I did immunohistochemistry for brain capillaries and I am looking for an accurate way to quantify the number of capillaries and if they are affected by a certain treatment.
Which software I should use and how should I do it.
Thanks
There are a few options for software. I like using ImageJ because it is versatile, well documented/published, easy to learn, and open source. Because you are doing IHC and therefore stain a specific cell type of interest, you can use the software to isolate those features and/or colors you want. You can, of course, count or trace and measure the features in each image yourself. Time consuming but doable. However, if you code the software to isolate and measure parameters based in basic stereology, then you can reduce your processing time, increase your output, and decrease bias. Sounds straightforward, but you will likely go through some trial and error to get a code that consistently works.
I agree with Natalia about looking in to stereology. This will help you to refine your methods, define your parameters and design your code.
I recommend:
Howard, C. V. and M. G. Reed (2004). Unbiased stereology: Three-dimensional measurement in microscopy. New York, NY, Garland Science.
Russ, J. C. (1986). Practical stereology. New York, NY, Plenum Press, New York & London.
Good luck!
-Melissa
also:
https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html
Article ImageJ for Microscopy
Article Stereological tools in biomedical research
Article Stereology, morphometry, and mapping: The whole is greater t...
Article Stereology: A Method for Analyzing Images
Article Image Processing with ImageJ
The best way is to perform stereology, if you sample the tissue in the particular manner.
Dear Dr.Schlabritz-Loutsevitch
Thanks for your recommendation I will check the stereology technique since I'm not familiar with it and I guess I need to have a special stereology sysyem at our lab but thank you.
There are a few options for software. I like using ImageJ because it is versatile, well documented/published, easy to learn, and open source. Because you are doing IHC and therefore stain a specific cell type of interest, you can use the software to isolate those features and/or colors you want. You can, of course, count or trace and measure the features in each image yourself. Time consuming but doable. However, if you code the software to isolate and measure parameters based in basic stereology, then you can reduce your processing time, increase your output, and decrease bias. Sounds straightforward, but you will likely go through some trial and error to get a code that consistently works.
I agree with Natalia about looking in to stereology. This will help you to refine your methods, define your parameters and design your code.
I recommend:
Howard, C. V. and M. G. Reed (2004). Unbiased stereology: Three-dimensional measurement in microscopy. New York, NY, Garland Science.
Russ, J. C. (1986). Practical stereology. New York, NY, Plenum Press, New York & London.
Good luck!
-Melissa
also:
https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html
Article ImageJ for Microscopy
Article Stereological tools in biomedical research
Article Stereology, morphometry, and mapping: The whole is greater t...
Article Stereology: A Method for Analyzing Images
Article Image Processing with ImageJ
Thank you very much Melissa for this very informative and helpful response.
I will go through the papers since I have the ImageJ and hopefully I can get an access to the stereology techniques.
Thanks and best wishes
Sweilem
Stereology is the best way to go as suggested above. The articles suggested above is too broad for you to perform stereology of capillaries. Some articles which deal with your specific question are:
- Nature Protocols, VOL.10 NO.1, 2015 pg. 53
- Journal of Microscopy, Vol. 206, Pt 1 April 2002, pp. 54–64
You have to follow certain steps for stereology:
- Systematic random section selection
- Systematic random sampling (imaging) within you region of interest
- Estimation of reference volume of your region of interest (you can use stereology for this too)
- Tissue shrinkage (very important factor while estimating length (L) or Length per unit volume (Lv)
There is fantastic information on the web from various vendors:
- https://www.stereologyresourcecenter.com/src-stereology-innovations/space-balls/
- http://www.stereology.info/length/
Let me know if you need some guidance with the estimation. Good luck.
Sathya Srinivasan
ONPRC
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