Bitan:MicrogliaIHC: Difference between revisions
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| Line 4: | Line 4: | ||
Acquisition → Acquire Image | Acquisition → Acquire Image | ||
*Click on the image, but not on the section | |||
Image → Add Scalebar | Image → Add Scalebar | ||
*Set to 100 microns | |||
Apply Scalebar | *Apply Scalebar | ||
To get to the next section revert back to Live Image and Select New Data File | To get to the next section revert back to Live Image and Select New Data File | ||
| Line 22: | Line 21: | ||
Both Images: | Both Images: | ||
*Image → Type → 8-bit | |||
One Image: | One Image: | ||
*Analyze → Set Scale | |||
70 | **70 pixels → 100 μm | ||
Process → Subtract Background | *Process → Subtract Background | ||
50 pixels rolling ball radius | **50 pixels rolling ball radius | ||
Check Light Background | **Check Light Background | ||
Use color picker to pick a color closest to white | *Use color picker to pick a color closest to white | ||
Use pencil to whiteout all dark areas not part of the spinal section or are “spots” on the section | *Use pencil to whiteout all dark areas not part of the spinal section or are “spots” on the section | ||
Image → Adjust → Brightness and Contrast | *Image → Adjust → Brightness and Contrast | ||
Adjust min/max bars until microglia are clearly contrasting the background | **Adjust min/max bars until microglia are clearly contrasting the background | ||
Compare with other 8 bit image | **Compare with other 8 bit image | ||
Image → Adjust → | *Image → Adjust → Threshold | ||
Adjust Threshold value until microglia are selected and as much background is removed as possible | **Adjust Threshold value until microglia are selected and as much background is removed as possible | ||
Use zoom tool to compare with 8-bit image | **Use zoom tool to compare with 8-bit image | ||
Analyze→ Analyze Particles | *Analyze→ Analyze Particles | ||
Select Summarize and DIsplay Measurement | **Select Summarize and DIsplay Measurement | ||
Mark down: Count, Total Area, and Average Size | **Mark down: Count, Total Area, and Average Size | ||
Revision as of 04:30, 5 September 2015
Bring section under focus in StereoInvestigator
Acquisition → Acquire Image
- Click on the image, but not on the section
Image → Add Scalebar
- Set to 100 microns
- Apply Scalebar
To get to the next section revert back to Live Image and Select New Data File
Open Image in ImageJ
Open a second image
Both Images:
- Image → Type → 8-bit
One Image:
- Analyze → Set Scale
- 70 pixels → 100 μm
- Process → Subtract Background
- 50 pixels rolling ball radius
- Check Light Background
- Use color picker to pick a color closest to white
- Use pencil to whiteout all dark areas not part of the spinal section or are “spots” on the section
- Image → Adjust → Brightness and Contrast
- Adjust min/max bars until microglia are clearly contrasting the background
- Compare with other 8 bit image
- Image → Adjust → Threshold
- Adjust Threshold value until microglia are selected and as much background is removed as possible
- Use zoom tool to compare with 8-bit image
- Analyze→ Analyze Particles
- Select Summarize and DIsplay Measurement
- Mark down: Count, Total Area, and Average Size
