Hello everyone,
I want to do SIM with three different proteins in living cells. Therefore, I need three different fluorescence proteins with high photostability and high brightness? Could anyone suggest a combination (blue, green and red)? Especially I need a good advice for choosing a blue and red FP.
Thanks for your help and your suggestions! Best Andreas
You won't have any luck finding a usable blue fluorescent protein. I would use CFP, YFP, (or their newer variants) and mCherry for three channel live cell imaging with SIM.
Dear Christopher, your suggestions are not really perfect, because they bleach very fast. Do you have tried them in SIM on your own? Andreas
It is difficult to find a suitable blue candidate. Maybe mCerulean3 or dTurquoise will work. For green channel, I would say EGFP is enough for most of the cases, or mNeonGreen (better). For red channel, you can pursue mScarlet.
http://www.nature.com/nmeth/journal/v10/n5/abs/nmeth.2413.html
http://www.nature.com/nmeth/journal/v14/n1/full/nmeth.4074.html
Good luck!
I've done a lot of SIM, but never 3 colors with fluorescent proteins. In fact, I don't know anyone who has done this because the acquisition times of several commercial SIM systems are slow (with the exception of OMX) combined with the light dosage from 3 channels, each with 15 images per plane to get a 3D-SIM reconstruction, means you will take 45 images to get one image of each channel. You will need strong expression in all three channels to have any chance of success and this is hard to do.
I would look at some of the modern variants of CFP and YFP, one of which was pointed out by Bei, to find ones resistant to photobleaching.
If you do go with green, check out mMaple. It was published to work well with SIM, but EGFP will also be OK.
thanks ... maybe it is better to start first with two fluroescence proteins. Does anyone have used mRuby3 for SIM?
Find the "brightest" FPs and choose those - http://www.fpvis.org/FP.html
mNeonGreen looks good.
Modern CFPs are mTurquoise2 and/or mCerulean3, CyPet or mVenus for yellow. mCardinal or mSmurFP maybe for red
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