Red fluorescent proteins suitable for two-color two-photon microscopy and protein tagging

[sjh]

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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?

[Cole, Sara]

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I've had good luck with mCherry but admittedly little experience with 2P excitation. A quick lit search suggests that it's possible but how good the results are, I'm not certain.

Sara

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steven Henle
Sent: Monday, July 01, 2013 10:57 AM
To: [hidden email]
Subject: Red fluorescent proteins suitable for two-color two-photon microscopy and protein tagging

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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?

[Tobias Rose]

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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:[hidden email]] On Behalf Of Steven Henle
Sent: Monday, July 01, 2013 16:57
To: [hidden email]
Subject: Red fluorescent proteins suitable for two-color two-photon microscopy and protein tagging

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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?

[Steffen Dietzel]

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A note of caution concerning the sequential imaging approach,  930->1040
or 850->1000: Most objectives won't be color corrected in that range, so
it is quite possible that the excitation with these two wavelengths may
be several microns apart in z.

For testing this, I'd recommend quantum dots dried on a glass surface:
Excitable with any wavelength and giving a bright signal, of which the
peak is easy to determine in a z-stack. I used 665 nm emission dots, but
others should work to.

Steffen


On 02.07.2013 10:18, Tobias Rose wrote:
(deleted)


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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

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[sjh]

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Thanks Tobias,

I will have to give mRuby2 a second chance. I chose it initially because it
seemed perfect, but was really surprised at how dim it was. Even in single
photon it didn't seem particularly bright, maybe slightly brighter than what I
have seen with mCherry.

To your point about tdTomato not aggregating, the only time I have seen it
was using a CAAX-tag to send it to the membrane, in which case it seems to
get stuck in the golgi or ER. Since the golgi is of interest to me I have by a
little worried.

Also things move too fast for sequential scanning so I am stuck try to excite
both. I mention CFP/YFP because I have seen papers doing this previously. It
may require a little bit of unmixing afterwards though.


Steffen,

I had assumed there would be some difference, but didn't realize it could be
multiple microns off. Thanks for the tip.


Steve

[Tobias Rose]

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I cannot say anything quantitatively right now - it serves my purpose, though. tdTomato is tough to beat - I may compare it more thoroughly at some point. I just cannot use tdtomato because of size issues, otherwise I would.

BTW: Did you use this mRuby2 version from addgene?
http://www.addgene.org/40260/

That thing has 34 AAs worth of tags at its N-terminus (His, T7, Xpress). I got rid of those and start with the GFPized MVSKGEE N-terminus. Not sure if that does anything with the expression but I certainly don't need the tags.

And yes: Steffen is right - one surely should not use sequential imaging if precise subcellular colocalization is an issue.

Tobias

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steven Henle
Sent: Tuesday, July 02, 2013 15:58
To: [hidden email]
Subject: Re: Red fluorescent proteins suitable for two-color two-photon microscopy and protein tagging

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Thanks Tobias,

I will have to give mRuby2 a second chance. I chose it initially because it seemed perfect, but was really surprised at how dim it was. Even in single photon it didn't seem particularly bright, maybe slightly brighter than what I have seen with mCherry.

To your point about tdTomato not aggregating, the only time I have seen it was using a CAAX-tag to send it to the membrane, in which case it seems to get stuck in the golgi or ER. Since the golgi is of interest to me I have by a little worried.

Also things move too fast for sequential scanning so I am stuck try to excite both. I mention CFP/YFP because I have seen papers doing this previously. It may require a little bit of unmixing afterwards though.


Steffen,

I had assumed there would be some difference, but didn't realize it could be multiple microns off. Thanks for the tip.


Steve

[sjh]

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I did use the same mRuby2 construct from addgene, and like you started and the
normal GFP start site. I just rechecked the sequence too and everything looks
perfect. I am setting up to test it again on the 2-photon. Part of the issue may
be the filter I am using (565-610), which is probably not far enough red to be
optimal, but should catch over half of the signal. What filter do you use?

Steve

[Tobias Rose]

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This is indeed a bit on the short side. However, in your test this should hurt mCherry even more than mRuby2; tdtomato should be favored  (emission: mCherry 610 vs mRuby2 600 vs. tdtomato 580).

I use this filter:
http://www.semrock.com/FilterDetails.aspx?id=FF01-607/70-25

Tobias



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steven Henle
Sent: Tuesday, July 02, 2013 18:10
To: [hidden email]
Subject: Re: Red fluorescent proteins suitable for two-color two-photon microscopy and protein tagging

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I did use the same mRuby2 construct from addgene, and like you started and the normal GFP start site. I just rechecked the sequence too and everything looks perfect. I am setting up to test it again on the 2-photon. Part of the issue may be the filter I am using (565-610), which is probably not far enough red to be optimal, but should catch over half of the signal. What filter do you use?

Steve