fluorescence intensity measurement

[ylckyoto08]

Hi all,

I am trying with ImageJ to measure red fluorescent spots (against black background) in order to compare the intensity between several samples.  Could someone kindly tell me the detailed protocol how to do that?  I have searched through the ImageJ Manual but there isn`t specific instruction on fluorescence measurement.  Your help would be greatly appreciated.  Thanks!

[Rina]

Hello,
I used to that using this way:

duplicate image, to get (1) and (2)
select (2) => process => substract background
with (2): set threshold => apply
with (2): edit => selection => create selection
with (2): edit => selection => add to manager
go to (1) => click measure at the ROI manager window
“Mean” shows the gray level of the particels
with (2) => edit => selection => make inverse
delete number in ROI manager
with (1) => edit => selection => select none
go to ROI and click measure (shows the gray level of the background, so if you substract it from the first number you got your intensity)