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.
- Image> Color> Split Channels
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).
- Image> Adjust> Threshold
- 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) .
- Image> Adjust> Threshold
- Set measurements:
- Analyze > Set measurements
- Select ‘mean gray value’, then click on ‘OK’.
- Analyze > Set measurements
- 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.
- Analyze> Analyze Particles