For informations about installation of the image analysis tool, access the “Simulator†tab
Step 1: Open ImageJ by double clicking Image J icon from the desktop.
ImageJ will open in a new window as shown below.
Step 2: To analyze an Image into ImageJ, we have to import the image into ImageJ. To perform that, Go to files ---> Open. A popup window will appear. Use Choose button to choose your respective images into ImageJ software.
The imported picture will shown in a new window.
Step 3: The image will be in the RGB format. Convert the RGB image into a gray-scale image: Image --->Type ---> 8-bit
Step 4: Click on Process ---> Subtract Background. This is done to remove the uneven back ground.
Step 5: Click on Image ---> Adjust ---> Threshold. The "thresholding" of the image is done by setting all pixels above certain intensity value to black and leaving background as white. This is called image segmentation.
Step 6: Click on Process ---> Binary ---> Watershed. If fluorescent particles are touching each other, thresholding will recognize them as one object. Watershed segmentation is a way of automatically separating the adjacent particles.
Step 7: To count the objects, Open: Analyze ---> Analyze particles.
To perform this, Adjust the size: “0- infinityâ€ÂÂ, show: “Outlines†and tick on all boxes except “Record Startsâ€ÂÂ
Step 8: The result will display on four separate windows. The count of fluorescent objects is show on the first window while the numerical data (area and integrated density) will be on the third window. The number of nuclei in your image, and their outlines displayed in the fourth window.
Result Interpretation:
The count and the total area covered by the fluorescent labelled cells shown in the first window. The Area Mean and Integrated density of the fluorescently labelled cells shown in the third window. The number of fluorescently labelled nuclei and their outlines displayed in the fourth window.