Re: Spectrally unmixing mCherry and tdTomato

[Wendy Salmon]

*****
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 get good separation on our Zeiss 710 without unmixing simply by exciting the tdTomato at 514nm and mCherry at 561nm and imaging sequentially.  We have the spectral emission selection, so there's likely a slight shift in our detection wavelengths, too (I'll have to power up the system to check), but the biggest difference is the excitation efficiency.  This assumes similar fluorophore concentrations, of course.  

Good luck!
Wendy

~~~~~~~~~~~~~~~~~~~~~~~
Wendy Salmon
Light Microscopy Specialist
Whitehead Institute for Biomedical Research
W.M. Keck Imaging Facility
9 Cambridge Center, Rm 447
Cambridge, MA 02142
c: 617-429-0158
e: [hidden email]
w: http://staffa.wi.mit.edu/microscopy/


On Mon, Apr 7, 2014 at 12:55 PM, G. Esteban Fernandez <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.=

> *****
>
> Hi everyone,
>
> A user is planning an experiment where spectral separation of mCherry and=

> tdTomato in the same mouse tissue will be needed (single photon).  I know=

> that their peak emissions should be ~25 nm apart so this seems very doabl=
e
> on my LSM 710, but I wanted to check...has anyone actually spectrally
> unmixed mCherry and tdTomato successfully?
>
> Thanks,
> Esteban
>

[jerie]

*****
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.
*****

You can feed spectral info from both, excitation and/or emission into
unmixing algorithms.

Cheers,  Jens
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Am 16.04.2014 11:41 schrieb "Wendy Salmon" <[hidden email]>:

> *****
> 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 get good separation on our Zeiss 710 without unmixing simply by
> exciting the tdTomato at 514nm and mCherry at 561nm and imaging
> sequentially.  We have the spectral emission selection, so there's likely a
> slight shift in our detection wavelengths, too (I'll have to power up the
> system to check), but the biggest difference is the excitation efficiency.
>  This assumes similar fluorophore concentrations, of course.
>
> Good luck!
> Wendy
>
> ~~~~~~~~~~~~~~~~~~~~~~~
> Wendy Salmon
> Light Microscopy Specialist
> Whitehead Institute for Biomedical Research
> W.M. Keck Imaging Facility
> 9 Cambridge Center, Rm 447
> Cambridge, MA 02142
> c: 617-429-0158
> e: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/
>
>
> On Mon, Apr 7, 2014 at 12:55 PM, G. Esteban Fernandez <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.=
>
> > *****
> >
> > Hi everyone,
> >
> > A user is planning an experiment where spectral separation of mCherry
> and=
>
> > tdTomato in the same mouse tissue will be needed (single photon).  I
> know=
>
> > that their peak emissions should be ~25 nm apart so this seems very
> doabl=
> e
> > on my LSM 710, but I wanted to check...has anyone actually spectrally
> > unmixed mCherry and tdTomato successfully?
> >
> > Thanks,
> > Esteban
> >
>