Re: advice with far red probe to distinguish from mRFP/mCherry **tech support response**

[Roy Edward-2]

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Dear Michael,
This may seem a prosaic alternative but I would suggest swapping out DAPI with DRAQ5. That can be detected by non-coincidental excitation (647nm) to mCherry and in a longer wavelength detection channel, that have been described as used together elsewhere.  That would then free up the “DAPI” channel for a UV/Vio excitation xFP, perhaps eBFP, that can be excitation-separated from GFP?

I hope that helps a little.
Regards,
Roy

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Roy Edward
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> Date:    Fri, 9 Oct 2020 18:36:30 +0000
> From:    "Cammer, Michael" <[hidden email]>
> Subject: advice with far red probe to distinguish from mRFP/mCherry
> *****
> 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]>

[Tim Feinstein]

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We have separated GFP and YFP before using spectral detection.  Spectral deconvolution works a lot better for live cells if you use the PMT-array method (ie Nikon or Zeiss), but if you have one of those it's an option.  

Best,


T

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

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Roy Edward
Sent: Saturday, October 10, 2020 10:54 AM
To: [hidden email]
Subject: Re: advice with far red probe to distinguish from mRFP/mCherry **tech support response**

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Dear Michael,
This may seem a prosaic alternative but I would suggest swapping out DAPI with DRAQ5. That can be detected by non-coincidental excitation (647nm) to mCherry and in a longer wavelength detection channel, that have been described as used together elsewhere.  That would then free up the “DAPI” channel for a UV/Vio excitation xFP, perhaps eBFP, that can be excitation-separated from GFP?

I hope that helps a little.
Regards,
Roy

Sent from my iPhone
Roy Edward
Biostatus Ltd.
Off. [phone]
Fax [phone]
https://nam12.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.biostatus.com%2F&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7C3bb16a9d5932497fb23508d86d46a4e6%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637379497472656232&amp;sdata=XMXQF%2BwgFrM5g1gE3fFFl0iP9vFqlouckuR1EPej0g0%3D&amp;reserved=0
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> Date:    Fri, 9 Oct 2020 18:36:30 +0000
> From:    "Cammer, Michael" <[hidden email]>
> Subject: advice with far red probe to distinguish from mRFP/mCherry
> *****
> 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]>