Hi!
Have anyone ever tried single protein tracking (let's say a secretory protein) but also see its relative position to other organelles such as ER and nucleus (labelled by other fluorophores)?
I read a few papers but they only track the protein without visualizing organelles. e.g. Liu et al. PNAS 2018.
P.S. I'd like to do it 3D in live mammalian cells but (I guess) I don't really need super-resolution as I just want to roughly see the localization of single proteins?
Thank you!
HY
Hy Wo, It looks you are intended to do tracking and co-localization. This can be done with confocal. There is no need to use super resolution as you can scan X,Y and Z coordinates in confocal. Please note that super resolution imaging has color limitations (depends on manufacturer/model) and use high power laser. You may have to compromise the viability of the live cell in confocal as well as super-resolution imaging, however, confocal imaging is faster and better for co-localization studies. In case you need precise molecular co-localization (below 100nm) you may use self healing SRM dyes and 3D-SIM or 3D-STROM technique.
Hi Venkatesh Rengaswamy ,
Thank you! I later found some papers which allow overlaying single particle trajectory with organelles, such as Perillo et al. 2014 Nat Comm. Their setups are indeed based on confocal microscopes.
However, such setups deploy motorized stage/laser beam that receive feedback from previous image frames to "lock-on" the target particle. I'm not so sure if I can get this in my institute. I know there's a lab in nearby department which has this setup but need to check if they'll collaborate with us.
On the other hand, I have easier access to 3D-SIM and 3D-STORM microscopes, although I don't need precise co-localization at this moment. Do you know if they can be used for real-time co-localization? Thanks!
HY
Hy Wo, you can definitely use SIM or STORM for the tracking and co-localization analysis. Real time 3D live cell imaging is possible with both the methods, although 3D-STORM or N-STORM would give precise axial and lateral resolutions. You may try with activator-reporter dye pair or SNAP-tag. The success of live cell STROM imaging is dependent on the right buffer.
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