I have infected BHK21 cells with a virus that expresses GFP. I want to count the percentage of GFP positive cells and the intensity of the GFP. How do I do this with fluorescent microscope pictures and imagej?
Hi
I only know how to quantify the cell fluorescence, but I think maybe even that could already help you?
- Firstly, open ImageJ.
- Select the cell of interest using any of the drawing/selection tools (i.e. rectangle, circle, polygon or freeform)
- From the Analyze menu select “set measurements”. Make sure you have area integrated intensity and mean grey value selected (the rest can be ignored).
- Now select “Measure” from the analyze menu. You should now see a popup box with a stack of values for that first cell.
- Now go and select a region next to your cell that has no fluorescence, this will be your background.
- Repeat this step for the other cells in the field of view that you want to measure.
I hope i could help
Art
You may be able to automate a bit the procedure proposed by Art.
-Duplicate the image you want to analyse.
-Image/Adjust/Threshold opens a dialogue for automatic setting of threshold. It offers a few algorithms, if the Default doesn't work well for your images, try others. It will result in binary image, where the GFP expressing cells are white and the rest is black. To reduce noise (that would create small white speckles outside of cells and small black speckles inside of cells) apply first a Gaussian blur (Process/Filter/Gaussian blur), play with the radius to remove noise without blurring everything our.
- You may have problems if you have high cells density, so several cells will appear as a single white region. Watershed (Process/Binary/Watershed) may help to some extent. In that case you won't be able to measure intensities in individual cell, but rather get an average over all cells that form one cluster.
-Analyse/Analyse particles will find all the cells (white regions) and perform measurements on them. Check the Add to Manager option to save each cell as an ROI in the ROI manager. You can also set limits on the cells area to avoid some residual white speckles outside of cells (e.g. from fluorescent cell debris).
-Select the original (not binary) image as active and select all ROIs in the list in ROI manager and run Measure. You will get a list of average all ROIs with corresponding areas and average intensity in the Results window.
-The whole process can be automated by recording it as a Macro and then running in batch on all images, which can help if you have a lot of images to process.
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