Am using ImageJ to count my GFP cells. Is there some information online or from someone who can help guide me in the right direction to getting it setup. Ideally the software should be able to detect false positives and fully fluorescing cells.
Dear Daniel,
please take a look here:
http://imagejdocu.tudor.lu/doku.php?id=video:analysis:particle_counting_-_automated_and_manual
To differentiate between all cells and GFP positive ones you might use Hoechst33342 to mark all cells. I'm not shure what you ment with false positive onces? After thresholding your cells your might also include a size filter. I would also recommend to take (depending on your microscope also automatic) a number of pictures with the same exposure time and then process them as stack. I ussually also keep the out lines as a control of my counting of partikels (cells or anything)
May be you want to provide example data (images) to rule out some problems.
Best Soenke
Dear Daniel,
I wonder why you chose to go that route? Counting GFP cells is very easy with flow cytometry and you should be able to get access to one of the cytometers at UCL easily (they were going to set up a core FACS unit at the Cruciform Iast I heard about this a year or two ago). This would give you much more accurate data...
First you will need to set your threshold for your 8bit grey-scale images (image--> adjust-->threshold), which should be held consistent throughout your images. (make sure you use your negative control to set your false positive settings both for imaging and analysis). Tell ImageJ what you want to analyze in the "set measurements" dialog box (you can capture area, perimeter, IntDen, basic morphology...). Determine what size and shape (circularity) all of your true GFP+ cells are and set these metrics in the "analyze particle" tool (Analyze--> analyze particles). You can also decide if you want summary data of the entire field/ROI or info on every cell analyzed. This will also work in a ROI if you outline a specific area. Once you know what you want and set everything up you can record the process as a macro and run in quickly through multiple images (just be sure your automated version is giving you what you want; I suggest counting a subset by eye for comparison).
Thanks for the replies. I should have rephrased my question. Its e.coli expressing gfp, so my false positives are when e.coli is growing on agar with antibodies but the vector it took in did not contain the gfp gene.
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