PE APC crosstalk?

Mario Roederer Roederer at Darwin.Stanford.EDU
Mon Nov 24 17:30:43 EST 1997

>    Why is it necessary to have compensation [utilizing a
>   FACScalibur] between FL4 and other fluor channels with 2
>   spatially distinct excitation sources?

Finally!  A question I am qualified to answer!  

Compensation is required whenever two detectors can detect signal from multiple
fluorochromes.  Most commonly, this happens because of emission spectrum overlap
when different fluors are excited by the same laser (for example, FITC and PE).
However, it can also happen when different fluors have overlapping emission
spectra when excited by different lasers.

For example, Cy5PE (the tandem) is efficiently excited by 488 nm laser and emits
at about 680 nm.  This is because the PE is excited by 488 (the Cy5 is not), and
the transfers the energy through a non-radiative process to the covalently
linked Cy5 molecules.  This process, resonance energy transfer, can be very
efficient and results in virtually all of the energy in the Cy5 molecule.  The
Cy5 then emits with its standard emission (i.e., 680 nm).

However, there is no reason that the Cy5 molecules bound to the PE cannot
directly be excited.  Thus, when the Cy5PE tandem enters the second (red) laser,
while PE is not excited, the Cy5 is.  It then emits (again) with its
characteristic spectrum.  The emission of Cy5 is very similar to that of APC;
thus, the Cy5PE tandems look a lot like APC.

However, APC is not excited by the first laser--thus, by compensation between
FL3 and FL4, we can remove the Cy5PE contribution to the APC channel leaving
only APC fluorescence.


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