TagRFP-T performance in vivo 2p

[Tobias Rose]

Dear all,

I am desperately looking to exchange our currently used tdtomato with a smaller
and more bleach-resistant red protein (2p excitation, in vivo imaging, cortex).
Tsien's TagRFP-t (<http://goo.gl/hTUR2>http://goo.gl/hTUR2) is the obvious choice - however so far
there is surprisingly little published with this protein. Even though it looks perfect
on paper I am therefore a little reluctant to generate expensive viruses with it.

Does anyone of you have experience concerning performance and especially
toxicity of TagRFP-T - ideally with expression in sensitive mammalian neurons?

Thanks,
Tobias

[Urs Utzinger]

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Tobias
Using wavelengths >1100nm for excitation (TiSaph & OPO) would be advantageous compared to excitation with TiSaph alone.
Also bleaching of some (perhaps all) red FPs is increased with 2p compared to confocal imaging when you consider bleaching in a single imaging plane alone.
Sorry I can not advise about which red FP to choose as replacement.
Urs Utzinger
Two papers about using OPO&TiSaph for red fluorophores
http://www.ncbi.nlm.nih.gov/pubmed/19324541
http://www.ncbi.nlm.nih.gov/pubmed/19654740

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tobias Rose
Sent: Saturday, August 06, 2011 9:44 AM
To: [hidden email]
Subject: TagRFP-T performance in vivo 2p

Dear all,

I am desperately looking to exchange our currently used tdtomato with a smaller and more bleach-resistant red protein (2p excitation, in vivo imaging, cortex).
Tsien's TagRFP-t (<http://goo.gl/hTUR2>http://goo.gl/hTUR2) is the obvious choice - however so far there is surprisingly little published with this protein. Even though it looks perfect on paper I am therefore a little reluctant to generate expensive viruses with it.

Does anyone of you have experience concerning performance and especially toxicity of TagRFP-T - ideally with expression in sensitive mammalian neurons?

Thanks,
Tobias


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[Tobias Rose]

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Dear Urs,
Yes: an OPO would be nice - according to this paper tdtomato (or in this case
tdiner2(12)) excited at 1100nm is more bleachresistant than GFP: http://goo.gl/xwerL
However, an OPO would add >100K EUR to a single setup...
I am looking into a different option right now. The solid state Ytterbium lasers
are mug cheaper (limited to 1050nm, though) and one could choose one with a low
repetition rate (20Mhz instead of 80). Since I am using a fast 8KHz reonant scanner this
should bring me roughly into the DREX regime (http://goo.gl/A65lF)... Hopefully I can get
my hands on a test unit to check it out.
Best,T