-beam scanning electron microscopy (FIB-SEM)</i></b>Cells were adhered to the surface of a gridded MatTek dish and fixed with 2.5% glutaraldehyde (TAAB) in 0.1 M PHEM-buffer. All samples were processed using a Pelco Biowave Pro+ microwave tissue processor (Ted Pella, Redding, CA) according to <sup>81</sup> with minor modifications: no calcium was used during fixation and the contrasting steps with lead aspartate was omitted to reduce the risk of overstaining. Samples were detached from the glass using liquid nitrogen and glued to an SEM-stub with epoxy and silver glue. Samples were further coated with 5 nm platinum to reduce charging. Volumes were acquired using a Scios dual-beam (Thermo Fischer Scientific) with the electron beam operating at 2 kV/0.2 nA detected with a T1 In-lens detector. To automate volume acquisition, we used the Auto Slice and View 4 software provided with the microscope. A 700 nm protective layer of platinum was deposited on the selected area before milling. A FIB-SEM volume of 1780 slices of <i>A. flamelloides</i> BUSSELTON2 was acquired close to isotropic resolution (6.7 × 6.7 × 7 nm). Volumes were further registered and processed using the ImageJ plugins Linear alignment by SIFT and Multistackreg. After registration the volumes were converted to mrc-files and header was modified to recover the pixel-sizes that got lost during conversion.<i>Segmentation and visualization of cell structures</i>We segmented eight cell structures (nucleus, symbionts, hydrogenosomes, symbiosome-membrane, dense granules, other prokaryotes, plasma membrane, and the acentriolar centrosome with individual microtubules) using Microscopy Image Browser v2.84 <sup>82,83</sup>. The nucleus was segmented using the Graphcut semi-automatic segmentation function in MIB. Symbionts, symbiosome-membrane and hydrogenosomes were segmented by the deep-learning segmentation tool in MIB (DeepMIB). Briefly, symbionts and hydrogenosomes were manually annotated in a 50-slice segment of the FIB-SEM volume abundant in symbionts and hydrogenosomes. Image segments (patch size 256x256) were extracted and used to train DeepLabV3 ResNet50 model using default settings. The trained model was then used to predict segmentations for symbionts and hydrogenosomes. The resulting symbiont model was manually refined using the MIB segmentation tools. The hydrogenosomes and dense granules were predicted together by the classifier and were manually separated by hand using the MIB segmentation tools. Other prokaryotes were partially annotated by the symbiont+hydrogenosome model and were separated manually for additional curation by hand using the MIB segmentation tools. The symbiosome-membranes were predicted by training a model that segments whole symbionts (symbiont+symbiont subcompartment and membrane) as described as for symbiont and hydrogenosomes above. The outer membranes were obtained by eroding the whole symbiont predictions by 3 pixels (approximated thickness of the symbiosome-membrane) and using this selection as a mask to cut out the outermost 3 pixels of the whole symbiont model. The plasma membrane was manually annotated using black-white thresholding of brush-traced selections every 5-10 slices and shape interpolation was applied between those slices. In segments where the membrane was sharply shifting between slices individual slices were segmented by hand using black-white thresholding of brush selections. The acentriolar centrosome was segmented using Graphcut and individual microtubules were manually annotated using the brush tool. Symbiosome subcompartment connectivity was manually traced, annotated and visualized in MIB v2.84 <sup>82,83</sup>. The volumes were rendered using ORS Dragonfly v2022.2.0.1399.<b><i>FIB-SEM extended results</i></b>Using light microscopy of the fixed amoebae, we observed cells that had cleared a path through the bacterial lawn. Only cells that had cleared a lawn of bacteria were mounted as these were likely to be viable upon fixation. A FIB-SEM volume of 1780 slice of <i>Anaeramoeba</i> was acquired close to isotropic resolution (6.7 × 6.7 × 7 nm). We segmented eight cell structures (nucleus, symbionts, hydrogenosomes, symbiosome-membrane, dense granules, other prokaryotes, plasma membrane and the acentriolar centrosome with individual microtubules) using Microscopy Image Browser (MIB) v2.84 / 09-12-20222. The different approaches used for segmentation is described below.<i>Nucleus</i>The nucleus was segmented using the Graphcut semi-automatic segmentation function in MIB. Symbionts were segmented by the deep-learning segmentation tool in MIB (DeepMIB).<i>Symbiont, hydrogenosomes, granules and other prokaryotes</i>Symbionts and hydrogenosomes were manually annotated in a 50-slice segment of the FIB-SEM volume abundant in symbionts and hydrogenosomes. Image segments (patch size 256x256) were extracted and used to train DeepLabV3 ResNet50 model using default settings. The trained model was then used to predict segmentations for symbionts and hydrogenosomes. The resulting symbiont model was manually refined using the MIB segmentation tools. The hydrogenosomes and dense granules were predicted together by the classifier and were manually separated by hand using the MIB segmentation tools. Other prokaryotes were partially annotated by the symbiont+hydrogenosome model and were separated manually for additional curation by hand using the MIB segmentation tools.<i>Symbiosome-membrane</i>The symbiosome-membranes were predicted by training a model that segments whole symbionts (symbiont+symbiont subcompartment and membrane). Whole symbionts were manually annotated in a 50-slice segment of the FIB-SEM volume and 256x256 pixel patches were used to train a DeepLabV3 ResNet50 model in DeepMIB. The predicted model was manually refined using the MIB segmentation tools. Finally, the outer membranes were obtained by eroding the whole symbiont predictions by 3 pixels (approximated thickness of the symbiosome-membrane) and using this selection as a mask to cutout the outermost 3 pixels of the whole symbiont model. Manual refinement of the symbiosome-membrane segmentation was done in regions of special interest.<i>Plasma membrane</i>The plasma membrane was manually annotated using black-white thresholding of brush-traced selections every 5-10 slices and shape interpolation was applied between those slices. In segments where the membrane was sharply shifting between slices individual slices were segmented by hand using black-white thresholding of brush selections.<i>Acentriolar centrosome and microtubules</i>The acentriolar centrosome was segmented using Graphcut and individual microtubules were manually annotated using the brush tool.<i>General volume statistics</i>Visualization of segmentations are shown in Figure 2 and Figure S3. The cell contained a single drop-shaped nucleus that occupies 2.80 % of the cell volume. The cell is bounded by a plasma membrane (0.91% of cell volume). Microtubules radiate outwards from the single acentriolar centrosome at the ventral side of the cell. The symbiont and hydrogenosomes form a densely packed mass close to but not in direct contact with nucleus. There are 185 symbionts (183 intact and 2 degenerating) that account for 7.95 % of the total cell volume. The symbionts are housed in a membrane system whose volume was 1.92% of the cell volume. The hydrogenosomes consist of individual organelles but many form a reticulated network that altogether encompass 4.85% of the cell volume. Abundant dense granules (5.40%) of unknown composition are found in the cytoplasm. Dense granules of this type have previously not been observed in Anaeramoebae and might be due to a difference in culturing conditions (ASW+LB instead of SW802 as growth media). We reconstructed additional prokaryotes (1.21%) in the volume that did not have a tight connection to hydrogenosomes and whose enclosing membranes were less structured than those harboring the symbiont population. These cells were invariably inside of membrane vacuoles and some were in the process of being digested.<br><b>Files: FIB-SEM image files. mrc-files can be opened in Microscopy Image Browser</b><i>8640_2.mrc</i><i>8640_2_levels.mrc</i><i>Aligned 1780 of 1780.tif</i><i>SEM Image - SliceImage 001 - 1878.zip</i><b>Model files - </b><b>ORSObject-format that can be opened in Dragonfly</b><i>230718_Whole_cell_volume.ORSObject</i><i>granuels.ORSObject</i><i>hydrogenosomes_clean.ORSObject</i><i>Microtubules.ORSObject</i><i>Nucleus.ORSObject</i><i>other_prokaryotes.ORSObject</i><i>Plasma_membrane.ORSObject</i><i>Symbiont_clean.ORSObject</i><i>Symbiont_membranes.ORSObject</i>