Photoshop is great for image processing, and I have been using it since the late 1990s, but I would recommend using ImageJ, or another dedicated image analysis software, for analysis. You can download it for free. Do you want to measure fluorescence intensity?

Some free image analysis software tools links:

https://digitalpathologyplace.com/6-free-open-source-software-programs-for-image-analysis-of-pathology-slides/

I highly recommend QuPath (https://qupath.github.io/) to analyze your IHC images. It is a free, open-source program for histology image analysis, and includes tools for cell segmentation and IHC stain quantification.

Hello Muhammad Ameen Jamal

I agree with John Hardy Lockhart

QUPATH software is most relevant one for the quantification of Immunohistochemistry(IHC) images.

You can also try ImageJ (https://imagej.nih.gov/ij/download.html)

For Reference:

https://doi.org/10.1038/s41598-017-17204-5

http://hdl.handle.net/10380/3211

I hope this would be helpful.

Bundle of Thanks

@John Hardy Lockhart@Charles Immanuel David@Bhairavi Vajaria@Alexandros A Lavdas

Yes it is quite easy. First separate the 3 channels of color and save as grey scale image. Then click on the intended grey scale image to get the grey scale value of at least 30 points. Get the average and you can compare t-values from these 30 points in microsoft excel. you can click on different regions of the same image to compare differences. For separate images you have to normalize the grey scale value. That is find a background grey scale value and divide the whole images with this background and then get the new grey scale values and then compare. I have done the color channel separation and grey scale image by Adobe photoshop 7.0 (free) and then calculate the grey scale values with imageJ (free). I have been using the same Adobe photoshop 7.0 (free) for the last 25 years without ever upgrading! Hope this helps. Regards

You cannot quantify fluorescence intensity in any meaningful way. There is not a linear relationship between protein levels and intensity of the signal produced by your fluorescent markers. THIS IS BAD SCIENCE. Please see Article The 39 Steps: A Cautionary Tale of Quantitative 3-D Fluoresc...

John Griffin Bundle of Thanks Sir......

If I may may add something after reading Mr Griffin's comment: of course one can quantify fluorescence intensity in a meaningful way, and that is for comparisons between different conditions. And - as this introductory paper that he shared alludes to - this should be done with all other parameters unchanged. I agree that there is no linear relationship between protein levels and intensity of the signal produced by your fluorescent markers, but this is important only if one is trying to quantify protein levels. In such a case, you would need a curve that would help you interpret your results. But if you have cells treated with agent A or agent B, or carrying a mutation A and a mutation B and you want to compare between A and B, you can. It's fine as long as you are keeping all parameters unchanged, which means not just that they have the same optical and digitization settings, but also that they have been processed together in the wet lab.

The problem, Alexandros A Lavdas, as noted in that paper, is that there are too many variables to control adequately. First and foremost you will have photobleaching from simply locating your samples and this will be different from sample to sample and can be significant even if you know how to mount your samples properly or if you use oxygen scavengers. You can compare morphology, but it is not proper to compare fluorescence intensity. There is no curve that can fit all of the many variables, which Dr. Pawley admits are too many to enumerate. All you can do is perhaps set a threshold and say that any signal above that arbitrary level counts as signal and anything below as background. This is also part of why colocalisation studies are not actually valid. How do you account for resonant energy transfer that is then transformed to thermal modes, for instance?

I know this throws a spanner in the works of many researchers, but we are meant to be getting at the truth here. The truth is that these methods were developed by and large by biologists who don't consider the chemistry and physics underlying these tools.

Mr Griffin, the paper is a useful list of the variables, for people who are new to imaging. It does not in any way invalidate comparative studies. Maybe the best way to describe such studies is "semi-quantitative". They don't tell you how much protein you have but they can tell you in which direction a change is taking place, after statistical validation. Same with colocalization, and there are algorithms for that, for example in Imaris. Thinking that all colocalization studies are useless because they do not account for "resonant energy transfer that is then transformed to thermal modes", is missing the forest for the trees. We agree that caution is warranted, but let's not go to extremes, that's all I am saying.

Alexandros A Lavdas Bundle of thanks

Alexandros A Lavdas, having read the article several times myself over the years, the conclusion that the author draws is significantly different to the one you draw. What he says is that you cannot possibly account for all of the uncontrolled variables, that the list of variables he provides is far from exhaustive, and that due to this fluorescence brightness levels are non-quantifiable. Just because people have written deeply flawed analysis code and it is accepted by the Biology community is not proof that it is correct. Rather, I see that it is proof that most people have no idea what they're doing. Where is the research that establishes that, despite Dr. Pawley's assertions, fluorescence levels are indeed quantifiable? With respect to genetically encoded reporters, how do you even know how many copies of the gene each cell has? Surely that has an effect on fluorescent brightness. Finally, there is a better word for "semi-quantitative": qualitative.

John Griffin. Well, you are certainly free to believe that "most people have no idea what they're doing".