Agilent Technologies NovoCyte Quanteon, Operator'S Manual, Page: 35 / 148

Introduction
NovoCyte Quanteon Flow Cytometer Operator's Guide 35
Figure 7. Emission Spectra of Commonly Used Fluorochromes
Fluorescence Compensation Theory
Fluorochromes emit light over a range of wavelengths (Figure 7). Optical filters are
used to limit the range of wavelength measured by a given detector. However,
when two or more fluorochromes are used in the same test, the overlap in
wavelength ranges often makes it impossible to isolate light from a given
fluorochrome from another fluorochrome using optical filters. As a result, light
emitted from one fluorochrome will appear in a detector intended for detecting the
other fluorochrome (Figure 8). This is known as fluorescence spillover. Spillover
can be corrected mathematically by using a method called compensation.
Figure 8. Spillover from the FITC Fluorochrome to the PE Detector