Hello All,
I would like to ask if anyone know if measuring mitochondria number in cells could be feasible by using STORM microscopy to detect a specific fluorescent mitochondrial marker. If yes is there any particular marker that you recommend using? Or alternatively anyone has good suggestions to enumerate mitochondria in cells using other methods? Many thanks in advance.
Tom70 and Tom 20 may be good as markers.
Theoretically it is possible, but practically it is going to be quite challenging. First, 3D imaging is required (this increases the data load and complexity of STORM). Then you have very huge data to register and segment. Then the counting is unlikely to be possible with automation.
Rather than high-end imaging, the old fashioned biochemical mitochondria isolation (followed by counting under a sufficiently powerful microscope) could well be more feasible and less expensive. You compensate for the low-tech aspect by repeating to improve confidence.
protocols for isolated mitochondria in FACS exist.
Besides, STORM microscopy would require to fix the sample, therefore you will not be able to measure the number of mitochondria over time, but only in the specific moment in which you would fix your sample. Since mitochondria undergo many processes of fusion and fission, I'm not sure how reliable that measure would be.
Correct me if i am wrong, but wouldn't live cell storm observer the mitochondria number over time?
http://www.imaging-git.com/science/light-microscopy/live-cell-super-resolution-dstorm
It depends on many things, like choosing the right fluorophore which allows for long acquition together with maintaining live cell conditions (buffer and such) and low phototoxicity. Besides, there's also the fact that you would still need to acquire many images (e.g. hundreds/thousands) in order to obtain a super resolution image, therefore the frame rate could not be feasible for this application.
In principle it is possible, but it will be rather challenging
You are right Lorenzo, live STORM is significantly more difficult compared to imaging fixed cells. But it can be done at video rates.
http://www.nature.com/nmeth/journal/v10/n7/full/nmeth.2488.html
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