In this figure, absorbance and fluorescence emission spectra are shown with peak wavelengths, optimal excitation wavelengths, and filter edge choices; all color coded according to the visible spectrum.

For single wavelength excitation, the red-edge wavelength is used (see Stark Effect) along with long-pass dichroic and emission filters to collect as much emission as possible for signal-to-noise considerations, while eliminating any bleedthrough excitation light.

For dual-excitation ratiometric imaging, a blue-edge excitation light source is added by time multiplexing and this wavelength must also be reflected by the dichroic.