*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Steven,

I have some qualitative experience with mRuby2 now (chronic cytosolic expression [several months] and 2p imaging in cortical neurons in vivo) and can say that I'm so far quite happy. In contrast to your experience I find it very bright - especially at 1040 where it should be. In my hand it compares nicely to tdtomato in the same application. mRuby2 has the huge advantage, though, of being much smaller (AAV packaging!)  and of actually being a red protein (emission is more like mCherry and not tdtomato) . In terms of bleaching I find it slightly superior to tdtomato. I used tdtomato extensively in vitro (cytosolic and tagged expression) and never saw any aggregates there. I did not do the same tests with mRuby2 but will do so soon and report back. Concerning cytotoxicity it looks good so far - I would not feel comfortable saying the same about tag-RFP-t, though...

For simultaneous dual color imaging using only one excitation wavelength it is not ideal but quite doable: GFP/mRuby2 can be co-excited around 960nm - however, mRuby2 is far from optimal excitation there. Still: I find it bright enough for my purpose. If you can afford sequential imaging, then this does not apply, of course (930->1040). I cannot say how much light you lose by using a narrow filter CFP/YFP coexcitation approach. If you can afford doing two imaging runs: mTurquoise2 and mCitrine may be good candidates for sequential blue->yellow dual color imaging (850->1000) using a very broad single filter / single channel approach.

Cheers,
Tobias

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Steven Henle
Sent: Monday, July 01, 2013 16:57
To: [log in to unmask]
Subject: Red fluorescent proteins suitable for two-color two-photon microscopy and protein tagging

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Does anyone know of a good monomeric fluorescent protein in the red range that is also good for two photon excitation with a green/yellow protein. The bright red fluorescent proteins I have used (DSred and tdTomato) have some tendency to aggregate what tagged, although tdTomato may be ok. I tried mRuby2, but going all the way out to 1040nm, I could barely see it, and can't find any previous work using mRuby or mRUby2. I have considered using mOrange2 or TAGRFP-t, does anyone have experience with these. I have seen papers reporting the two photon spectra, but they are in solution and don't always match what I have seen in vivo. Alternatively is better to just invest in CFP and YFP filters, since I know there are good monomeric proteins to use for dual-color two-photon imaging there?