Processed Deerinck-stained samples were mounted onto a Gatan specimen pin and coated with silver conductive epoxy adhesive (TAAB laboratories). The pin was sputtered with 8 nm of gold using the Leica ACE 200 and placed inside the Zeiss Sigma VP FEG SEM equipped with a Gatan 3View system. Automated serial sectioning was undertaken of the block face surface every 50 nm until a dataset of 1000 images was acquired. 3-D reconstructions of the datasets using the automated volren segmentation function were composed using Amira 6.4 software (FEI, Mérignac, France).
Gatan specimen pin
Manufactured by Ametek
Sourced in United States
The Gatan specimen pin is a core component for use in electron microscopy. It is designed to securely hold and position a specimen sample within the instrument. The specimen pin provides a reliable and stable interface between the sample and the microscope's sample stage.
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4 protocols using gatan specimen pin
1
Automated Serial Sectioning and 3D Reconstruction
2
3D Reconstruction of Cellular Ultrastructure
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Samples were mounted onto a Gatan specimen pin and coated with silver conductive epoxy adhesive (TAAB laboratories). The pin was sputtered with gold and placed inside the Zeiss Sigma VP FEG SEM equipped with a Gatan 3View system. Automated serial sectioning was undertaken of the block face surface every 50 nm. A dataset of 1000 images was acquired. 3-D reconstructions of the datasets were produced using either the manual segmentation function (for larger cell and membrane structures) or the automatic isosurface function (for the finer elastic fibre network where manual segmentation was not practical) using Amira 6.4 software (FEI, Mérignac, France).
3
Serial Block-Face SEM Imaging of Tissue
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Three samples for each age were mounted onto a Gatan specimen pin and coated with a silver conductive epoxy adhesive (TAAB laboratories). Each pin was sputtered with gold to a thickness of 8 nm using a Leica ACE 200 gold coating unit and placed inside the Zeiss Sigma VP FEG SEM equipped with a Gatan 3View system. The block face surface was imaged on the SEM at 3.5 kV at a pixel resolution of 4 nm and a dwell time of 8 μs and approximately 1000 image slices were acquired from the specimen block every 50 nm by automated serial sectioning. The data sets were recorded at 4096 x 4096 (4K) in Gatan format dm4 files then batch converted to TIFF format.3-D reconstructions of the datasets using the isosurface or segmentation functions were composed using Amira 6.4 software (FEI, Mérignac, France).
4
3D Ultrastructural Imaging of Samples
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Samples were placed onto a Gatan specimen pin (Gatan Inc., Pleasanton, CA, USA) and covered with silver conductive epoxy adhesive (TAAB Laboratories, Aldermaston, UK). The pin was sputtered with 7 nm of gold using an ACE200 (Leica Biosystems, Buffalo Grove, IL, USA) and placed inside the Zeiss Sigma VP FEG SEM (Zeiss Group, Oberkochen, Germany) equipped with a Gatan 3View system (Gatan Inc.). The block face surface was imaged on the SEM at 3.5KV at a pixel resolution of 4 nm and a dwell time of 8 µs, and approximately 1000 image slices were acquired from the specimen block every 50 nm by automated serial sectioning. The data sets were recorded at 4096 × 4096 (4K) in Gatan format dm4 files (Gatan Inc.) then batch converted to TIFF format. Three-dimensional reconstructions of the datasets with the isosurface or segmentation functions were composed using Amira 6.4 software (FEI, Mérignac, France).
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