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Reconstruction
Display image description
Single computed slice through a tomographic reconstruction of a spine synapse from the molecular layer of the cerebellar cortex from tissue that was prepared from a combination of chemical fixation and high pressure freezing. The contrast was adjusted and the image downsampled to 8 bits from the submitted data for display purposes.
Full resolution image description
Tar archive file containing TxBR combined (double tilt) tomographic volume of high pressure frozen cerebellar tissue in IMOD format. Two versions of the volume are included: the full resolution version HPFcere2_full.rec; a trimmed version HPFcere2_proj_trim.rec and a trimmed downsampled version HPFcere2_proj_trim_rot_trim_bin2.rec. Volume dimensions and scales given in CCDB are for the full resolution version. Also included are various animation files that show different types of projections through the volume. Total size of sitx file ~6 Gb.
Volume_dimension
1839, 1740, 591
Volume scale
0.0005, 0.0005, 0.0005
Animation description
Animation of a tomographic reconstruction of a spine synapse from the molecular layer of the cerebellar cortex from tissue that was prepared from a combination of chemical fixation and high pressure freezing.

Image 2D
Display image description
Zero degree tilt electron micrograph from a double tilt series of a spine synapse from the molecular layer of cerebellar cortex tissue prepared using a combination of conventional chemical fixation followed by high pressure freezing, imaged using intermediate voltage electron microscopy
Full resolution image description
Tar file containing original digitized TIFF images (in folder marked A and B), IMOD files (*com, *log, st, preali, fid, rawtlt,...), and TxBR files (*mat, *txt, preali, rawtlt, fid, ...) of high pressure frozen Cerebellar tissue, showing post synaptic density. Note total file size is ~ 11 Gb, so this will take a long time to download.
Animation description
Aligned tilt series (one of two orthogonal tilt series from a double tilt tomogram) of a spine synapse from the molecular layer of cerebellar cortex tissue prepared using a combination of conventional chemical fixation followed by high pressure freezing, imaged using intermediate voltage electron microscopy.

Segmentation
Display image description
Segmentation of post synaptic density (PSD) using automatic segmentation
Segmentation file description
Sitx (zipped) archive of segmentation files generated by Amira (.hx, .am, surf) of the post synaptic density. Also included are animation files showing the surfaced PSD (PSDamira_surface_rot_noimage2.mpg) and a the surfaced PSD visualized along with computed slice through the tomographic volume (PSDamira_surface_rot.mpg).

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:3684*
Project: P1243
Project name
High Pressure Freezing and Freeze Substitution
Description
This project is designed to achieve ultimate ultrastructure of animal tissues.
Funding agency
NIH
Leader(s)
Mark Ellisman
Gina Sosinsky
Ying Jones
Start date
01-01-2004
End date
unspecified
 
Experiment
Experiment ID
3396
Experiment date
08-17-2006
Title
HPF/FS on fixed rat brain and nerve tissues of rat
Purpose
To achieve better ultrastructure of brain and nerve tissue.
Experimenter(s)
Ying Jones
Microscopy product
Microscopy product ID
3684
Instrument
JEOL 4000#1
Microscopy type
IVEM
Product type
DOUBLE TILT
Image basename
HPFcere2
Spatial Axis Image Size Pixel Size
X 2242px
Y 3340px
Subject
Species
rat sprague dawley
Scientific name
rat
Strain
sprague dawley
Group by
Type of fixation
Treatment
Chemical fixation followed by high pressure freezing
Age
21 days
Age class
young adult
Tissue section
Anatomical location
cerebellum
Microtome
Ultramicrotome
Thickness
0.5 µm
Specimen description
Organ
brain
System
central nervous
Structure
synapse
Imaging parameters
Type
Electron microscopy product
Recording medium
film
Magification
50000
Accelerating voltage
400 kV
Specimen preparation
Protocol used
1. Fixation: A 21 day old rat was perfused with a solution of 2% of paraformaldehyde/2.5% of glutaraldehyde in 0.15 M cacodylate buffer according to the protocol described in (Giepmans, 2005 see original publication for details). Brain was taken out and post-fixed in same fixative for 2 hrs at 4C. For brain tissue, 100mm thick sections were kept in the fixative solution until HPF. 2. High Pressure Freezing: Brain sections were cut with 1.8 mm tissue puncher. This step ensures that the proper size of brain tissue will fit into the shallow side of a 100 mm-deep well in the type A HPF brass planchette. For peripheral nerve tissue, the nerves were carefully trimmed to a proper length in order to fit into the type A freezing hats. Trimmed brain or nerve tissue was loaded into the planchettes and the well was filled with 1-hexadecene. The planchette was then covered with the flat side of brass type B planchette, quickly loaded into a freezing holder and frozen with the Bal-Tec HPM 010. Freeze-substitution procedure: After freezing, the planchette sandwiches were separated under liquid nitrogen and the specimen/type A hats were placed into cryo-vials and stored under liquid nitrogen or subsequently placed into the freeze substitution device. The first substitution media was a solution of filled with freshly made 0.1% tannic acid (EM grade, from Polysciences Inc., Warrington, Pennsylvania) in acetone (EM grade from Fullam Inc., Latham, New York). After 24 hours, samples were then washed three times in cold acetone over a 2 hour period. The solution was changed to 2% osmium tetroxide in acetone for 48 hrs. The temperature was slowly raised to 20 deg C. The specimens were rinsed three times at room temperature for 10 minutes in acetone. Tissues were removed from the planchettes after the last wash step. The total time for this procedure is 113 hrs.Infiltration and embedding: Infiltration was conducted over 3 days followed by embedding in Durcupan ACM resin (Electron Microscopy Science Inc., Hatfield, PA). Samples were infiltrated in 30% Durcupan in acetone for 4 hours and 50% Durcupan overnight. The next day, the specimens were placed into 70% Durcupan for 4 hours, 90% over 2 hours and were placed in 100% Durcupan for overnight incubation. After two incubations in fresh 100% Durcupan, the sample was then polymerized at 60deg C for 2 days.
Imaging product type
Type
Double tilt
X min range
-60 degrees
X max range
60 degrees
X tilt increment
2 degrees
Y min range
-60 degrees
Y max range
60 degrees
Y tilt increment
2 degrees
Description
double-tilt tomographic dataset of high pressure frozen cerebellum tissue


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