Quantification of Fluorescent images - (Feb/13/2013 )
Does any one have some experience in the use of softwares for quantitative comparison of images taken using fluorescence microscope.
I have no experience using these software ever.
what is the best and easiest software?
I wonder if u could share ur experience with me, or share any books or papers for beginners like me?
Thanks in advance guys
Fiji is what I use. It is ImageJ with all the necessary add ons. I provided a link below, detailing a general measurement method. Your method will depend on what you are trying to determine. The program is easy to use and depending on the quality of the images, it is even easier to find your total fluorescence. Let me know if you need any help with the program.
*One thing I do differently is load the same image twice (into Fiji) and then convert one of the images to black and white. With this black and white image, you can adjust the threshold of the image to obtain a very definitive border around each cell. Merge these two images and then outline all of the threshold cells (magic wand, shift + mouse click). Scroll over to your other image and you should have a very clean outline of each cell. You may do the same thing with nuclear vs. cytoplasmic staining if you have stained your nucleus.
You can also try the MBF set of imageJ plugins
Thank u very much this blog is very helpful,
I have a question for u:
If I want quantify the Reactive oxygen species using Fluorescence agent, I would want to calculate fluorescence inside the whole cell .
My question which is more meaningful, using high magnification images (100X, 40X) or using low magnification (20X).
from my point of view and my pic: low magnification is better, give me a lot of cells in the same pic, besides I can see fluorescence better in low magnfication pic, but not details, as I dont choose fluorescent spot but the whole cells
or it is more meaningful to use high mag pictures and analyze.
Thank u for ur advice, I already download this program, but as I have no experience in using it, I wanted some advice, links, PdF or tutorials regarding how to use this program, I have found a couple of tutorials online, but they have not cover all my question yet.
I would honestly try both. Analyze your low images to show that your staining shows a high amount of staining in all your cells, indicating your staining method is successful. Get a total cell fluorescence of all your cells at the low magnification and then go in for a snap shot at high magnification. If your staining seems to be clustered at one point of the cell, it would be extremely interesting for you to measure the high degree of fluorescence at that position within the cell and then correlate it to the staining at other portions of your cell.
I would just measure the staining that you feel expresses your overall point. Definitely look at both magnifications, as one magnification may lead to a more intriguing result.
Edit: If you are looking for additional resources to learn about ImageJ staining, use Youtube.
Thanks for helpful advice