In harmonic generation, multiple photons interact simultaneously with a molecule with no absorption events. Because n-photon harmonic generation is essentially a scattering process, the emitted wavelength is exactly 1/n times the incoming fundamental wavelength. When the excitation color is changed, the emission color changes also. In contrast the wavelength of fluorescence emission is Stokes-shifted to a longer wavelength; the line shape is determined strictly by the molecular energy levels. Practically it is relatively easy to design multicolor MPM by tuning the harmonic generation so that it is well separated from the fluorescence (see mouse ovary image above -- acquired in collaboration with Alexander Nikitin, Biomedical Sciences, Cornell).

Another major difference is that for scattering microscopies, the emitted light is coherent and therefore must satisfy phase matching constraints. For example, the SHG profile in MPM retains phase information and emits with a directionality dependent on the nature of the scatterers (Moreaux, L., Sandre, O. & Mertz, J. (2000) J Opt Soc Am B 17, 1685-1694. Mertz, J. & Moreaux, L. (2001) Opt Commun 196, 325-330.). Fluorescence in general emits isotropically.