Far Red Dye for Nucleoplasm

[Alison Dun]

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Hi,

Wondering if anyone can help me source a far red dye that stains the nucleoplasm (but not the nucleolus) of live cells. The main issue we are having is that when exciting mCherry in the same sample the emission excites the far red dye if its spectrum overlaps too much. So we need a far red dye with excitation ideally above 680 nm. Any other suggestions about how to overcome this problem would be much appreciated.

Thanks
Ali

Dr. Alison Dun
Technology and Facility Manager
Edinburgh Super Resolution Imaging Consortium (ESRIC)
Heriot-Watt University
Edinburgh
EH14 4AS

Tel: [phone]

http://www.esric.org

[Avi Jacob]

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I use DRAQ5 to stain nucleus in cells with RFP. While it's close, with good
filters I find it to be a good combination. Can't say about the nucleoli
If you use di or trichroic filters rather than a sedat set, might be
trickier to avoid bleed through. Been awhile since I looked at the spectra
so I don't rememeber off hand the exact wavelengths.

Avi
--
Abraham I. Jacob, Ph.D.
http://lifefaculty.biu.ac.il/shav-tal/



On Tue, Aug 6, 2013 at 6:37 PM, Alison Dun <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> Wondering if anyone can help me source a far red dye that stains the
> nucleoplasm (but not the nucleolus) of live cells. The main issue we are
> having is that when exciting mCherry in the same sample the emission
> excites the far red dye if its spectrum overlaps too much. So we need a far
> red dye with excitation ideally above 680 nm. Any other suggestions about
> how to overcome this problem would be much appreciated.
>
> Thanks
> Ali
>
>

[mcammer]

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We routinely use DRAQ5 with 633 to 647 nm excitation and separate it from mCherry, RFP, Alexa568 etc.  Nuclei look great.  Also, one of our users likes using a label for histones to fill in the volume between the DNA but this is an antibody method.

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Avi Jacob [[hidden email]]
Sent: Tuesday, August 06, 2013 12:02 PM
To: [hidden email]
Subject: Re: Far Red Dye for Nucleoplasm

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I use DRAQ5 to stain nucleus in cells with RFP. While it's close, with good
filters I find it to be a good combination. Can't say about the nucleoli
If you use di or trichroic filters rather than a sedat set, might be
trickier to avoid bleed through. Been awhile since I looked at the spectra
so I don't rememeber off hand the exact wavelengths.

Avi
--
Abraham I. Jacob, Ph.D.
http://lifefaculty.biu.ac.il/shav-tal/



On Tue, Aug 6, 2013 at 6:37 PM, Alison Dun <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> Wondering if anyone can help me source a far red dye that stains the
> nucleoplasm (but not the nucleolus) of live cells. The main issue we are
> having is that when exciting mCherry in the same sample the emission
> excites the far red dye if its spectrum overlaps too much. So we need a far
> red dye with excitation ideally above 680 nm. Any other suggestions about
> how to overcome this problem would be much appreciated.
>
> Thanks
> Ali
>
>

[Tim Feinstein-2]

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If using DRAQ5 for live cells keep in mind that this is an imperfect solution.  It does not kill the cells right away, but they will not divide.  If you have spectral detection, a fluorescent protein with a NLS might work well in this case; with modern spectrals the emission spectra do not have to be all that different as long as you have clean spatial separation.  Nikon likes to show three-color pictures of fixed cells using dyes that are all 'green'.  If not then one of the new generation of infrared proteins could work.  Keep in mind, though, that the efficiency of most detectors goes way down in the non-visible reds.  

One final idea: for the chemists out there, would be possible to bind a suitable dye to, say, a poly-basic cell-permeant peptide (CPP) with a NLS?  Since lysine, used for conjugation, and NLS's are both basic it might be crazy enough to work.  

All the best,


TF

Timothy Feinstein, Ph.D.

On Aug 6, 2013, at 2:04 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
> *****
>
> We routinely use DRAQ5 with 633 to 647 nm excitation and separate it from mCherry, RFP, Alexa568 etc.  Nuclei look great.  Also, one of our users likes using a label for histones to fill in the volume between the DNA but this is an antibody method.
>
> _________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: [phone]  Cell: [phone]
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on behalf of Avi Jacob [[hidden email]]
> Sent: Tuesday, August 06, 2013 12:02 PM
> To: [hidden email]
> Subject: Re: Far Red Dye for Nucleoplasm
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I use DRAQ5 to stain nucleus in cells with RFP. While it's close, with good
> filters I find it to be a good combination. Can't say about the nucleoli
> If you use di or trichroic filters rather than a sedat set, might be
> trickier to avoid bleed through. Been awhile since I looked at the spectra
> so I don't rememeber off hand the exact wavelengths.
>
> Avi
> --
> Abraham I. Jacob, Ph.D.
> http://lifefaculty.biu.ac.il/shav-tal/
>
>
>
> On Tue, Aug 6, 2013 at 6:37 PM, Alison Dun <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi,
>>
>> Wondering if anyone can help me source a far red dye that stains the
>> nucleoplasm (but not the nucleolus) of live cells. The main issue we are
>> having is that when exciting mCherry in the same sample the emission
>> excites the far red dye if its spectrum overlaps too much. So we need a far
>> red dye with excitation ideally above 680 nm. Any other suggestions about
>> how to overcome this problem would be much appreciated.
>>
>> Thanks
>> Ali
>>
>>

[Steffen Dietzel]

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Ali,

by definition, the nucleo*plasm* would be the solution surrounding but
excluding chromatin. Is that what you mean? if so, you would need to get
a dye in the nucleus that does not bind to DNA. I am not aware of any
specific stainable substances in that solution, but some people managed
to inject large molecules in the nucleus that are excluded from the
chromatin and thus 'stain' the surroundings. If memory serves me right
is was dextran. The mentioned NLS-FP seems to be another option.

If you have access to the equipment, you might also want to have a look
at 2-photon excitation spectra, sometimes they are surprisingly distinct
for dyes having overlapping 1-photon excitation.

Steffen



On 06.08.2013 17:37, Alison Dun wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> Wondering if anyone can help me source a far red dye that stains the nucleoplasm (but not the nucleolus) of live cells. The main issue we are having is that when exciting mCherry in the same sample the emission excites the far red dye if its spectrum overlaps too much. So we need a far red dye with excitation ideally above 680 nm. Any other suggestions about how to overcome this problem would be much appreciated.
>
> Thanks
> Ali
>
> Dr. Alison Dun
> Technology and Facility Manager
> Edinburgh Super Resolution Imaging Consortium (ESRIC)
> Heriot-Watt University
> Edinburgh
> EH14 4AS
>
> Tel: [phone]
>
> http://www.esric.org
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

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