Quantifying fluorescence in zebrafish

[johnmoreman]

Hi

I wondered if anyone had any experience using ImageJ to provide a quantitative/semi-quantitative value for fluorescence. I have some transgenic zebrafish which express GFP in different organs and ideally wanted a fairly simple and cheap method to compare the intensity of fluorescence between individual fish, using either captured images of the whole organism or more detailed pictures of specific organs. I'm very new to ImageJ it was just suggested to me that it might be possible to do something like this, though I have found no clear solution as of yet.

Any help would be greatly appreciated

Thanks

John

[John Oreopoulos]

Hi John,

Your question is a very general one that gets asked about 2-4 times a year on the ImageJ email listserver. (There really should be a permanent link on the ImageJ website FAQ for this question.) Quantifying and comparing fluorescence intensities acquired with a microscope is possible using ImageJ, but you have to make sure the data is collected carefully and properly first (and this is usually not trivial!). I would highly recommend you get your hands on and read the following published articles on this topic:

Pawley, J., The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. Biotechniques, 2000. 28(5): p. 884-888.

North, A.J., Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. Journal of Cell Biology, 2006. 172(1): p. 9-18.

Waters, J.C., Interpreting fluorescence microscopy images and measurements. Cell, 2008.

Waters, J.C., Accuracy and precision in quantitative fluorescence microscopy. Journal of Cell Biology, 2009. 185(7): p. 1135-1148.

Then, after that, take a look at the excellent tutorials on the ImageJ Wiki and the FIJI website (and others as well):

http://imagejdocu.tudor.lu/doku.php?id=video:start
http://pacific.mpi-cbg.de/wiki/index.php/Colocalization#Colocalization_Analysis_-_What_is_Colocalization_anyway.3F
http://rsbweb.nih.gov/ij/docs/examples/index.html
http://www.fmhs.auckland.ac.nz/sms/biru/_docs/Image_Analysis_Basics.pdf

There was another excellent link someone posted a while back about basic intensity measurements via ImageJ, but now I can't find it. Try searching the email archive and you might find it.

Unfortunately, there is no one recipe for analyzing the data that you collect with your microscope system since every imaging situation and sample is different and usually the researcher has very specific questions they want to answer with the analysis. You'll have to find out what methods and plugins work best for your data. Search the plugin list and see if someone has developed a specific tool for your application already (in this case specific organs labeled with GFP in zebrafish).

Cheers, and good luck!


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada


On 2011-08-18, at 7:50 AM, johnmoreman wrote:

> Hi
>
> I wondered if anyone had any experience using ImageJ to provide a
> quantitative/semi-quantitative value for fluorescence. I have some
> transgenic zebrafish which express GFP in different organs and ideally
> wanted a fairly simple and cheap method to compare the intensity of
> fluorescence between individual fish, using either captured images of the
> whole organism or more detailed pictures of specific organs. I'm very new to
> ImageJ it was just suggested to me that it might be possible to do something
> like this, though I have found no clear solution as of yet.
>
> Any help would be greatly appreciated
>
> Thanks
>
> John
>
> --
> View this message in context: http://imagej.588099.n2.nabble.com/Quantifying-fluorescence-in-zebrafish-tp6699068p6699068.html
> Sent from the ImageJ mailing list archive at Nabble.com.