Small pieces of block including glomerulus were trimmed and mounted on aluminum specimen pins (Gatan, Pleasanton, CA) using CircuitWorks Conductive Epoxy (Chemtronics, Kennesaw, GA). The entire surface of the specimen was coated with a thin layer of heavy metal. New surface for serial block-face imaging was generated using a 3View in-chamber ultramicrotome (Gatan) within a ΣIGMA/VP SEM (Carl Zeiss Microscopy, Jena, Germany). Block-face images were obtained every 70-nm depth with a backscattered electron detector at an acceleration voltage of 1.1 kV. The pixel size was 21.0 nm/pixel wide, 21.0 nm/pixel height, 70 nm/pixel depth, and the pixel dimensions of a recorded image were 4096 × 4096 pixels. The contrast of the images was inversed.
3view in chamber ultramicrotome
Manufactured by Ametek
Sourced in Germany
The 3View in-chamber ultramicrotome is a piece of lab equipment designed for high-resolution 3D imaging of samples. It is capable of cutting thin sections from a sample and imaging the exposed surface using electron microscopy techniques.
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Lab products found in correlation
4 protocols using 3view in chamber ultramicrotome
1
Serial Block-Face Imaging of Glomeruli
2
Ultrastructural Analysis of Neural Cells
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Blocks from each group were trimmed and mounted on aluminium rivets with conductive glue (Chemtronics, Kennesaw, GA, USA). The surfaces of the trimmed samples were sputtered with gold to increase the conductivity and then imaged under various imaging conditions in a MERLIN or SIGMA/VP SEM instrument (Carl Zeiss Microscopy, Jena, Germany) equipped with a 3View in-chamber ultramicrotome (Gatan Inc., Pleasanton, CA, USA). Imaging in the MERLIN instrument was performed under a constant probe current (150 pA) and in the crossover-free mode. Imaging in the Sigma instrument was performed using a 30-μm aperture. The serial images obtained were processed with ImageJ and Fiji plugins (https://fiji.sc/wiki/index.php/Fiji ), and segmentation and image analyses were performed in TrakEM251 (link), Amira version 5.6 (FEI Visualisation Science Group, Hillsboro, OR, USA), and Microscopy Image Browser (https://mib.helsinki.fi/ ), as shown in Fig. 2 . Axons, synaptic vesicles, mitochondria, and lysosomes were semi-automatically and manually traced using these software packages.
3
3D Reconstruction of Proximal Tubule Mitochondria
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Epon blocks were trimmed and mounted on aluminum rivets using a conductive adhesive (Chemtronics, Kennesaw, GA, USA). Surfaces of trimmed specimens were sputtered with gold to increase conductivity, and specimens were observed using a MERLIN or SIGMA/VP SEM system (Carl Zeiss AG, Jena, Germany) equipped with a 3 View in-chamber ultramicrotome (Gatan Inc., Pleasanton, CA, USA). The images of observed samples were captured under various imaging conditions by microscopy. Serial section images from SBF-SEM of mouse proximal tubules were taken at every 50-nm thickness over 500 slices and processed using Fiji/ImageJ (http://fiji.sc/wiki/index.php/Fiji ) incorporated with TrakEM2 [21 (link)] (Cardona A, et.al. 2012), Amira (Thermo Fisher Scientific), and Simpleware (Synopsys, Mountain View, CA, USA), and then reconstructed into 3D images. Mitochondria were randomly selected in each segment of the proximal tubule. The mitochondrial area was measured using the area list and the polyline tool in the TrakEM2 software. Mitochondrial volume was calculated using TrakEM2.
4
Serial Block-Face Scanning EM Imaging of Myelinated Axons
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Serial blockface scanning EM imaging was performed at Renovo Neural Inc (Cleveland, OH) using standard protocols. Briefly, tissue preparation and imaging were performed as previously described (Ohno et al., 2011 ) using the staining methods described by Deerinck et al. (West et al., 2010 ). Sample blocks of approx 0.5 × 0.5 mm were examined in a Zeiss Sigma VP scanning EM fitted with a Gatan 3View in‐chamber ultramicrotome and using a Gatan low‐kV backscattered electron detector. At low magnification, regions of interest with longitudinally oriented axons were identified and image stacks covering 60 µm wide and 150–200 µm in length generated. Images were collected at 2.25 kV, x3.5 K with pixel size of 6 nm (x,y) using the 30 μm aperture in high current mode. Two stacks, of 250 and 500 slices were generated with 100 nm slice thicknesses. Myelin was segmented based on density thresholding, downsampled to 100 nm voxels and 3D surface visualizations generated using the ImageJ 3D Viewer plugin. Single slice views of axons were generated automatically from multiple slices using an algorithm that assembled multiple ROIs obtained along a user‐defined path that followed the axon centerline through the stack (x, y, z coordinates). Video sequences were generated in ImageJ and converted to standard codecs using winFF software.
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