Measuring Fluorescence

Note:  We suggest you create a copy (duplicate) of your image and work on the duplicate instead of the original image. Most functions on Image J are not reversable.

• Multiple channels:  If you have more than one channel in your image, you’ll need to split them into separate images:
  • Image> Color> Split Channels
    • Close the images (channels) that you will not be using to obtain measurements.

• Thresholding:  Instead of manually drawing a line around your regions of interest, (e.g. cells, nuclei), you can use thresholding:
  • Image> Adjust> Threshold
    • Make sure ‘Dark background’ is checked.
    • Adjust the upper slider to a position where all the fluorescence pixels are selected (areas of interest are highlighted in red)
    • Click ‘Apply’.
    • This will create a binary image (black and white pixels only)
  • (Optional) Process> Binary> Watershed (This will split cells that are too close).
• Measure area(s) of interest: Obtain area measurements using the binary image:
  • Analyze> Analyze Particles
  • Select ‘Add to Manager’. This will open up a ROI manager window along with the result windows.
  • Area measures will appear in the Results window under the ‘Area’ column

NOTE: To compare the fluorescence intensity between different samples, you must acquire images using identical settings (e.g. laser power, gain, exposure time).

• Thresholding:  Instead of manually drawing a line around your regions of interest (e.g. cells, nuclei), you can use thresholding:
  • Image> Adjust> Threshold
    • Make sure ‘Dark background’ is checked.
    • Move the upper slider to a position where all the fluorescence pixels are selected. (areas of interest are highlighted in red)
    • Do not click apply (i.e. do not make the image binary) .
• Set measurements: 
  • Analyze > Set measurements
    • Select ‘mean gray value’, then click on ‘OK’.
• Measure Intensity: Obtain mean gray values for the selected areas: 
  • Analyze> Analyze Particles
    • Select ‘Add to Manager’. This will open up a ROI manager window along with the result windows.
    • Select ‘Display results’ and ‘Summarize’, then click on ‘OK’.
    • The  fluorescence intensity value will appear in the ‘Results’ window in the ‘Mean’ column.

NOTE: To compare the fluorescence intensity between different samples, you must acquire images using identical settings (e.g. laser power, gain, exposure time).

• Define area(s) of interest: Outline region of interest (e.g. cell, nucleus, etc.) with Freehand ROI tool or use the threshold method below.
  • Image> Adjust> Threshold
    • Make sure ‘Dark background’ is checked.
    • Move the upper slider to a position where all the fluorescence pixels are selected (areas of interest are highlighted in red)
    • Do not click apply (i.e. do not make the image binary)
• Set measurement: 
  • Analyze > Set Measurements.
  • Select ‘Area’, ‘Integrated density’, and ‘Mean gray value’, then click on ‘OK’
• Measure Intensity: Obtain integrated density values for the selected areas.
  • Analyze> Analyze Particles
    • Select ‘Add to Manager’. This will open up a ROI manager window along with the result windows.
    • Select ‘Display results’ and ‘Summarize’, then click on ‘OK’.
    • The  Integrated intensity values will appear in the ‘Results’ window in the ‘IntDen’ column.
• Background measurement: taking mean gray value measurements of background areas outside of your ROI.
  • Using the rectangular tool, select an area from the background (outside the region of interest)
    • Analyze > Measure
    • The mean gray value will appear in the ‘Results’ window in the ‘Mean’ column.
    • If you want to be more accurate, take three or more background mean gray value measurements.
• Calculate CTCF:
  • • Copy your data from the results window and paste into a spreadsheet
    • Enter the following formula = Integrated Density – (Area of Selected Cell x Average Mean gray value of Background )