CytoFlex Flow Cytometer Application Notes

Absence of Laser Cross Talk and Infrared Light Spillover Reduces Compensation in Multicolor Experiments One advantage of Infrared excitation is to expand the number of colors that can be used for multiparametric analysis. The long wavelengths of the IR laser do not exhibit cross talk into traditional detection channels. Alex Fluor 790 and PromoFlour 840 are two commercially available Infrared excited dyes (Figure 4). A compensation study was performed to evaluate the spillover of Alexa Fluor 790 (AF790) and PromoFluor 840 (PF840) fluorochromes into other channels and the spillover of other dyes into the two IR channels. The study showed that there is minimal spillover of AF790 and PF840 into the detection channels of other lasers (Figure 5) and that there is minimal spillover of other single color dyes into the IR channels (Figure 6). Altogether, these results confirm that IR excitation and detection can be incorporated into flow cytometry instruments and thus allow users additional options for multi-color design and biological applications.

100

Alexa Fluor 790

4A

808 808

80

60

40

20

0

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300

350

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Wavelength (nm)

PromoFluor 840

4B

Absorbance / Intensity

250

300

350

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Wavelength (nm)

Figure 4: Excitation and Emission spectra for (a) Alexa Fluor 790 and (b) PromoFluor 840. Alexa Fluor has an excitation maximum of 784 nm and an emission maxima of 814 nm. PromoFluor 840 has an excitation maximum of 844 nm and an emission maxima of 884 nm.

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