Calculation of BrdU positive cells using the software ImageJ and MBF plugin:
· Select the area of interest:
o open the new file
o select an even area with ellipses (round proliferative layer) or rectangles (columnar layer) and copy it to a new file (named after the original section and the type of area analyzed)
o go to Image, Adjust, Brightness/Contrast, Auto and save
· Detect the total number of cells:
o go to Process, Binary, Make Binary (this will make a binary black and white image)
o go to Process, Binary, Watershed (this divides two adjacent cells that were previously identified as one)
o go to Analyze, Analyze particles and select the following options
§ Show ellipses (so you can see where and which are the cells that the program identifies. Different “minimum pixel” options have different sensitivity on what are cells and what is just random noise. Make sure all the cells selected are the cell you see and that there are not cells missing)
§ Select minimum pixel (usually from 2 to 5 pixel so that noise is elimintated)
§ Results (save on excel file)
o don't save and open the image again
· Find the number of positive cells:
o go to Image, Adjust, Window bar (you want to move the Window bar so that you can visualize only positive cells. The line is usually vertical)
OR
o go to Plugins, Segmentation, Color Based Thresholding
§ set brightness at usually 190-160 (you need to see only the positive cells and as low noise as possible. Be consistent with this setting on all the pictures, otherwise it might change the counting. Ideally the pictures from the microscope should have the same brightness and contrast level.)
o go to Process, Binary, Make Binary
o go to Process, Binary, Watershed
o go to Analyze, Analyze particles and select the following options
§ Show ellipses
§ Select minimum pixel (usually from 7 to 10 pixels. You should be able to eliminate all the noise and highlight only the positive cells that usually are bigger than the others. Be consistent with the number of minimum pixel as this might change the counting.)
§ Results (save on excel file)
§ Always check that you have all and only the positive cells you see in the original image. If some cells disappear, diminish the number of minimum pixel
Updated by EA on Jan 26th, 2010