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Home • hpfnode • Segmentation Information
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  Specimen preparation
  Microscopy product
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  2D image
  Reconstruction
  Segmentation

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Project Information
Microscopy product ID: 3696
Image basename: hpfnode
Project information
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Experiment
Subject group
Subject
Organism
Tissue section or block

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Project (P1704)
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Experiment (179)
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Subject group (167)
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Subject (172)
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Organism (179)
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Tissue section or block (202)
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Specimen Preparation Information
Microscopy product ID: 3696
Image basename: hpfnode
Specimen Preparation
Tissue group type High Pressure Frozen
Ext file name
Protocol used Rats were first anaesthetized by intraperitoneal injection of Nembutal (35 mg per kg body weight), and whole blood was flushed out with oxygenated rat Ringers solution at 37 C. They were then fixed by intracardial perfusion with an aldehyde mixture composed of 2 percent paraformaldehyde and 2.5 percent glutaraldehyde in 0.15 M cacodylate buffer (pH 7.4) at 37 C. The spinal root was dissected out, immersed in the same fixative and transferred to the high-pressure freezing (HPF) unit. These samples were trimmed and blotted (to remove excess water) before placing in the carrier. Samples were loaded into the carriers of the Bal-Tec HPM010 HPF unit (Bal-Tec, Liechtenstein) and operated according to manufacturers instructions. Carriers with a depth of 0.2 mm were used with thin bundles of spinal root to provide uniform ultrastructural preservation. The inert liquid, hexadecene, was added to the carrier to fill in the well, excluding air from surrounding the sample.

Freeze Substitution: After the samples were high-pressure frozen, they were freeze-substituted in dry acetone containing 0.1 percent tannic acid for 24 h at negative 90 degrees C in a Leica EMAFS freeze substitution unit (FSU, Leica Microsystems, Bannockburn, IL), then in acetone containing 2 percent OsO4 and 0.1 percent of uranyl acetate for the remaining time of the procedure. Both tannic acid and uranyl acetate were added not only to help stain the material but also to aid in membrane preservation. During this procedure, the water in the sample is completely extracted and replaced by the fixative, i.e., freeze-substituted. The temperature for substitution was maintained at minus 90 C for 3 days to ensure that the biological structures were cryo-immobilized during this substitution. The temperature was raised gradually at a rate of 2 degrees C per hour to minus 60 degrees C and held there for 8 h because OsO4 does not start to cross-link until around minus 70 degrees C. The samples were then warmed to minus 20 degrees C at a rate of 5 degrees C per hour and held at this temperature for 12 h. The carriers were then taken from the FSU, the tissue removed from the carriers and quickly washed several times in dry acetone at room temperature (RT) to remove the OsO4 and uranyl acetate. Acetone above 70 percent can extract lipids at RT. Hence, the exposure time to it was minimized. The tissues were then incubated in a 50:50 mixture of acetone/Durcupan for 4 h at RT followed by 100 percent Durcupan resin of 24 h infiltration with three changes. Resin polymerization was accomplished in a vacuum oven at 65 to 70 degrees C for 48 h. The resin-embedded samples were removed from the oven and sections were cut using a Leica ultramicrotome. Sections were then stained 30 min in 2 percent aqueous uranyl acetate, followed by 15 min in lead salts. Fiducial cues consisting of 15 and 20 nm colloidal gold particles were deposited on opposite sides of the section.









Microscopy Product Information
Microscopy product ID: 3696
Image basename: hpfnode
Microscopy product
Microscopy product id 3696
Image basename hpfnode
Instrument JEOL 4000
X Size 0
Y Size 0
Z Size
X Resolution
Y Resolution
Z Resolution
Create date
PURL
Product type SINGLE TILT
Microscope type IVEM
Session name
Grid box count
Grid info. N/A
Slide info.
Plane count 61
SRB ID 0
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Imaging Product Types
Microscopy product ID: 3696
Image basename: hpfnode
Single tilt
Description Tilt Series
Range min -60.0 degrees
Range max 60.0 degrees
Tilt increment 2.0 degrees
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Specimen Description
Microscopy product ID: 3696
Image basename: hpfnode
Specimen description (3696, hpfnode)
System peripheral nervous system
Tissue nerve
Organ spinal root
Region
Subregions
Cell type Schwann cell
Cell id
Structure Node of Ranvier
Atlas coordinates
Atlas
Map location
Notes


Imaging Parameters
Microscopy product ID: 3696
Image basename: hpfnode
Electron microscopy product
Magnification 20000.0
EXT filename
Spot size
Recording medium film
Accelerating voltage 400.0 kv
Energy filter type
Energy filter slit
Energy filter dispersion
Notes
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2D Image Information
Microscopy product ID: 3696
Image basename: hpfnode

Downloadable data file description
Full-resolution version Original tilt images in suprim format (*.f). Also contains fid, preali, rawtlt, st, suprim files needed for alignment. According to the scanning dimensions given in the paper, the images (hpf*.f) submitted represent cropped and aligned images used for the reconstruction. The raw tilt images are contained in the .st file as a multifile image stack.
Down-sampled version N/A
Raw data info
Bit depth N/A
Digitized by N/A
Digitizing platform N/A


Animation
No animation available. N/A
Data files
Action File type File size Description
Download 512x512 image Unknown Single image taken on an intermediate voltage electron microscope at zero degrees tilt from a single axis tilt series through the Node of Ranvier from rat peripheral nerve prepared by chemical fixation followed by high pressure freezing and free substitution. Image is shown in negative contrast. Bright spots are colloidal gold particles applied to the section surface to serve as fiducial markers for subsequent alignment.
Download Data file 1.4GB Original tilt images in suprim format (*.f). Also contains fid, preali, rawtlt, st, suprim files needed for alignment. According to the scanning dimensions given in the paper, the images (hpf*.f) submitted represent cropped and aligned images used for the reconstruction. The raw tilt images are contained in the .st file as a multifile image stack.

Description: 
Single image taken on an intermediate voltage electron microscope at zero degrees tilt from a single axis tilt series through the Node of Ranvier from rat peripheral nerve prepared by chemical fixation followed by high pressure freezing and free substitution. Image is shown in negative contrast. Bright spots are colloidal gold particles applied to the section surface to serve as fiducial markers for subsequent alignment.

Launch WIB viewer (in a new window).


Description: 
Single image taken on an intermediate voltage electron microscope at zero degrees tilt from a single axis tilt series through the Node of Ranvier from rat peripheral nerve prepared by chemical fixation followed by high pressure freezing and free substitution. Image is shown in negative contrast. Bright spots are colloidal gold particles applied to the section surface to serve as fiducial markers for subsequent alignment.



Reconstruction Information
Microscopy product ID: 3696
Image basename: hpfnode

Reconstruction description
Reconstruction description
Full-resolution reconstruction file Zip file contains the volume (yinghpf_good.img/hdr) in Analyze 7.5 format.
Down-sampled version of the reconstruction file
Image map file  
Reconstruction file format MRC
Volume dimension (1792.0, 2671.0, 250.0) pixels
Voxel scale (0.0014, 0.0014, 0.0014) µm
More details
Animation
Annotated movie through the computed slices of a tomographic reconstruction of a Node of Ranvier from rat peripheral nerve, along with surface renderings of structures segmented from the reconstruction. See the Segmentation section for a details about the structures and the segmentation process.
Data files
Action File type File size Description
Download 512x512 image Unknown Single computed slice through a tomographic reconstruction of a Node of Ranvier from rat peripheral nerve, prepared by a combination of chemical fixation, high pressure freezing and freeze substitution
Download Reconstruction data file 1.11GB Zip file contains the volume (yinghpf_good.img/hdr) in Analyze 7.5 format.
Download Animation video 226.49MB Annotated movie through the computed slices of a tomographic reconstruction of a Node of Ranvier from rat peripheral nerve, along with surface renderings of structures segmented from the reconstruction. See the Segmentation section for a details about the structures and the segmentation process.

Description: Single computed slice through a tomographic reconstruction of a Node of Ranvier from rat peripheral nerve, prepared by a combination of chemical fixation, high pressure freezing and freeze substitution

Launch WIB viewer (in a new window).

Description: Single computed slice through a tomographic reconstruction of a Node of Ranvier from rat peripheral nerve, prepared by a combination of chemical fixation, high pressure freezing and freeze substitution



Segmentation Information
Microscopy product ID: 3696
Image basename: hpfnode

Segmentation description
Segmentation description Manual segmentation of paranodal loops, microtubules, neurofilaments, sepjate junctions and cross bridges using Xvoxtrace v 2.9. Different parts of the myelin sheath were segmented as different objects. Objects were surfaced using both Synu and Amira
Segmented by Sassan Ghassemzadeh
Object type Surface
Analysis description Segmented objects were surfaced using Synu and then converted into Amira format
Description for the downloadable segmentation data file Tar archive containing the trace file hpfnode_backup.trace and the segmented objects in synu (*.synu) and Amira (*.inventor) format.
View list of segmented objects
Data files
Action File type File size Description
Download 512x512 image Unknown Manual segmentation of paranodal loops, microtubules, neurofilaments, sepjate junctions and cross bridges using Xvoxtrace v 2.9. Different parts of the myelin sheath were segmented as different objects. Objects were surfaced using both Synu and Amira
Download Segmentation data file 215.16MB Tar archive containing the trace file hpfnode_backup.trace and the segmented objects in synu (*.synu) and Amira (*.inventor) format.

Description: Surface rendering of structures segmented from the Node of Ranvier of a rat peripheral nerve. Blue=Paranodal loops, Orange=microtubules, Green=neurofilaments, Magenta=mitochondria, Red=cross bridges and Yellow=septate junctions.



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