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Compensation necessary when using different excitation lasers? - (Jun/06/2008 )

I am analyzing cells labelled with both Cy5 (detected on APC channel), and Cy3 (detected on PE or PE-Texas Red channel) on a BD LSR II cytometer. A labmate explained to me that since APC and PE are excited with different lasers, compensation isn't necessary. Based on my perfunctory understanding of spectral overlap and compensation, this seems to make sense (eg. with FITC/PE channels, the same excitation laser will cause emission that will be detected by both the FITC and PE detector, but different excitation lasers for APC/PE mean detection occurs at different times). However, I've noticed some discussions online about compensation when using the PE and APC pair. So in practice, is compensation necessary for this fluorophore pair?



I haven't worked with these two fluorochromes, so I will just try to answer in general.

Some fluorophores are excited efficiently with one laser, but (to a lesser extent) also by another one. If then you use two fluorophores with overlapping emission spectra, you will need to do some compensation.
Best example would be quantum dots, that are very effiently excited with UV or near-UV lasers, but are also excited by 488nm and 633/635 lasers (and all in between), so if you would use Qdot 585 you would get some signal in the PE-channel. This you can however easily compensate for, since excitation by the 488nm laser is a lot less than with the 405 (or other) laser you would preferentially use for your Qdot585 excitation.

Since you are working with an LSRII, you have FACSDiva software running, so you can do compensation after your acquisition. Take a negative control sample, and 2 single stained samples and see if you get spectral overlap of one of your fluorophores, and if so: do compensation. If not: leave it as it is.


Please DO NOT follow the advice of your lab mate. No matter what laser line is used to excite the fluorophore, and no matter how far apart the emission peaks of two fluorophores are. For every single flow cytometry experiment that you do YOU MUST USE SINGLE COLOR CONTROLS and compensate the instrument.

There are several reasons for this that have been explained in great detail in other flow cytometry forums (see or Any self-respecting flow cytometrist would turn you away at their door if you brought them samples to run with no single color controls.

In fact if you actually look at the full emission spectra for the two dyes that you are using (Cy3 and Cy5) you will actually see that there is a very small tail from Cy3 that bleeds into the Cy5 emission spectrum. Furthermore, PE (and PE-Texas Red to an even greater extent) does overlap significantly with APC.

A note on the LSRII ...
because of the optical set-up on this instrument (which is one of the novelty's of this cytometer), it is possible to use what is called 'cross-beam compensation' in theory but in practice this is difficult to do if you are a novice user.

good luck and happy flowing

-JE UMass IVP-