ImageJ nuclear morhoplogy/intensity analysis

ImageJ nuclear morhoplogy/intensity analysis

Joanna Y. Lee | Chaudhuri Lab

Dec. 2018

For images from 2D culture (page 2)

For images from 3D culture (page 4)

For images from patient IHC samples (page 6)

2D culture ImageJ nuclear morhoplogy/intensity analysis

Joanna Y. Lee | Chaudhuri Lab

Dec. 2018

Create Macro:

Plugins -> Macros -> Record

1. Find image scale.

Draw line over embedded scale bar. Go to Analyze -> Set Scale, enter known length of scale bar to get pixels/µm.

2. Set scale

Analyze -> Set Scale (6.9 pixels/µm, for our 63x, 1024 x 1024 confocal images) *depends on microscope/image acquisition; calibrate to image stack as described above

3. Filter to remove the noise.
   Process -> Filters -> Gaussian Blur (2.00)

4. Subtract the background.
   Process -> Subtract Background (50)

5. Threshold your image.
   Image -> Adjust -> Threshold (used 4), then check “dark background” - background should be black and nuclei white

6. Fill in any holes in the nuclei.
   Process -> Binary -> Fill Holes

7. Separate “Touching” nuclei.
   Process -> Binary -> Watershed

8. Find Edges
   Process -> Find Edges

9. Count nuclei

Analyze -> Analyze Particles (Size (pixel^2): 1000-infinity; Circularity: 0-1; check Pixel Units, Add to Manager, Exclude Edges)

10. Close DAPI image.

Save Macro:

Save As “2D_FindNuclei.txt”

11. Open corresponding YAP image.

Create 2nd Macro:

Plugins -> Macros -> Record

12. Set Measurements

Analyze -> Set Measurements, select Area, Center of Mass, Bounding Rectangle, Shape Descriptors, Integrated Density (Mean Gray Value times Area), Skewness, Mean Gray Value (sum of all gray values of all pixels divided by number of pixels), Perimeter

13. Set scale

Analyze -> Set Scale (6.9 pixels/µm)

14. Overlay outlines of nuclei into YAP image.

Open ROI manager (if not already open)

    Analyze -> Tools -> ROI Manager

In ROI Manager, select Show All

In ROI Manager, select Measure

15. Save results

File -> Save As, save to Desktop as “Results”

Save Macro:

Save As “2D_AnalyzeIntesityinNuclei.txt”

To run:

16. Open DAPI image.

17. Run Macro

Plugins -> Macros -> Run “IF_FindNuclei.txt”

18. Open corresponding YAP image.

19. Run Macro

Plugins -> Macros -> Run “IF_AnalyzeIntesityinNuclei.txt”

20. Rename “Results” on Desktop.
    
    

3D culture ImageJ nuclear morhoplogy/intensity analysis

Joanna Y. Lee | Chaudhuri Lab

Dec. 2018

Create Macro:

Plugins -> Macros -> Record

1. Find image scale.

Draw line over embedded scale bar. Go to Analyze -> Set Scale, enter known length of scale bar to get pixels/µm.

2. Set scale

Analyze -> Set Scale (3.45 pixels/µm, for our 63x, 512 x 512 confocal images). *depends on microscope/image acquisition; calibrate to image stack as described above

3. Filter to remove the noise.
   Process -> Filters -> Gaussian Blur (2.00)

4. Subtract the background.
   Process -> Subtract Background (50)

5. Threshold your image. *image acquisition-dependent; calibrate to image stack
   Image -> Adjust -> Threshold (used 17), then check “dark background” - background should be black and nuclei white

6. Fill in any holes in the nuclei.
   Process -> Binary -> Fill Holes

7. Separate “Touching” nuclei.
   Process -> Binary -> Watershed

8. Find Edges
   Process -> Find Edges

9. Count nuclei

Analyze -> Analyze Particles (Size (pixel^2): 100-infinity; Circularity: 0-1; check Pixel Units, Add to Manager, Exclude Edges)

10. Close DAPI image.

Save Macro:

Save As “3D_FindNuclei.txt”

11. Open corresponding YAP image.

Create 2nd Macro:

Plugins -> Macros -> Record

12. Set Measurements

Analyze -> Set Measurements, select Area, Center of Mass, Bounding Rectangle, Shape Descriptors, Integrated Density (Mean Gray Value times Area), Skewness, Mean Gray Value (sum of all gray values of all pixels divided by number of pixels), Perimeter

13. Set scale

Analyze -> Set Scale (3.45 pixels/µm)

14. Overlay outlines of nuclei into YAP image.

Open ROI manager (if not already open)

    Analyze -> Tools -> ROI Manager

In ROI Manager, select Show All

In ROI Manager, select Measure

15. Save results

File -> Save As, save to Desktop as “Results”

Save Macro:

Save As “3D_AnalyzeIntesityinNuclei.txt”

To run:

16. Open DAPI image.

17. Run Macro

Plugins -> Macros -> Run “IF_FindNuclei.txt”

18. Open corresponding YAP image.

19. Run Macro

Plugins -> Macros -> Run “IF_AnalyzeIntesityinNuclei.txt”

20. Rename “Results” on Desktop.
    
    

Patient IHC ImageJ nuclear morhoplogy/intensity analysis

Joanna Y. Lee | Chaudhuri Lab

Dec. 2018

Create Macro:

Plugins -> Macros -> Record

1. Find image scale.

Draw line over embedded scale bar. Go to Analyze -> Set Scale, enter known length of scale bar to get pixels/µm.

2. Set scale

Analyze -> Set Scale (4.9 pixels/µm, for our 20x IHC images). *depends on microscope/image acquisition; calibrate to image stack as described above

3. Filter to remove the noise.
   Process -> Filters -> Gaussian Blur (2.00)

4. Subtract the background.
   Process -> Subtract Background (50)

5. Threshold your image.
   Image -> Adjust -> Threshold (used 69), then check “dark background”, hit “Apply” - background should be black and nuclei white

6. Separate “Touching” nuclei.
   Process -> Binary -> Watershed

7. Find Edges
   Process -> Find Edges

8. Count nuclei

Analyze -> Analyze Particles (Size (pixel^2): 100-800; Circularity: 0-1; check Pixel Units, Add to Manager, Exclude Edges)

9. Close DAPI image.

Save Macro:

Save As “Patients_Find Nuclei.txt”

10. Open corresponding YAP image.

Create 2nd Macro:

Plugins -> Macros -> Record

11. Set Measurements

Analyze -> Set Measurements, select Area, Center of Mass, Bounding Rectangle, Shape Descriptors, Integrated Density (Mean Gray Value times Area), Skewness, Mean Gray Value (sum of all gray values of all pixels divided by number of pixels), Perimeter

12. Set scale

Analyze -> Set Scale (4.9 pixels/µm)

13. Overlay outlines of nuclei into YAP image.

Open ROI manager (if not already open)

    Analyze -> Tools -> ROI Manager

In ROI Manager, select Show All

In ROI Manager, select Measure

14. Save as “Results” to Desktop.

Save Macro:

Save As “Patients_MeasureIntesityinNuclei.txt”

To run:

15. Open DAPI image.

16. Run Macro

Plugins -> Macros -> Run “Find Nuclei.txt”

17. Close DAPI image. Open corresponding YAP image.

18. Run Macro

Plugins -> Macros -> Run “MeasureIntesityinNuclei.txt”

19. Rename “Results” on Desktop.