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| 2D Image |
Reconstruction |
Segmentation |
Map Location |
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Add to MyLabBench
Download dataset
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Show product in Pivot
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Specimen Preparation Information
Microscopy product ID: 41
Image basename: datko_g6
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Specimen Preparation
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Tissue group type
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NA
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Ext file name
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NA
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| Protocol used |
Experiment #1 DAT KO mouse 01/09/03
Description: Photoconverted dye-filled striatal medium spiny neurons for EM
Animal Info: ID# 980
Weight: 27g
DOB: 7/8/02
Protocol
1. Perfusion (at Duke)
Nembutal; 4% paraformaldehyde + 0.1% gluteraldehyde
2. Sectioned on Vibratome (at NCMIR)
Thickness = 100 microns
Store in 1X PBS in fridge
3. Fill cells with Lucifer yellow
4. Store slices with filled cells in 4% para in fridge
5. Wash 6x with PBS 1X (on ice)
6. When ready to begin photoconversion, turn on the chiller in confocal room. Set at ~4?C. The refrigerator unit should be set at TEMP < 45?C. 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 minutes
0.8 ml 25% gluteraldehyde
2 ml 5x PBS
6.2 ml ddH20
7. Briefly wash slices in PBS
8. Place slices in PBS/glycine for a few minutes
38 mg glycine
10 ml 1x PBS
9. Follow instructions for Photoconversion of Lucifer Yellow-filled cells
10. 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 20?C, ~ RT): water bath (left hand side) will fill.
* Insert temperature probe
* Fill other T-beaker with water
* Set temperature to 35?C
* Open new bottle of 100% ethanol and prepare following dilutions:
90% ethanol
70% ethanol
50% ethanol
* Make up osmium solution under fume hood and chill on ice
* 1% osmium tetroxide in PBS on ice.
2.0 ml PBS 5X
then 5.5 2x distilled H2O
2.5 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 50?C.
* 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 @ 35?C)
1st
2nd
50% EtOH
70% EtOH
90% EtOH
100% EtOH
100% 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 @ 50?C):
With a 50/50 mixture of resin and acetone:
1 x 15 min
1:1 Resin:acetone
* Check rear water load at 7.5 minutes
Switch to 100% resin for 3 x 10 minutes:
1st
2nd
3rd
100% Resin
*Periodically check rear water load
* Flat embed samples between mould release slides and place in embedding oven under vacuum.
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Microscopy Product Information
Microscopy product ID: 41
Image basename: datko_g6
Microscopy product
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| Microscopy product id |
41 |
| Image basename |
datko_g6 |
| Instrument |
Hitachi UHVEM
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| X Size |
1024
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| Y Size |
1024
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| Z Size |
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| X Resolution |
0.021 um/pixel
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| Y Resolution |
0.021 um/pixel
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| Z Resolution |
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| Create date |
2004-03-09 |
| PURL |
NA
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| Product type |
single axis tilt series
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| Microscope type |
UHVEM
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| Session name |
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| Grid box count |
6
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| Grid info. |
N/A
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| Slide info. |
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| Plane count |
0
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| SRB ID |
1
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Specimen Description
Microscopy product ID: 41
Image basename: datko_g6
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Specimen description (41, datko_g6)
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| System |
central nervous system
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| Tissue |
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| Organ |
brain |
| Region |
neostriatum |
| Subregions |
dorsal
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| Cell type |
medium spiny neuron |
| Cell id |
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| Structure |
spiny dendrite |
| Atlas coordinates |
(1.375, -3.375, 0.62)
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| Atlas |
Paxinos and Franklin |
| Map location |
Show map location
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| Notes |
corresponding atlas plate#26 |
Imaging Parameters
Microscopy product ID: 41
Image basename: datko_g6
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Electron microscopy product
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| Magnification |
3000.0
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| EXT filename |
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| Spot size |
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| Recording medium |
film |
| Accelerating voltage |
3.0 Mev |
| Energy filter type |
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| Energy filter slit |
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| Energy filter dispersion |
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| Notes |
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Back to top
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2D Image Information
Microscopy product ID: 41
Image basename: datko_g6
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Downloadable data file description
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Full-resolution version
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a .tar file containing processing files for an IMOD volume reconstruction from a tilt series taken on an EM. Specifically, the .ali, .fid, .preali, and the .seed files are all found on in this tar file.
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Down-sampled version
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N/A
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Raw data info
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Bit depth
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N/A
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Digitized by
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N/A
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| Digitizing platform |
N/A
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| Animation |
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a .mpg movie file of the tilt series taken of this spiny dendrite specimen
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| Data files |
| Action |
File type |
File size |
Description |
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Download
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512x512 image |
Unknown
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a 512 by 512 image of a spiny dendrite specimen
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Download
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Data file |
178.0MB
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a .tar file containing processing files for an IMOD volume reconstruction from a tilt series taken on an EM. Specifically, the .ali, .fid, .preali, and the .seed files are all found on in this tar file.
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Download
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Animation video |
3.84MB
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a .mpg movie file of the tilt series taken of this spiny dendrite specimen
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Description:
a 512 by 512 image of a spiny dendrite specimen
No WIB image found.
Description: a 512 by 512 image of a spiny dendrite specimen
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Reconstruction Information
Microscopy product ID: 41
Image basename: datko_g6
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Reconstruction description
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Reconstruction description
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Full-resolution reconstruction file
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Reconstruction of selectively stained spiny dendrite from single axis tilt tomography
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Down-sampled version of the reconstruction file
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| Image map file |
View | Download
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Reconstruction file format
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| Volume dimension |
(424.0, 955.0, 181.0) pixels
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| Voxel scale |
(0.021, 0.021, 0.021) µm
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| Animation
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maximum intensity projection of selectively stained spiny dendrite rotated along the y axis |
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| Data files |
| Action |
File type |
File size |
Description |
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Download
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512x512 image
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Unknown
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Maximum intensity project of a tomographic reconstruction of a spiny dendrite from a medium spiny neuron of dopamine transporter knock out mouse
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Download
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Reconstruction data file
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Unknown
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Reconstruction of selectively stained spiny dendrite from single axis tilt tomography
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Download
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Animation video
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Unknown
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maximum intensity projection of selectively stained spiny dendrite rotated along the y axis
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Download
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Fiducial mark file
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Unknown
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File containing the set of fiducial markers.
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Description: Maximum intensity project of a tomographic reconstruction of a spiny dendrite from a medium spiny neuron of dopamine transporter knock out mouse
No WIB image found.
Description: Maximum intensity project of a tomographic reconstruction of a spiny dendrite from a medium spiny neuron of dopamine transporter knock out mouse
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Segmentation Information
Microscopy product ID: 41
Image basename: datko_g6
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