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, 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: Convert the RGB image into a gray-scale image: Image ---> Type ---> 8-bit.
Step 4: Click on Analyze ---> Set Measurement. Select Area, Mean Gray Value and Integrated Density. Click “OK”.
Step 5: Select the cell which one want to know fluorescence using the freehand selection tool.
Step 6: Select Analyze ---> Measure. The results box containing area, mean and integrated density of that cell.
Step 7: Select a region next to that cell using rectangular selection tool. This will be considered as your background. Also calculate the area, mean and integrated density on the same way.
Step 8: The results window is obtained like this.
Step 9: Repeat this step for a few small boxes around and near of the cell. The results windows getting like this.
Step 10: Use Microsoft excel to calculate corrected density ( total fluorescence per cell)
Open the file “Excel Sheet for Bkgd Correct.xls”
Step 11: Copy the first row of the Results window obtained from ImageJ
Step 12: Paste it on cell A2
Step 13: Likewise, copy the data in the row 2 to onwards in the results window and paste it on the cell A5. The corrected integrated density get in the the D15 cells of the excel sheet or one can use the fillowing equation to calculate this.
Corrected Integrated Density (CTCF) = Integrated Density - (Area of selected cell X Mean fluorescence of background readings)
In this example, the area and integrated density of selected fluorescetly stained cell is 7009 and 249888 respectively. The corrected integrated density ( amount of total fluorescence per cell) of the cell is obtained as 246888.