This software is great - just what I needed for my project. I monitor liposome-loaded rhodamine accumulation in cells, and I’d need to measure the change in intensity over time. So far others in my lab only measured the average pixel intensity of an area which was selected so that the same amount of cells were present in each sample.
I think it’s not a good solution.
I was playing around with imageJ, but it turned out to be a nightmare to quantify each and every one of my images, not to mention that most of the technical terms are not very clear to me -so I’m not even sure if this was done right. (I got some books ordered from the library on image analysis, but it will take time to catch up, and I have to work till then.)
I need general advice on how to proceed (practically right now I’m the only one who does this kind of work, and it’s not in the profile of the lab), and some specifics about the use of cellprofiler. I really apologize, but right now I have no one else to ask. (I’ve spent the last 3 days trying to figure things out with some success.)
which magnification should I use on the microscope for this kind of work: lower, which shows many small cells or higher which only shows 20-40 cells?
I usually start from black to gradual increase in fluorescence; so in order to detect all the cells even when stained weakly I have to use the DAPI images, right? But how? When I only use the rhodamine signal, only 4-9 cells are recognized from about 30. How can I tell the software that the cells detected by the blue signal are there at the red as well?
Should I use the average fluorecence on the image or localize the cells, and get their average? Can I correct it later with the number of cells? (Obviously 45 cells will give off more signal, than 20.)
By setting the treshold to my cells, I eliminate the area of background -so from that on I only measure my cells, right? I tried to do that in cellprofiler, but I can’t see beforhand if the values are good. (In imageJ you can use a slider and see your image change)
I need some help making sense of the results as well: where do I find the meaning of the terms used there?
how do you report the results? What format is acceptable?
it sound stupid, but how do I know how many pixels are the diameter of an object? (The microscope can put a scale on the image; how can I measure it in pixels?)
How do you think my pipeline should look like? (General description; I really don’t expect anyone to do my job for me.) What kind of modules do I need?
I really appreciate anyone’s imput. This is a very exciting field, but an unknown one for me. (I was lookin for a book like “image analysis for dummies” on amazon, but no luck so far…) Thank you very much.