Serial blockface scanning electron microscopy (SBF SEM) data was collected using a 3View2XP (Gatan, Pleasanton, CA) attached to a Sigma VP SEM (Zeiss, Cambridge). Flat embed vibratome slices were cut out and mounted on pins using conductive epoxy resin (Circuitworks CW2400). Each slice was trimmed using a glass knife to the smallest dimension in X and Y whilst retaining all the tissue, and the surface polished to reveal the tissue before sputter coating with a 2 nm layer of platinum, and loading in the 3View2XP. Two SBF SEM datasets were collected, both of which fully contained the fluorescence microscopy volume. Backscattered electron images were acquired using the 3VBSED detector at 8,192∗8,192 pixels with a dwell time of either 5 or 4 μs (10 nm reported pixel size for a horizontal frame width of 81.92 μm) and 50 nm slice thickness. The SEM was operated in variable pressure mode at a chamber pressure of either 10 or 5 pascals, with high current mode inactive. The 30 μm aperture was used, with an accelerating voltage of 2.5 kV. Dataset 1 comprised a total of 1,180 images, representing a depth of 59 μm, and volume of 395,942 μm3; dataset 2 comprised a total of 1,296 images, representing a depth of 64.8 μm, and volume of 434,865 μm3.
3view2xp
Manufactured by Ametek
Sourced in United States
The 3View2XP is a lab equipment product from Ametek. It is designed for high-resolution imaging and data acquisition. The core function of the 3View2XP is to enable the user to capture detailed 3D images and data.
Automatically generated - may contain errors
Lab products found in correlation
Sigma vp sem, by Zeiss (15 mentions)
3view detector, by Ametek (2 mentions)
Q150r s sputter coater, by Quorum Technologies (2 mentions)
Digital micrograph, by Ametek (1 mentions)
Sigma vp scanning electron microscope, by Zeiss (1 mentions)
Conductive silver epoxy, by Ted Pella (1 mentions)
Gemini scanning electron microscope, by Zeiss (1 mentions)
Merlin scanning electron microscope, by Zeiss (1 mentions)
19 protocols using 3view2xp
1
Serial Blockface SEM Datasets
2
Sputter Coating and 3View SEM Imaging
3
Integrated Light and 3D EM Imaging
4
Ultrastructure Analysis of Tactile Bristle Differentiation
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Samples were sputter coated with a 10 nm layer of platinum (Q150S, Quorum Technologies) and loaded into a 3View2XP (Gatan) attached to a Sigma VP SEM (Zeiss) with focal charge compensation (FCC, Zeiss), and data were collected using a BSE detector (3View detector, Gatan). Imaging parameters for each stage of tactile bristle differentiation can be found in Supplementary Table 2 . The morphology of the neuronal dendrite from bristles lacking the F-Cell was visualized in the genotype neur > spi/EGFKD, and no obvious defects were detected in the neuron and surrounding sheath cell membrane relative to wild-type controls.
5
SBF SEM Imaging of HeLa Cells
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HeLa cells were prepared for SBF SEM following the method of the National Centre for Microscopy and Imaging Research (NCMIR) [51 ]. SBF SEM data were collected using a 3View2XP (Gatan, Pleasanton, CA) attached to a Sigma VP SEM (Zeiss, Cambridge). In total, 517 images of 8192 × 8192 pixels were acquired. Voxel size was 10 × 10 × 50 nm with intensity [0—255] (Figure 1 ). Initially, the data were acquired at higher bit-depth (32 bit or 16 bit) and, after contrast/histogram adjustment, reduced to 8 bit. For this paper, seven individual cells were manually cropped as volumes of interest. For each cell, the centroid was manually selected as the centre of a sub-volume of 300 slices with dimensions (nh, nw, nd) = (2000, 2000, 1) and were saved as single channel TIFF files. Images are openly accessible via the EMPAIR public image database (http://dx.doi.org/10.6019/EMPIAR-10094 ).
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HeLa Cell Ultrastructure Imaged by SBF SEM
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HeLa cells were were prepared, embedded in Durcupan and observed with SBF SEM following the method of the National Centre for Microscopy and Imaging Research (NCMIR) [80 ]. SBF SEM data were collected using a 3View2XP (Gatan, Pleasanton, CA, USA) attached to a Sigma VP SEM (Zeiss, Cambridge, UK). In total, five-hundred eighteen 518 images of 8192 × 8192 pixels were acquired. The voxel size was nm with intensity [0–255]. Figure 1 a shows six EM images positioned within the 3D stack. Initially, the data were acquired at a higher bit-depth (32 bit or 16 bit), and after contrast/histogram adjustment, this was reduced to 8 bit [69 (link)]. Images are openly accessible via the EMPIAR [81 (link)] public image database (http://dx.doi.org/10.6019/EMPIAR-10094 Accessed on 28 May 2021).
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Serial Blockface Scanning Electron Microscopy Protocol
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Once the cells were prepared, the samples were imaged using Serial Blockface Scanning Electron Microscopy (SBF SEM) with a 3View2XP (Gatan, Pleasanton, CA) attached to a Sigma VP SEM (Zeiss, Cambridge). The resolution of each image was 8, 192 × 8, 192 pixels corresponding to 10 × 10 nm (Fig 1a ). In total, the sample was sliced 517 times and corresponding images were obtained. The slice separation was 50 nm. The images were acquired with high-bit contrast (16 bit) and after contrast/histogram adjustment, the intensity levels were reduced to 8 bit and therefore the intensity range was [0 − 255]. Then, one cell was manually cropped by selecting its estimated centroid and a volume of 2,000 × 2,000 × 300 voxels was selected (Fig 1b ). Images are openly accessible via the EMPIAR public image database (http://dx.doi.org/10.6019/EMPIAR-10094 ).
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Correlative SBF-SEM Imaging Protocol
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The block was trimmed with a razor blade, removed, and mounted onto an aluminum pin using conductive epoxy glue (ITW Chemtronics, Enschede, The Netherlands) as previously described (38 (link)). The glue was then hardened at 60°C overnight and the block sputter coated with 2 nm platinum using a Q150R S sputter coater (Quorum Tech, East Sussex, UK).
Correlative SBF-SEM data were collected using a 3View2XP (Gatan, Pleasanton, CA) attached to a Sigma VP scanning electron microscope (Zeiss, Cambridge) as previously described (38 (link)). The scanning electron microscope was operated at an accelerating voltage of 2 kV with high current mode active, using a 20 μm aperture and chamber pressure of ∼8 Pa. A per pixel dwell time of 2 μs was used with a slice thickness of 50 nm. Images were acquired at 8,192 × 8,192 pixels with 8.7 nm pixel resolution (horizontal frame width of 71 μm). The entire volume comprised 420–520 slices to give an ∼1,500 μm3 image volume as inFig. 4 .
Correlative SBF-SEM data were collected using a 3View2XP (Gatan, Pleasanton, CA) attached to a Sigma VP scanning electron microscope (Zeiss, Cambridge) as previously described (38 (link)). The scanning electron microscope was operated at an accelerating voltage of 2 kV with high current mode active, using a 20 μm aperture and chamber pressure of ∼8 Pa. A per pixel dwell time of 2 μs was used with a slice thickness of 50 nm. Images were acquired at 8,192 × 8,192 pixels with 8.7 nm pixel resolution (horizontal frame width of 71 μm). The entire volume comprised 420–520 slices to give an ∼1,500 μm3 image volume as in
9
Multiscale Imaging of Cryptococcus Cells
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For standard scanning electron microscopy (SEM), sample preparation and visualization were performed as described in reference 41 (link). For SBF-SEM, a single block was produced for analysis by mixing together aliquots from strains known to produce appreciable numbers of the different cell phenotypes (H99E, H99W, H99O, H99S, and KN99a ) following capsule induction. Cells were harvested and washed with 0.1 M phosphate buffer (PB), covered with 1 mL of fixative (2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M PB), and left overnight at 4°C. Secondary fixation, staining, ethanol dehydration, resin infiltration, and embedding were performed as described in reference 18 (link). The final sample block contained approximately 5,000 cells. The sample block was then loaded onto a Zeiss Sigma variable-pressure SEM equipped with Gatan 3View 2XP. Inverted backscattered electron images (8,192 by 8,192 pixels, 16-bit) were acquired with an xy pixel size of 100 nm and z pixel size (slice thickness) of 10 nm.
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Correlative SBF-SEM Imaging of Cell-Cell Contacts
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Prior to SBF-SEM, the block was sputter-coated with 5–10 nm platinum using a Q150R S sputter coater (Quorum Technologies). SBF-SEM data was collected using a 3View2XP (Gatan Inc.) attached to a Sigma VP SEM (Zeiss). The microscope was operated at 1.8kV with 30-μm aperture, using Focal Charge Compensation mode (Deerinck et al., 2018 (link)). Inverted backscattered electron images were acquired every 50 nm, at a resolution of 8.44 nm/pixel. Acquired images were imported into Fiji (Schindelin et al., 2012 (link)) and aligned using the Register Virtual Stack Slices (Arganda-Carreras et al., 2006 ) and in TrakEM2 using manual landmarks (Cardona et al., 2012 (link)). 63 × Z stack was matched to the aligned SBF-SEM data using BigWarp (Bogovic et al., 2016 ) based on nuclei positions and overlaid to the EM data after applying thin-plate spline transformation. Cells in the region of expanded cell-cell contacts were selected and segmented using the TrakEM2. Cell-cell contacts were segmented if the plasma membranes of two neighboring cells were closely apposed with spacing less than or equal to 25 nm based on the length of homo dimer of PECAM extracellular domains (Jiang et al., 2016 (link)). 3D reconstructions were made in the 3dmod program.
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