I agree with Abdullah Sheikh, Image J is really a good option for doing what you asked for. You can easily define a line or an area of interest and just press the Ctrl and M keys simultaneously to obtain the fluorescence intensity along that line or in this area. A "measure" window will then pop up with the results, and from there you can also adjust exactly the type of measurements you wish to have (min and max or only the mean, etc...). There are a lot of tutorial online if you need, for example here: http://sciencetechblog.com/2011/05/24/measuring-cell-fluorescence-using-imagej/

You can easily quantify the fluorescence inteisities using Image J. It is a free soft, just follow the instructions.

Have a look here

https://www.researchgate.net/publication/256472880_Arques_et_al._Standardized_Relative_Quantification_of_Immunofluorescence_Tissue_Staining_Protocol_Exchange-2012?ev=prf_pub

Data Standardized Relative Quantification of Immunofluorescence T...

I agree with Abdullah Sheikh, Image J is really a good option for doing what you asked for. You can easily define a line or an area of interest and just press the Ctrl and M keys simultaneously to obtain the fluorescence intensity along that line or in this area. A "measure" window will then pop up with the results, and from there you can also adjust exactly the type of measurements you wish to have (min and max or only the mean, etc...). There are a lot of tutorial online if you need, for example here: http://sciencetechblog.com/2011/05/24/measuring-cell-fluorescence-using-imagej/

ImageJ is from NIH and you can find many citations.

some tutorial: http://imagejdocu.tudor.lu/

I also use ImageJ (now its update as FIJI) for quantifying pixels on my pics. It's a free powerfull and smart software. Here is the link to download the soft. http://fiji.sc/Fiji. Have fun with your analysis. Thomas

Like many others have said, there really isn't anything better than Image J for image analysis of this type. There are other software that cost a lot of money (1000$ +), and have a really slick user interface, but after using various image analysis software, Image J appears to be more consistent even if it is not very user friendly.

If you know how to program the macros, it's very useful and saves a lot of time if you have to analyse hundreds of images. One of the advantages of the expensive software is that it has a lot of automation built in. With Image J you have to either program the macros your self, or find/download macros from other researchers.

I also suggest to use ImageJ / Fiji! A huge community of ImageJ users can also help you a lot in finding solutions to your problem. As reported by others, automation of the measuring process is possible, but of course it's not so easy to do that, as a beginner!

Let me add something about the images you already have: to get a reliable quantification, it is necessary that all of them are taken with the same camera settings (exposure time, gamma, saturation... ), or at least that you know exactly which parameters you used, and that you don't have saturated pixels (or at least not so many).

Then I guess that in the same picture you have more than a single C. elegans, so that you can't simply measure the whole fluorescence intensity from one picture, and compare with other pictures (unless you normalize the fluorescence per number of C.elegans in the picture). You will then need to select an area corresponding to a single C.elegans within the image (and that you can easily do with ImageJ/Fiji), and then measure the fluorescence intensity.

I have cultured and immunofluorescence stained the HEPM cell line on 3D particles of newly developed bone graft, how can I quantify fluorescence intensity in photos? is the Image beneficial here please?