Wavelength is the distance between two consecutive crests of a wave. In the optical range it is usually measured in nanometers (nm). In optical microscopy involving fluorescence, two wavelengths are important: the Excitation Wavelength and the Emission Wavelength.

The lower the WaveLengths involved in the measurement, the higher the resolution. This dependency was described by Ernst Karl Abbe and later in an equation describing the diffraction limit. For a good measurement you must adapt the Sampling Density accordingly to the Nyquist Rate.

Huygens Essential will warn you if the difference between the reported Excitation Wavelength and Emission Wavelength is larger than 20%. This is just a reminder based on that many Fluoro Chromes have the difference in wavelength inside this range. But other, as DAPI, have a larger difference. To make a proper deconvolution the correct values must be reported, despite the warning message.