Spectrally unmixing mCherry and tdTomato

[G. Esteban Fernandez]

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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 doable
on my LSM 710, but I wanted to check...has anyone actually spectrally
unmixed mCherry and tdTomato successfully?

Thanks,
Esteban

[Craig Brideau]

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We've unmixed Calcium Green and Newport Green, (Nikon C1Si, 32 channels) so
if your algorithm is up to the task and you have sufficient spectral
resolution, you should be fine.

Craig


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=confocalmicroscopy
> 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 doable
> on my LSM 710, but I wanted to check...has anyone actually spectrally
> unmixed mCherry and tdTomato successfully?
>
> Thanks,
> Esteban
>

[Laure Plantard]

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We have set up a practical with students where we unmix AF555 and AF568 on a Zeiss LSM780. It works well as long as the intensities of the two fluorophores are comparable.

Best regards,
Laure



________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Craig Brideau [[hidden email]]
Sent: 07 April 2014 21:07
To: [hidden email]
Subject: Re: Spectrally unmixing mCherry and tdTomato

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We've unmixed Calcium Green and Newport Green, (Nikon C1Si, 32 channels) so
if your algorithm is up to the task and you have sufficient spectral
resolution, you should be fine.

Craig


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=confocalmicroscopy
> 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 doable
> on my LSM 710, but I wanted to check...has anyone actually spectrally
> unmixed mCherry and tdTomato successfully?
>
> Thanks,
> Esteban
>

[Feinstein, Timothy]

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You can do that.  It will work best if they have comparable emission
intensities and you minimize co-localization between the two fluorophores.
 In tricky cases we sometimes get better results when we record the
emission spectrum of a fluorophore ourselves rather than use the spectrum
provided with the scope software.

Spectral unmixing often has a number of options such as different
algorithms and whether to include a Œremainder¹ channel for fluorescence
that the software does not think comes from any of the predicted
fluorophores.  Sometimes different setting combinations can produce
dramatically different results.  Using a Nikon A1-S and Elements we have
learned to leave the remainder channel option permanently off.  A little
trial and error can be worth it in tricky cases like yours.

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 4/7/14, 2:55 PM, "G. Esteban Fernandez" <[hidden email]>
wrote:

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>roscopy
>Post images on
>http://scanmail.trustwave.com/?c=129&d=rvTC0-cLjwAq9zuzY55SO5WNrf1yFZMBW93
>MfK6Ysg&u=http%3a%2f%2fwww%2eimgur%2ecom 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 doable
>on my LSM 710, but I wanted to check...has anyone actually spectrally
>unmixed mCherry and tdTomato successfully?
>
>Thanks,
>Esteban