Cardiac cells were incubated in various experimental conditions. Cells were stained with fluorescent mitotracker and nuclear dye.
I am having difficulty quantifying mitochondria from these images. Is there a way I can use these images to compare mitochondrial differences between conditions?
I will attach an example pictures at various magnifications.
I appreciate any suggestions.
Hi Jacob,
You could use image analysis to identify mithocondrial area and nuclear area. This would allow you to measure area, area ratios and combine this with intensity measures for stain expression (Area ratio x intensity). This would allow you to compare samples where the reaction is expressed as an intensity shift or area increase/decrease. Let me know if it is something you want to try out with our solutions.
Best regards Martin
The 2nd one (lower magnification) has multi-layer. I think it is too difficult to adjust the intensity when cells are in different layer. Optical section using confocal microscopy may be better than that of compound microscope.
It depends on your purpose, if you want to see the oxidation stress on mitochondria, MitoSOX is better (sharp image) than mitotracker. Some of mitotracker is fixable, but both mitotracker and mitoSOX are usually fixation-sensitive.
I prefer to use Image J. You can label cells by DiO. Because your cells have various shapes, so nuclei staining doesn't help the quantification. Well, you can know the number of the cells.
Using Green channel, you can cut out only cell area. Later you could know how many perticles/ (Area/cell numbers=single cell area). Alternatively, you can remove cell debris manually. Then, using Red channel, you could quantify like this:
1. Set scale > Measure several particle size, then use "Analyze particle"
You could do thresholding by size of signals. You may need to adjust "circularity" to avoid miss-counting of the particles.
2. Threshold by intensity > Measure area
Interpretation of data is up to you.
If you can use transfection of virus or whatever, then you can use MitoBFP, it is much easier to quantify activation of mitochondria.
Hello
Kindly look at links here
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3007120/
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.21061/full
http://www.sciencedirect.com/science/article/pii/S0167488914003917
http://www.nature.com/nprot/journal/v11/n9/abs/nprot.2016.094.html
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence