Re: advice with far red probe to distinguish from mRFP/mCherry

Posted by abhijit debroy on
URL: http://confocal-microscopy-list.275.s1.nabble.com/advice-with-far-red-probe-to-distinguish-from-mRFP-mCherry-tp7591381p7591384.html

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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:

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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]
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> http://nyulmc.org/micros  http://microscopynotes.com/
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