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.

Area of Fluorescence

Determine the area of fluorescence labeling using thresholding.

  • 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

 

Fluorescence Intensity

Measuring the mean gray value (i.e. fluorescence intensity) for selected areas using thresholding.

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.