Measuring Fluorescence

Fluorescence Area: This method can be used for a quick determination of the area of fluorescence labeling.

• If you have more than one channel in your image, you’ll need to split them into separate images:
  • Image>Color>Split Channels
• Instead of manually drawing a line around your regions of interest, (e.g. cells, nuclei), you can use thresholding:
  • Image>Adjust>Threshold
    • Move the slider to a position where all the fluorescence pixels are selected.
    • Make sure “Dark background” is checked.
    • Click Apply
    • This will create a binary image (white and black pixels only)
• Obtain area measurements using the binary image:
  • Analyze>Analyze Particles
  • Select “Display results” and “Summarize” and then click on “OK”.

Fluorescence Intensity: This method measures the mean gray value (i.e. fluorescence intensity) for selected areas.

• Instead of manually drawing a line around your regions of interest (e.g. cells, nuclei), you can use thresholding:
  • Image>Duplicate.
  • Using the duplicated image:
    • Image>Adjust>Threshold
      • Move the slider to a position where all the fluorescence pixels are selected.
      • Make sure “Dark background” is checked.
      • Click Apply
      • This will create a binary image (white and black pixels only)
      • (Optional) Process>Binary>Watershed (This will split cells that are too close).
• Obtain areas to measure using the binary image:
  • Analyze>Analyze Particles
    • Select “Add to Manager”. This will open up a ROI manager window along with the result windows.
• Obtain mean gray values for the selected areas:
  • Analyze> Set measurements. Select “Mean gray value”
  • Click over (“activate”) your original image.
  • In the ROI manager click “Measure”.
    • This will result in a popup window with a table listing mean gray values (labeled as “mean”) for the regions of interest that you outlined in the binary image.

Corrected Total Cell Fluorescence: This method determines total cell fluorescence intensity by subtracting out background signal and correcting for intensity differences due to area. For more information see this webpage.

• Outline region of interest (e.g. cell, nucleus, etc.) with Freehand ROI tool (or use the threshold method above).
• Set desired parameters by Analyze > Set Measurements.
  • Make sure “Area”, “Integrated density” and “Mean gray value” are checked.
• Obtain measurements by Analyze > Measure.
  • • A window will pop up with your measurements. Copy data into spreadsheet.
    • Repeat for all regions of interest.
• Select a small area of your image that has no fluorescence for a background measurement.
  • • Analyze > Measure for that region. Copy data into spreadsheet.
      • Repeat for several more background regions.
      • Calculate the mean fluorescence of background readings.
• Calculate corrected total cell fluorescence (CTCF) = Integrated Density – (Area of Selected Cell x Mean Fluorescence of Background readings)