To qualitatively evaluate how the mouse skull deforms under dorsal-ventral compression, a mouse was euthanized using carbon dioxide inhalation, then analyzed by micro-CT on an ImTek micro CT system (ImTek, Knoxville, TN), at the Cincinnati Children’s Hospital Medical Center Imaging Research Center. Image analysis was performed using the Amira software package (FEI, Hillsboro, OR). Post-mortem scans were done at rest, then repeated using a wooden clamp to cause dorsal-ventral skull compression, in approximately the same plane as impact from the TBI device.
Amira software package
Manufactured by Thermo Fisher Scientific
Sourced in United States
AMIRA software package is a visualization and analysis platform developed by Thermo Fisher Scientific. It provides tools for processing, visualizing, and analyzing data from various imaging modalities, including electron microscopy, X-ray computed tomography, and light microscopy.
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Lab products found in correlation
6 protocols using amira software package
1
Mouse Skull Deformation Under Compression
2
Volumetric Analysis of Nuclei
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In order to confirm the reliability of the data obtained using the ImageJ Volumest software, the same dataset was also analyzed with the AMIRA software package (FEI, Hillsboro, OR, United States). For the latter, nuclei were segmented according to standard procedures and correspondingly volume rendered. Briefly, to enhance the signal to noise ratio while preserving edges, images were first filtered with a 2D-median rolling ball, then nuclei were segmented by applying a white Top-Hat filter. Sorted out nuclei from gall sections were then filled to avoid holes within the selection. GC and NGC nuclei were individually labeled via the AMIRA segmentation tool and analyzed for morphologic traits.
3
High-Resolution 3D Ultrastructural Imaging
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Tissues were dissected and fixed using 2.5% glutaraldehyde [Electron Microscopy Sciences (EMS)] and 2% paraformaldehyde (EMS) in sodium cacodylate buffer 0.15 M (pH7.4) (EMS). Liver pieces (1 mm3) have been postfixed for an additional 24 hours at 4°C. The samples were prepared as previously described (57 (link)). The blocks were imaged and cut by a Thermo Fisher Scientific–FEI Teneo Volumescope scanning electron microscopy (SEM), operating at an accelerating voltage of 2 kV, a beam current of 50 pA, upon low-vacuum conditions (30 Pa), and with a cutting step of 100 nm. Serial SEM images were acquired by collecting the backscattered electron signal by a dedicated detector, with a magnification of ×3.500, a dwell time of 10 μs per pixel, and an image resolution of 6144 × 4096 pixels, corresponding to a final voxel size of 18.7 nm × 18.7 nm × 100 nm. For 3D reconstruction, rendering, and analysis, serial SEM images were manually segmented using AMIRA software package (FEI Company).
4
Cryo-Electron Tomography Analysis of NPCs
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Cryo-electron tomograms that were acquired previously (Turgay et al., 2017 (link)) were further analyzed. The central coordinates of NPCs within cryo-tomograms of NE were determined manually, and subtomograms (340 nm × 340 nm × 20 nm) were reconstructed in MATLAB using the TOM toolbox (Nickell et al., 2005 (link)). The lamin filaments and NPCs in four selected subvolumes were segmented manually and rendered using the Amira software package (Thermo Fisher Scientific).
5
3D Reconstruction of Synaptic Structures
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Serial SEM images were assembled into volume files aligned using the FiJi software (Schindelin et al, 2009 ) plugin called linear stack alignment with SIFT (Lowe, 2004 ). Following the images acquisition, recording, and alignment, the 3D shape of samples peculiar features (in our case excitatory and inhibitory synapses) was reconstructed layer by layer by careful segmentation. For performing the latter, and the 3D model generation, electron microscopy image stacks were then converted to 8‐bit grayscale tiff format images and manually segmented using AMIRA software package (Thermo Scientific, Eindhoven, NL). Three‐dimensional structures in image stacks containing hundreds or thousands of 2D orthoslices were traced individually in each plane and automatically surface rendered. The excitatory and inhibitory synapse density (n° of synapse/μm3) was finally measured by using Ilastik‐0.5.12 software.
6
Cryo-ET Workflow for Membrane Segmentation
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MotionCor2 (Zheng et al., 2017 (link)) was used to align the frames and dose-weigh the tilt series according to the cumulative dose. Subsequent alignment of the tilt series was done in IMOD (Kremer et al., 1996 (link)) using patch-tracking in the absence of fiducials and the tomograms reconstructed using weighted back-projection method. For purposes of representation and segmentation, tomograms were binned 3× or 4×. Semi-automatic segmentation of the membranes was performed using TomosegmemTV (Martinez-Sanchez et al., 2014 (link)) followed by manual refinement in Amira software package (Thermo Fisher Scientific). FtsAZ filaments were manually traced in Amira.
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