Best FPs for 2-Photon

[Jens]

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Dear List,

a colleague of mine wants to do 2 channel imaging on a 2-photon system with a max 1040 nm laser.
What is the "best" fluorescent protein combination right now in terms of brightness, photostability and maturation time for this application?

Thanks so much!

Jens

--------------------------------------------------------------
Jens B. Bosse Ph.D.
Enquist Lab
Department of Molecular Biology
and
Princeton Neuroscience Institute
Princeton University
301 Schultz Lab
Washington Rd
08544 Princeton, NJ, USA

Phone: [phone]
Email: [hidden email]
Web: http://molbio.princeton.edu/labs/enquist/

This electronic communication, including any attached documents, may contain confidential and/or legally privileged information that is intended only for use by the recipient(s) named above.  If you have received this communication in error, please notify the sender immediately and delete the communication and any attachments.

[Douglas Richardson]

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We primarily use GFP and tdTomato, they are both excited very well at
940-980.  If your green is very bright and you see some bleed through, tune
further into the IR.

-Doug


On Wed, Jun 25, 2014 at 11:07 AM, Jens-Bernhard Bosse <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear List,
>
> a colleague of mine wants to do 2 channel imaging on a 2-photon system
> with a max 1040 nm laser.
> What is the "best" fluorescent protein combination right now in terms of
> brightness, photostability and maturation time for this application?
>
> Thanks so much!
>
> Jens
>
> --------------------------------------------------------------
> Jens B. Bosse Ph.D.
> Enquist Lab
> Department of Molecular Biology
> and
> Princeton Neuroscience Institute
> Princeton University
> 301 Schultz Lab
> Washington Rd
> 08544 Princeton, NJ, USA
>
> Phone:  [phone]
> Email:  [hidden email]
> Web:    http://molbio.princeton.edu/labs/enquist/
>
> This electronic communication, including any attached documents, may
> contain confidential and/or legally privileged information that is intended
> only for use by the recipient(s) named above.  If you have received this
> communication in error, please notify the sender immediately and delete the
> communication and any attachments.
>

[Craig Brideau]

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Most of the common FP's like anywhere from 850 to 950nm. Very blue
*emitting* fluorophores tend to prefer more towards 700nm while really red
ones tend towards 1000nm. The common ones like GFP and YFP both prefer
around 950 to 1000nm. A large swath of the Alexa dyes are also excited in
this range.

Craig


On Wed, Jun 25, 2014 at 9:33 AM, Douglas Richardson <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We primarily use GFP and tdTomato, they are both excited very well at
> 940-980.  If your green is very bright and you see some bleed through, tune
> further into the IR.
>
> -Doug
>
>
> On Wed, Jun 25, 2014 at 11:07 AM, Jens-Bernhard Bosse <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear List,
> >
> > a colleague of mine wants to do 2 channel imaging on a 2-photon system
> > with a max 1040 nm laser.
> > What is the "best" fluorescent protein combination right now in terms of
> > brightness, photostability and maturation time for this application?
> >
> > Thanks so much!
> >
> > Jens
> >
> > --------------------------------------------------------------
> > Jens B. Bosse Ph.D.
> > Enquist Lab
> > Department of Molecular Biology
> > and
> > Princeton Neuroscience Institute
> > Princeton University
> > 301 Schultz Lab
> > Washington Rd
> > 08544 Princeton, NJ, USA
> >
> > Phone:  [phone]
> > Email:  [hidden email]
> > Web:    http://molbio.princeton.edu/labs/enquist/
> >
> > This electronic communication, including any attached documents, may
> > contain confidential and/or legally privileged information that is
> intended
> > only for use by the recipient(s) named above.  If you have received this
> > communication in error, please notify the sender immediately and delete
> the
> > communication and any attachments.
> >
>

[George McNamara]

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I cannot speak for the second color, but the brightest FP ever published
(Aequorea and relatives, not PE) is V6 from Steven Vogel

http://www.addgene.org/27813/

Article: Structural rearrangement of CaMKIIalpha catalytic domains
encodes activation. <http://www.addgene.org/browse/article/4004> Thaler
et al (Proc Natl Acad Sci U S A. 2009 Apr 14. 106(15):6369-74. PubMed
<http://pubmed.org/19339497>)

A Drosophila lab recently published a G6 and a R6, but failed to cite
Steven so I'll just provide the PubMed number
http://www.ncbi.nlm.nih.gov/pubmed/24451596

Bulinski and colleagues had several 5xEGFP papers (ex. 1999-2001), including
http://www.ncbi.nlm.nih.gov/pubmed/11719555
but Venus is brighter. Steven's paper had data for dimerized V6's,
effectively V12.

See also Bulinski's 1999 US Patent, 5,985,577, though could be argued
that 2xEGFP is obvious from the first generation Blue-Green and
Cyan-Yellow FP FRET polypeptides.

I doubt Bulinski's patent would cover 6xUnaG, which will likely be
almost as bright as V6, but UnaG is 17 kDa vs V 27 kDa (Steven used a
short linker, so ignoring that), so more photons per amino acid (but you
never know until someone sues you for patent infringement and wins).
http://www.cell.com/cell/abstract/S0092-8674(13)00644-2

I have posted suggestions online on localizing FPs to multiplex more,
dark background, etc
http://works.bepress.com/gmcnamara/42/
Pat O'Farrell published an implementation, actually two color TALElights, in
http://www.ncbi.nlm.nih.gov/pubmed/24556431
and more references in the "42" download.


George


On 6/25/2014 10:07 AM, Jens-Bernhard Bosse wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear List,
>
> a colleague of mine wants to do 2 channel imaging on a 2-photon system with a max 1040 nm laser.
> What is the "best" fluorescent protein combination right now in terms of brightness, photostability and maturation time for this application?
>
> Thanks so much!
>
> Jens
>
> --------------------------------------------------------------
> Jens B. Bosse Ph.D.
> Enquist Lab
> Department of Molecular Biology
> and
> Princeton Neuroscience Institute
> Princeton University
> 301 Schultz Lab
> Washington Rd
> 08544 Princeton, NJ, USA
>
> Phone: [phone]
> Email: [hidden email]
> Web: http://molbio.princeton.edu/labs/enquist/
>
> This electronic communication, including any attached documents, may contain confidential and/or legally privileged information that is intended only for use by the recipient(s) named above.  If you have received this communication in error, please notify the sender immediately and delete the communication and any attachments.
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/