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miRFP670 or other near IR fluorescent protein?

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mcammer

miRFP670 or other near IR fluorescent protein?

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We are looking for a third genetically encoded fluorescent protein to use in live imaging with GFP and tdTomato.

New monometric red probes are described in a recent paper from the Verkhusha lab. http://www.nature.com/articles/ncomms12405

Has anyone used these, in particular miRFP670 https://www.addgene.org/80003/ which has emission spectra best fitting our existing Cy5/Alexa647 type filter set? Or would you know of or recommend another one?

Thank you very much for any info.

Best regards,
Michael

=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
Cell: 914-309-3270 Office: Skirball 2nd Floor main office, back right
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/

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=================================

Douglas Richardson

Re: miRFP670 or other near IR fluorescent protein?

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Hi Michael,

I prefer mtagbfp2 for three color if you're ok with using near UV light for excitation. We have also used mirfp670 for four color imaging and in thick embryos and are happy with it. It does need to be expressed at an above average level (worked great in H2B fusions as a nuclear marker) as you can see from the QY and EC it's not super bright.

Doug

> On Jan 10, 2017, at 3:01 PM, Cammer, Michael <[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 are looking for a third genetically encoded fluorescent protein to use in live imaging with GFP and tdTomato.
>
> New monometric red probes are described in a recent paper from the Verkhusha lab. http://www.nature.com/articles/ncomms12405
>
> Has anyone used these, in particular miRFP670 https://www.addgene.org/80003/ which has emission spectra best fitting our existing Cy5/Alexa647 type filter set? Or would you know of or recommend another one?
>
> Thank you very much for any info.
>
> Best regards,
> Michael
>
>
>
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell: 914-309-3270 Office: Skirball 2nd Floor main office, back right
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

Tim Feinstein

Re: miRFP670 or other near IR fluorescent protein?

In reply to this post by mcammer

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Post images on http://www.imgur.com and include the link in your posting.
*****

I know this is only semi-responsive to your question, but GFP and YFP are pretty easy to tell apart with a spectral confocal, and YFP excites well at 488 nm. We published GFP/YFP/tdTomato for pretty fine grained live-cell colocalization work in 2011.

Best,

Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

On 1/10/17, 3:01 PM, "Confocal Microscopy List on behalf of Cammer, Michael" <[hidden email] on behalf of [hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
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>Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7Ce903d577057444b3842908d43993b6ab%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=3sp%2BW81B7G350alzwhAJOg2AKJ0%2Bs%2BgmmoUdX1Bthpo%3D&reserved=0 and include the link in your posting.
>*****
>
>
>We are looking for a third genetically encoded fluorescent protein to use in live imaging with GFP and tdTomato.
>
>New monometric red probes are described in a recent paper from the Verkhusha lab. https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.nature.com%2Farticles%2Fncomms12405&data=01%7C01%7Ctnf8%40PITT.EDU%7Ce903d577057444b3842908d43993b6ab%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=c5Hg1nLknVAf5uDLUUQ2x6q%2BAhd3yEt%2BPmQBOCY%2B%2FJg%3D&reserved=0
>
>Has anyone used these, in particular miRFP670 https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.addgene.org%2F80003%2F&data=01%7C01%7Ctnf8%40PITT.EDU%7Ce903d577057444b3842908d43993b6ab%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=7F%2FgiZF40IfUa9p0TQPVqUC7m1c%2FKeLXcbwLwLZl7yo%3D&reserved=0 which has emission spectra best fitting our existing Cy5/Alexa647 type filter set? Or would you know of or recommend another one?
>
>Thank you very much for any info.
>
>Best regards,
>Michael
>
>
>
>
>=========================================================================
>Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell: 914-309-3270 Office: Skirball 2nd Floor main office, back right
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Focs.med.nyu.edu%2Fmicroscopy&data=01%7C01%7Ctnf8%40PITT.EDU%7Ce903d577057444b3842908d43993b6ab%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=hXiQdsMKcPk%2FJ2QrUyI3Yd8uTcwtXXEyRr1rccMTZNc%3D&reserved=0 & https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2F&data=01%7C01%7Ctnf8%40PITT.EDU%7Ce903d577057444b3842908d43993b6ab%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=KAhbTc%2Fxpbks1faBfO%2FmzxFDjG%2F7xdpT5N3UVctC3TI%3D&reserved=0
>
>
>------------------------------------------------------------
>This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
>=================================

sparker

Re: miRFP670 or other near IR fluorescent protein?

In reply to this post by mcammer

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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Michael,

I have used iRFP670 alone, N and C-terminally tagged on several proteins for live imaging and it works very well. We have a Cy5 filter set, as well as with a 640 laser, and they both work beautifully. It's far enough away from Cy3 to work with our go-to near-red mRuby2 (mCherry bleeds thru to Cy5 on our set-up). Actually, I had gotten into a disagreement with someone as to a tendency of iRFP to photobleach, and testing it myself, iRFP670 intensity reduced 40% after 2 minutes of continuous laser excitation, so that seems pretty stable to me! We’ve imaged iRFP670-Lifeact for 24 hours on a 10 minute interval without problems! Good luck!

Sara Parker
Postdoctoral Fellow, Mouneimne / Zinsmaier Labs
(520) 626-3860
Department of Neuroscience
University of Arizona

> On Jan 10, 2017, at 1:03 PM, Cammer, Michael <[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 are looking for a third genetically encoded fluorescent protein to use in live imaging with GFP and tdTomato.
>
> New monometric red probes are described in a recent paper from the Verkhusha lab. http://www.nature.com/articles/ncomms12405
>
> Has anyone used these, in particular miRFP670 https://www.addgene.org/80003/ which has emission spectra best fitting our existing Cy5/Alexa647 type filter set? Or would you know of or recommend another one?
>
> Thank you very much for any info.
>
> Best regards,
> Michael
>
>
>
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell: 914-309-3270 Office: Skirball 2nd Floor main office, back right
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

Sara Parker
Postdoctoral Fellow, Mouneimne / Zinsmaier Labs
(520) 626-3860
Department of Neuroscience
University of Arizona

sparker

Re: miRFP670 or other near IR fluorescent protein?

In reply to this post by mcammer

*****
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.
*****

Hi Michael,

I have used iRFP670 alone, N and C-terminally tagged on several proteins for live imaging and it works very well. We have a Cy5 filter set, as well as with a 640 laser, and they both work beautifully. It's far enough away from Cy3 to work with our go-to near-red mRuby2 (mCherry bleeds thru to Cy5 on our set-up). Actually, I had gotten into a disagreement with someone as to a tendency of iRFP to photobleach, and testing it myself, iRFP670 intensity reduced 40% after 2 minutes of continuous laser excitation, so that seems pretty stable to me! We’ve imaged iRFP670-Lifeact for 24 hours on a 10 minute interval without problems! Good luck!

Sara Parker
Postdoctoral Fellow, Mouneimne / Zinsmaier Labs
(520) 626-3860
Department of Neuroscience
University of Arizona

> On Jan 10, 2017, at 1:01 PM, Cammer, Michael <[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 are looking for a third genetically encoded fluorescent protein to use in live imaging with GFP and tdTomato.
>
> New monometric red probes are described in a recent paper from the Verkhusha lab. http://www.nature.com/articles/ncomms12405
>
> Has anyone used these, in particular miRFP670 https://www.addgene.org/80003/ which has emission spectra best fitting our existing Cy5/Alexa647 type filter set? Or would you know of or recommend another one?
>
> Thank you very much for any info.
>
> Best regards,
> Michael
>
>
>
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell: 914-309-3270 Office: Skirball 2nd Floor main office, back right
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

Romin, Yevgeniy/Sloan Kettering Institute

Re: miRFP670 or other near IR fluorescent protein?

In reply to this post by mcammer

*****
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.
*****

Hi Michael

Not sure if you have the setup for these, but we've been able to easily do live imaging on cells using mTurqois, GFP, tdTomato and Turbo FP635 in paralle. TurboF635 is closer to red, then near-infrared, but it was pretty stable and easily separated from tdTomato

Yevgeniy
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Cammer, Michael [[hidden email]]
Sent: Tuesday, January 10, 2017 3:01 PM
To: [hidden email]
Subject: miRFP670 or other near IR fluorescent protein?

*****
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 are looking for a third genetically encoded fluorescent protein to use in live imaging with GFP and tdTomato.

New monometric red probes are described in a recent paper from the Verkhusha lab. http://www.nature.com/articles/ncomms12405

Has anyone used these, in particular miRFP670 https://www.addgene.org/80003/ which has emission spectra best fitting our existing Cy5/Alexa647 type filter set? Or would you know of or recommend another one?

Thank you very much for any info.

Best regards,
Michael

=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
Cell: 914-309-3270 Office: Skirball 2nd Floor main office, back right
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/

------------------------------------------------------------
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================

Rosemary.White

Re: miRFP670 or other near IR fluorescent protein?

In reply to this post by Tim Feinstein

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

i agree. GFP and YFP are pretty easy to separate.

Another good alternative is mTurquoise2 - bright, stable, not readily bleached, at least in plants. It's the new CFP.

cheers,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
Canberra, ACT 2601
Australia

T 61 2 6246 5475
M 61 468 966 713
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Feinstein, Timothy N [[hidden email]]
Sent: Wednesday, 11 January 2017 7:20 a.m.
To: [hidden email]
Subject: Re: miRFP670 or other near IR fluorescent protein?

*****
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.
*****

I know this is only semi-responsive to your question, but GFP and YFP are pretty easy to tell apart with a spectral confocal, and YFP excites well at 488 nm. We published GFP/YFP/tdTomato for pretty fine grained live-cell colocalization work in 2011.

Best,

Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

On 1/10/17, 3:01 PM, "Confocal Microscopy List on behalf of Cammer, Michael" <[hidden email] on behalf of [hidden email]> wrote:

Kurt Thorn

Re: miRFP670 or other near IR fluorescent protein?

In reply to this post by mcammer

*****
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.
*****

What about (td)smURFP?
http://www.nature.com/nmeth/journal/v13/n9/full/nmeth.3935.html

It has the same ex / em peak as mIRFP670, but is quite a bit brighter,
at least on paper. I don't have direct experience with it, however.

Kurt

On 1/10/2017 12:01 PM, Cammer, Michael 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 are looking for a third genetically encoded fluorescent protein to use in live imaging with GFP and tdTomato.
>
> New monometric red probes are described in a recent paper from the Verkhusha lab. http://www.nature.com/articles/ncomms12405
>
> Has anyone used these, in particular miRFP670 https://www.addgene.org/80003/ which has emission spectra best fitting our existing Cy5/Alexa647 type filter set? Or would you know of or recommend another one?
>
> Thank you very much for any info.
>
> Best regards,
> Michael
>
>
>
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell: 914-309-3270 Office: Skirball 2nd Floor main office, back right
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================
>

--
Kurt Thorn
Associate Professor
Director, Nikon Imaging Center
http://thornlab.ucsf.edu/
http://nic.ucsf.edu/blog/

张莉莉

回复：Re: miRFP670 or other near IR fluorescent protein?

In reply to this post by Rosemary.White

*****
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.
*****

Hi, Be careful that miRFP670 is BphP-based and only used in mammals.

发自网易邮箱大师
在2017年01月11日 04:58，[hidden email] 写道:
*****
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.
*****

i agree. GFP and YFP are pretty easy to separate.

Another good alternative is mTurquoise2 - bright, stable, not readily bleached, at least in plants. It's the new CFP.

cheers,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
Canberra, ACT 2601
Australia

T 61 2 6246 5475
M 61 468 966 713
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Feinstein, Timothy N [[hidden email]]
Sent: Wednesday, 11 January 2017 7:20 a.m.
To: [hidden email]
Subject: Re: miRFP670 or other near IR fluorescent protein?

*****
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.
*****

I know this is only semi-responsive to your question, but GFP and YFP are pretty easy to tell apart with a spectral confocal, and YFP excites well at 488 nm. We published GFP/YFP/tdTomato for pretty fine grained live-cell colocalization work in 2011.

Best,

Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

On 1/10/17, 3:01 PM, "Confocal Microscopy List on behalf of Cammer, Michael" <[hidden email] on behalf of [hidden email]> wrote:

张莉莉

回复：回复：Re: miRFP670 or other near IR fluorescent protein?

*****
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.
*****

I have made a mistake before. I labeled my bacteria with miRFP670 and tried to inject in zebrafish embryos and it never worked...
Best regards,
Lili
发自网易邮箱大师
在2017年01月11日 06:15，张莉莉写道:
*****
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.
*****

Hi, Be careful that miRFP670 is BphP-based and only used in mammals.

发自网易邮箱大师
在2017年01月11日 04:58，[hidden email] 写道:
*****
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.
*****

i agree. GFP and YFP are pretty easy to separate.

Another good alternative is mTurquoise2 - bright, stable, not readily bleached, at least in plants. It's the new CFP.

cheers,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
Canberra, ACT 2601
Australia

T 61 2 6246 5475
M 61 468 966 713
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Feinstein, Timothy N [[hidden email]]
Sent: Wednesday, 11 January 2017 7:20 a.m.
To: [hidden email]
Subject: Re: miRFP670 or other near IR fluorescent protein?

*****
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.
*****

I know this is only semi-responsive to your question, but GFP and YFP are pretty easy to tell apart with a spectral confocal, and YFP excites well at 488 nm. We published GFP/YFP/tdTomato for pretty fine grained live-cell colocalization work in 2011.

Best,

Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

On 1/10/17, 3:01 PM, "Confocal Microscopy List on behalf of Cammer, Michael" <[hidden email] on behalf of [hidden email]> wrote: