This method was described previously in detail8 (link). Briefly, the glass-faced blocks were attached to 45° chamfered mounting stubs with conductive silver paste and placed into a Helios Nanolab 660 FIB-SEM (FEI, Systems for Research, Longueuil, QC, Canada). A sampling area 100 µm × 100 µm in size was selected and milled with gallium ions at rough (45 nA) followed by fine (9.4 nA) settings. Imaging was done with the through lens detector (TLD) and where possible with the in column detector (ICD). All FIB-SEM imaging was done using blocks prepared for sagittal (longitudinal) views of the enamel layer and associated enamel organ cells. Where possible, blocks from 3 different mice per genotype were examined. Charging on the surfaces of block faces was reduced by coating with a thin layer of platinum (3 nm) where required. Curtaining defects in TLD images were reduced using python language software modified from Schwartz et al.32 (link).
Helios nanolab 660 fibsem
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Helios NanoLab™ 660 FIBSEM is a focused ion beam scanning electron microscope (FIB-SEM) system designed for high-resolution imaging and nanofabrication. The instrument combines a SEM column with a focused ion beam column, enabling users to image and precisely mill samples at the nanoscale.
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Lab products found in correlation
5 protocols using helios nanolab 660 fibsem
1
FIB-SEM Imaging of Mouse Enamel
2
Ultrastructural Analysis of 3D Spheroids
3
Ultrastructural Analysis of 3D Spheroids
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Spheroids were fixed and processed as previously described52 (link). Samples were fixed for 1 hour at room temperature in a fixative solution containing 4% Paraformaldehyde (EMS), 2% Glutaraldehyde (EMS), and 0.1M Na Cacodylate (EMS). Samples were then osmicated, stained with uranyl acetate and embedded in EMbed 812 (EMS). Ultrathin sections (120 nm) from each spheroid sample were observed with a FEI Helios NanoLab™ 660 FIBSEM using extreme high resolution (XHR) field emission scanning electron microscope equipped with a Concentric (insertable) higher energy electron detector, all images were taken using 4 Kv and 0.2 current landing voltage at high magnifications (15000–35000x).
4
FIB-SEM Imaging of Microstructures
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Serial FIB-SEM images were obtained at 30 nm increments with a backscattered electron detector at 2.0-kV acceleration voltage using a Helios Nanolab 660 FIB-SEM (Thermo Fisher Scientific, Waltham, MA, USA). The pixel size of each FIB-SEM image was 13.5 × 17.1 nm (width × height × depth), and each recorded image was 3072 × 2048 pixels. Thus, the dimension of serial imaging by FIB-SEM was 41.5 × 35.0 μm (width × height). The new surface for serial FIB-SEM imaging was generated by FIB-milling using a 0.77-nA beam current, where gallium ions were accelerated with a voltage of 30 kV. For the detailed protocol of FIB-SEM tomography, see Kizilyaprak et al. (2014 (link)).
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Serial FIB/SEM Imaging of Microstructures
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Serial FIB/SEM images were obtained at 30-nm increments with a backscattered electron detector at a 2.0-kV acceleration voltage using a Helios Nanolab 660 FIB/SEM (Thermo Fisher Scientific, Waltham, MA, USA). The pixel size of each FIB/ SEM image was 13.5 × 17.1 × 50 nm/pixel (width × height × depth). The pixel dimensions for a recorded image was 3072 × 2048 pixels. Thus, the dimension of serial imaging by FIB/SEM was 41.5 × 35.0 × 20.0-35.0 μm (width × height × depth). The new surface for serial FIB/SEM imaging was generated by FIB milling using a 0.77-nA beam current, where gallium ions were accelerated with a voltage of 30 kV.
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