Now, I am using FIJI to analyze the fluoresence intensity in images from an images stack.At first, i use threshhold to select the ROI, then set the parameter of measurement, finally,click measure icon.However, there are follwoing problems:
1,Only one slice is to be done.However, the images constitute a image stack. Therefore, it should be repeated for several times to finish all the images(in one images stack) analysis. Is there shorcut for processing this?
2,The image to be analyzed is showing as the following.If i would like to analyze the fluoresence intensity.Is it required to substract the background fluoresnce intensity?If yes, how to do that?
Hello Ts (is this your real name?),
sorry, I have to revise my answer, you are of course right! If you wish to measure intensity, you want to use the "sum" not the "maximum" projection.
you can either make a z-projection of your stack and measure your ROI there. What projection you use depends on your task, the maximum projection takes the maximal pixel value found in z-direction for every pixel P(x/y) and is often used.
Images --> Stack --> Z-Project...
or if you really need to have the intensities of each slice, you could make a loop using the imageJ macro language and measure the slices separately.
background subtraction is found here:
Process --> Subtract Background...
However, I suggest you make a maximal z-projection and then follow the instructions from this link:
http://www.slu.se/PageFiles/388774/Pacho%20ImageJ%20measuring-cell-fluorescence.pdf
Hello Ts (is this your real name?),
sorry, I have to revise my answer, you are of course right! If you wish to measure intensity, you want to use the "sum" not the "maximum" projection.
yes, you are right! TS is the abbreviation of my first name.
According to your opinin, if the fluorescnece intensity from one image stacks is need to be analyzed. the first step is to sum the images into one image?it is not necessary to analyze one by one?
Another question...Apparently, the background of all images from confocal microscopy is dark. If there is no or not much noise signal in the background, so the intensity of the background can be ignored?it is not necessary to substract the value of backgound?
Thank you again
Usually the z-Projection is preferred over measuring one-by one; it gives you the same result but is much more comfortable. You would make it slice-by-slice if you wanted to know the intensities for each slice.
So, your signal-to-noise ratio seems to be optimal, then you can probably ignore the background subtraction. With the wrong settings it could anyway eliminate signal. In the protocol I sent you, they use a background ROI to estimate the amount of background signal
Good luck! :-)
Thanks a lot!
I have a misunderstaning on the second question. Actually, I use threshhold to select ROIs which make the outeside of ROI as background. Of course, the value of the "background" is zero!!!
Consequently, another question appear...
If the ROIs is selected by threshhold like the follwing images, so the background can be ignored?
Do I get you right? You want to measure the intensity within a mask that you obtained by thresholding? Do you want to measure the intensity of single particles or of the whole image?
I would do something like:
1) copy the original image - keep it open, you need it later to do the intensity measurements with.
2) with the copy you do a background subtraction, and maybe apply a slight gaussian filter. This will help you with the thresholding.
3) You threshold using an appropriate value - maybe also consider other threshold algorithms, otsu for example
4) After thresholding you should get a binary image
5) With this you maybe also want to apply a slight median filter and use a binary closure to get nicer outlines.
6) Then you convert your binary image to a mask and measure the particles. But first you need to define the original image from which you measure the intensity values.
The link above is out dated.
New link is
http://www.slu.se/contentassets/a454886b9b154b189ae2a7ded6baa4db/pacho-imagej-measuring-cell-fluorescence.pdf?si=3DB4E043D825F6E1513677D2512296DB&rid=1141616124&sn=sluEPi6-prodSearchIndex
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