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Reconstruction
Display image description
Single computed slice through a tomographic reconstruction of a selectively-stained spiny dendrite from a medium spiny neuron contained in a 4 um thick section from the neostriatum of a dopamine transporter knock out mouse.
Full resolution image description
Tar file containing the IMOD .rec format (datkoc2g26_full.rec) for the volume reconstruction and Amira .am/.hx format image stack of the labels used (datkoc2g26_full-labels.am, datkoc2g26.hx).
Volume_dimension
1122, 200, 1670
Animation description
Animation through the computed slices of a tomographic reconstruction of a selectively-stained spiny dendrite from a medium spiny neuron contained in a 4 um thick section from the neostriatum of a dopamine transporter knock out mouse.

Image 2D
Display image description
Electron micrograph of a selectively-stained spiny dendrite from a medium spiny neuron contained in a 4 um thick section from a dopamine transporter knock out mouse, imaged using ultra high voltage electron microscopy.
Full resolution image description
Tar file containing IMOD files (datkoc2g26.com/.log/.st/.preali/.fid/.rawtlt) used for the alignment and the original tiff images (in the TIFF folder in the format datkoc2g26000.tif.gz)
Animation description
Animation of aligned electron microscopic tilt series of a selectively-stained spiny dendrite from a medium spiny neuron contained in a 4 um thick section from a dopamine transporter knock out mouse, imaged using ultra high voltage electron microscopy. Tilt series was obtained at 2 degree increments through +/- 70 degrees of tilt.

Segmentation
Display image description
Manual tracing of dendritic spines from tomographic volume using IMOD version 3.7, followed by surfacing using IMOD with smoothsurf
Segmentation file description
a .tar file containing the segmentation files of a dendritic shaft and individual spines in IMOD's .mod format (datkoc2g26_full.mod)

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

CCDB:3561*
Project: P1207
Project name
Correlative microscopic characterization of dendritic spines in a transgenic mouse model of hyperdopaminergia: The dopamine transporter knockout mouse
Description
Multiscale characterization of DAT KO transgenic mouse
Funding agency
NIH
Leader(s)
Diana Price
Collaborator(s)
Aki Laakso
Michele Cyr
Maryann Martone
Naoko Yamada
Andrea Thor
Monica Berlanga
Start date
01-01-2003
End date
unspecified
 
Experiment
Experiment ID
3413
Experiment date
02-20-2003
Title
P1207 Exp 2
Purpose
Tomographic reconstruction of medium spiny dendrites from the neostriatum using UHVEM in a dopamine transporter knock out mouse
Experimenter(s)
Diana Price and Andrea Thor
Microscopy product
Microscopy product ID
3561
Instrument
Hitachi UHVEM
Microscopy type
UHVEM
Product type
SINGLE TILT
Image basename
datko2g26
Spatial Axis Image Size Pixel Size
X 1024px 0.021 nm/pixels
Y 1024px 0.021 nm/pixels
Subject
Species
mouse
Scientific name
mus musculus
Strain
B6;129-/Slc6a3 tm2Mca
Group by
genetic manipulation
Treatment
knock out of the dopamine transporter
Age
7.3 months
Age class
adult
Tissue section
Anatomical location
dorsal lateral striatum Cell 2
Microtome
ultramicrotome
Thickness
4 µm
Specimen description
Organ
brain
System
central nervous system
Structure
spiny dendrite
Cell type
medium spiny neuron
Imaging parameters
Type
Electron microscopy product
Recording medium
film
Magification
3000
Accelerating voltage
3 MeV
Specimen preparation
Protocol used
Experiment #1DAT KO mouse 02/20/03Description: Photoconverted dye-filled striatal medium spiny neurons for EMAnimal Info:ID# 1040Weight: 26gDOB: 7/12/02Protocol1. Perfusion (at Duke) Nembutal; 4% paraformaldehyde + 0.1% gluteraldehyde2. Sectioned on Vibratome (at NCMIR)Thickness = 100 micronsStore in 1X PBS in fridge3.Fill cells with Lucifer yellow4.Store slices with filled cells in 4% para in fridge5.Wash 6x with PBS 1X (on ice)6. When ready to begin photoconversion, turn on the chiller in confocal room. Set at ~4C. The refrigerator unit should be set at TEMP < 45C. Switch ON. Stage needs around 20 minutes to come to temperature. Pull unit out into hallway (to avoid increase in temperature).6.Place slices in 2% glut/PBS on ice for 15 minutes0.8 ml 25% gluteraldehyde2 ml 5x PBS6.2ml ddH207.Briefly wash slices in PBS8.Place slices in PBS/glycine for a few minutes38 mg glycine10 ml 1x PBS9.Follow instructions for Photoconversion of Lucifer Yellow-filled cells10. After photoconversion, remove DAB solution and wash slice 3x 10 minutes in generous volumes of PBS on ice. Must remove all DAB before beginning osmification.Microwaving protocol for osmication, dehydration, and embedding of photoconverted slices*Prepare Resin mix and let it sit covered and undisturbed until needed (instructions by fume hood in embedding area).*Rinse slices with a generous amount of cold 1X PBS on ice for ~ 10 min.*Turn on circulating bath (over 20C, ~ RT): water bath (left hand side) will fill. *Insert temperature probe*Fill other T-beaker with water*Set temperature to 35C*Open new bottle of 100% ethanol and prepare following dilutions:90% ethanol70% ethanol50% ethanol*Make up osmium solution under fume hood and chill on ice*1% osmium tetroxide in PBS on ice.2.0 ml PBS 5Xthen 5.5 2x distilled H2O2.5 ml Osmium 4%*Rinse w/ 2x distilled H2O 3 x 5min*Warm up microwave for 2 minutes on high*Label tubes & place in rack on ice*Fill tubes with osmium solution (w/ meniscus at 0.5)*Using glass hooks, transfer slices to tubes*Remove temperature probe & set temp above 50C.*Put rack w. tubes in for 40 sec at full power*Change rear water load in T-beaker*Change osmium solution on ice and microwave for another 40 seconds at full power*Rinse samples for 2 minutes in distilled water on benchtop (at RT)*Insert petri bath with H2O under rack*Dehydration steps (2 x 40 seconds per step; all @ 35C)1st2nd50% EtOH70% EtOH90% EtOH100% EtOH100% Acetone*All of the dehydration steps should be carried out in microcentrifuge tubes filled with 600 ml of solution. Temperature probe should be in petri dish and set for 35. Change water in rear water load when warm to touch.*Change from water to acetone in petri bath under rack check acetone bath level every 3 minutes*Infiltration steps (both @ 50C): With a 50/50 mixture of resin and acetone:1 x 15 min1:1 Resin:acetone* Check rear water load at 7.5 minutesSwitch to 100% resin for 3 x 10 minutes:1st2nd3rd100% Resin*Periodically check rear water load*Flat embed samples between mould release slides and place in embedding oven under vacuum.
Imaging product type
Type
Single tilt
Description
singlet_desc
Min range
-70 degrees
Max range
70 degrees
Tilt increment
2 degrees
Notes
Missing angles: -30, +22, +44, +56