advice with far red probe to distinguish from mRFP/mCherry

[Cammer, Michael-2]

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A lab is asking us for advice with a far red fluorescent probe.  They are already using Dapi, GFP, and mCherry or mRFP and want to add a fourth probe.  They suggested E2-Crimson.

I looked up E2-Crimson on FP Base  (a great website) and found that we could probably distinguish it well from mCherry by sequential excitations and clipping some of the emission spectra if controls showed bleed through.  However, with a 633 nm laser excitation efficiency would only be 40%.

I was thinking something more redshifted would be preferable and found miRFP670 which is reported to have 91% excitation efficiency with our laser, but only half as bright, so it might be a wash.

Any advice very much appreciated regarding:
Does anyone have experience with ER2-Crimson &/or miRFP670?
Is it as simple to separate from  mCherry or mRFP as I expect based on the reported ex and em data?
Any other suggestions for far red FPs?

Thank you!!

Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
http://nyulmc.org/micros  http://microscopynotes.com/



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[Craig Brideau]

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Some of Dylight's far red fluorophores are meant for high-throughput
screening so they are extremely bright. I've played with Dylight 680 and
managed to excite it reasonably well with 640nm.

Craig

On Fri, Oct 9, 2020 at 12:37 PM Cammer, Michael <
[hidden email]> wrote:

> *****
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> *****
>
>
>
> A lab is asking us for advice with a far red fluorescent probe.  They are
> already using Dapi, GFP, and mCherry or mRFP and want to add a fourth
> probe.  They suggested E2-Crimson.
>
> I looked up E2-Crimson on FP Base  (a great website) and found that we
> could probably distinguish it well from mCherry by sequential excitations
> and clipping some of the emission spectra if controls showed bleed
> through.  However, with a 633 nm laser excitation efficiency would only be
> 40%.
>
> I was thinking something more redshifted would be preferable and found
> miRFP670 which is reported to have 91% excitation efficiency with our
> laser, but only half as bright, so it might be a wash.
>
> Any advice very much appreciated regarding:
> Does anyone have experience with ER2-Crimson &/or miRFP670?
> Is it as simple to separate from  mCherry or mRFP as I expect based on the
> reported ex and em data?
> Any other suggestions for far red FPs?
>
> Thank you!!
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> Office: 646-501-0567 Cell: 914-309-3270  [hidden email]
> <mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/
>
>
>
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>

[Cammer, Michael-2]

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Thank you, the Dylight is great and we've also used Opal probes.

Sorry if I wasn't clear; they need a genetically encoded protein and don’t want to use a tagging system.

Best-
Michael

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Craig Brideau
Sent: Friday, October 9, 2020 2:51 PM
To: [hidden email]
Subject: Re: advice with far red probe to distinguish from mRFP/mCherry

[EXTERNAL]

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Some of Dylight's far red fluorophores are meant for high-throughput screening so they are extremely bright. I've played with Dylight 680 and managed to excite it reasonably well with 640nm.

Craig

On Fri, Oct 9, 2020 at 12:37 PM Cammer, Michael < [hidden email]> wrote:

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> *****
>
>
>
> A lab is asking us for advice with a far red fluorescent probe.  They
> are already using Dapi, GFP, and mCherry or mRFP and want to add a
> fourth probe.  They suggested E2-Crimson.
>
> I looked up E2-Crimson on FP Base  (a great website) and found that we
> could probably distinguish it well from mCherry by sequential
> excitations and clipping some of the emission spectra if controls
> showed bleed through.  However, with a 633 nm laser excitation
> efficiency would only be 40%.
>
> I was thinking something more redshifted would be preferable and found
> miRFP670 which is reported to have 91% excitation efficiency with our
> laser, but only half as bright, so it might be a wash.
>
> Any advice very much appreciated regarding:
> Does anyone have experience with ER2-Crimson &/or miRFP670?
> Is it as simple to separate from  mCherry or mRFP as I expect based on
> the reported ex and em data?
> Any other suggestions for far red FPs?
>
> Thank you!!
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> Office: 646-501-0567 Cell: 914-309-3270  [hidden email]
> <mailto:[hidden email]>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=oE3Cl5tIcbHXeIchIMEcTTqNQZuj2tf7Nyrt1_vD33A&s=zzlnkw_b6GAApf-qXdcvyrot-6McE1SGnDoouVrGfb4&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=oE3Cl5tIcbHXeIchIMEcTTqNQZuj2tf7Nyrt1_vD33A&s=iOVwztkuc58q6VI_mFf8bo7BQ9uFYq71uLS9jAzN4UI&e=
>
>
>
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[abhijit debroy]

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Hi Michael,

We use miRFP670 fairly regularly with 633 nm excitation and have not
observed significant overlap with mCherry. We typically increase the laser
power for miRFP670 compared to that for CFP, YFP or mCherry. Incubation
with biliverdin considerably increases the fluorescence of iRFP670 as
nicely documented in this paper below, and I can confirm that the increase
in fluorescence with biliverdin is quite significant.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519290/

I hope this helps.

Thanks
Abhi

Abhijit Deb Roy, PhD
Cell Biology Department
Johns Hopkins Medical Institute
Baltimore, MD



On Fri, Oct 9, 2020 at 2:37 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.
> *****
>
>
>
> A lab is asking us for advice with a far red fluorescent probe.  They are
> already using Dapi, GFP, and mCherry or mRFP and want to add a fourth
> probe.  They suggested E2-Crimson.
>
> I looked up E2-Crimson on FP Base  (a great website) and found that we
> could probably distinguish it well from mCherry by sequential excitations
> and clipping some of the emission spectra if controls showed bleed
> through.  However, with a 633 nm laser excitation efficiency would only be
> 40%.
>
> I was thinking something more redshifted would be preferable and found
> miRFP670 which is reported to have 91% excitation efficiency with our
> laser, but only half as bright, so it might be a wash.
>
> Any advice very much appreciated regarding:
> Does anyone have experience with ER2-Crimson &/or miRFP670?
> Is it as simple to separate from  mCherry or mRFP as I expect based on the
> reported ex and em data?
> Any other suggestions for far red FPs?
>
> Thank you!!
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> Office: 646-501-0567 Cell: 914-309-3270  [hidden email]
> <mailto:[hidden email]>
> http://nyulmc.org/micros  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.
> =================================
>

[Benjamin Smith]

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One other consideration, the tdTomato (orange) emission spectrum can be
readily deconvolved from the mCherry (red) emission spectrum as long as the
mCherry signal isn't significantly dimmer than the tdTomato signal.

-Ben Smith

On Fri, Oct 9, 2020 at 2:56 PM abhijit debroy <[hidden email]>
wrote:

> *****
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> 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,
>
> We use miRFP670 fairly regularly with 633 nm excitation and have not
> observed significant overlap with mCherry. We typically increase the laser
> power for miRFP670 compared to that for CFP, YFP or mCherry. Incubation
> with biliverdin considerably increases the fluorescence of iRFP670 as
> nicely documented in this paper below, and I can confirm that the increase
> in fluorescence with biliverdin is quite significant.
>
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519290/
>
> I hope this helps.
>
> Thanks
> Abhi
>
> Abhijit Deb Roy, PhD
> Cell Biology Department
> Johns Hopkins Medical Institute
> Baltimore, MD
>
>
>
> On Fri, Oct 9, 2020 at 2:37 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.
> > *****
> >
> >
> >
> > A lab is asking us for advice with a far red fluorescent probe.  They are
> > already using Dapi, GFP, and mCherry or mRFP and want to add a fourth
> > probe.  They suggested E2-Crimson.
> >
> > I looked up E2-Crimson on FP Base  (a great website) and found that we
> > could probably distinguish it well from mCherry by sequential excitations
> > and clipping some of the emission spectra if controls showed bleed
> > through.  However, with a 633 nm laser excitation efficiency would only
> be
> > 40%.
> >
> > I was thinking something more redshifted would be preferable and found
> > miRFP670 which is reported to have 91% excitation efficiency with our
> > laser, but only half as bright, so it might be a wash.
> >
> > Any advice very much appreciated regarding:
> > Does anyone have experience with ER2-Crimson &/or miRFP670?
> > Is it as simple to separate from  mCherry or mRFP as I expect based on
> the
> > reported ex and em data?
> > Any other suggestions for far red FPs?
> >
> > Thank you!!
> >
> > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> > NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> > 10016
> > Office: 646-501-0567 Cell: 914-309-3270  [hidden email]
> > <mailto:[hidden email]>
> > http://nyulmc.org/micros  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
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> > transmitted by this email.
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> >
>

--
Benjamin E. Smith, Ph. D.
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