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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: 616-234-5819 | Email: [log in to unmask]







On 4/7/14, 2:55 PM, "G. Esteban Fernandez" <[log in to unmask]>
wrote:

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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