Sigma vp feg sem

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).

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).

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).