I am trying to count the number of sperm cells in a fluorescent labelled image. After using the multi-point tool when I click measure the area column for all the selected cells is ZERO. I need that measure to calculate the CTCF(Corrected Total Cell Fluorescence) as
CTCF = Integrated Density - (Area of selected cell X Mean fluorescence of background readings)
I am neither using Thresholding (since I don't know why should I use it ) nor have I converted the image to binary. (Again. I don't know why should I use it )
Please Help.
P.S. Is this the correct way to quantify the fluorescent intensity?
Multi-point tool marks the locations where you click. It's literally a 'location' (with sub-pixel precision), not a area. You may want to try the combination of regular ROI tools (like rectangle, oval, polygon, etc) and ROI manager (under Analyze->Tools->ROI manager). If you really want to select only one pixel for each cell, try wand (tracing) tools next to the multi-point tool. After you draw a ROI, simply press t to add it to ROI manager. BTW, I'm using Fiji/ImageJ.
@Bei Liu Thank you, I did that and was able to click measure and obtain the intden as well as raw inden. However, on a different note could you please let me know where can we incorporate background fluorescence values? and Do I need to Threshold the images, and if yes please let me help understand it, and also to convert the the images (e.g. to binary), and if yes, please let me help understand it ?
Vipul Batra May be you will find this link useful:
http://vlab.amrita.edu/?sub=3&brch=278&sim=1474&cnt=1
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