A common challenge with multiparameter fluorescence is that excitation even by a narrow line will excite not only the targeted dye, but also other dyes in the sample. In most cases this is not wanted as the separation of various channels suffers from cross excitation making some applications, like FRET experiments, difficult to work with. Here again, the tunable wavelength of the white light laser source is an easy cure for cross excitation. The longer wavelength excitation can be moved out of the absorption of the blue dye. Here, an interactive optimisation for reduced cross excitation and efficient emission collection is easily done by dialing the excitation and adapting the emission band – online by a few scans only.
If separation is still not optimised, the system also offers sequential scan: that is recording a line with the first excitation and subsequently the same line with the next excitation for the next dye – and so on. Furthermore, the SP detector as a tunable device can be specified to collect sufficiently narrow bands for minimal crosstalk on the emission side. If the above procedures still do not provide optimal separation, linear unmixing for dye separation – emission or excitation – is also implemented.