Fig. 1

Excitation and emission processes of the same hypothetical molecule in the various microscopy regimes represents: a conventional confocal microscopy: where a single photon of wavelength equal to λ₀ excited molecule and produces a wavelength emission equal to λ₁, where λ₁ > λ₀; b two-photons excited fluorescence: where two photons with wavelength equal to \( \raisebox{1ex}{${\lambda}_0$}\!\left/ \!\raisebox{-1ex}{$2$}\right. \) excited the molecule, producing an emission wavelength equal to λ₂, where λ₂ < \( \raisebox{1ex}{${\lambda}_0$}\!\left/ \!\raisebox{-1ex}{$2$}\right. \); c second harmonic generation microscopy: where two photons with wavelength equal to \( \raisebox{1ex}{${\lambda}_0$}\!\left/ \!\raisebox{-1ex}{$2$}\right. \) excited the molecule, producing an emission equal to λ₃, where λ₃ = \( \raisebox{1ex}{${\lambda}_0$}\!\left/ \!\raisebox{-1ex}{$2$}\right. \); and d third-harmonic generation microscopy: where three photons with wavelength equal to \( \raisebox{1ex}{${\lambda}_0$}\!\left/ \!\raisebox{-1ex}{$3$}\right. \) excited the molecule, producing an emission equal to λ₄, where λ₄ = \( \raisebox{1ex}{${\lambda}_0$}\!\left/ \!\raisebox{-1ex}{$3$}\right. \). The color arrows have length proportional to the energy of the photons and black arrows indicate vibrational energy loss. Solid lines represent real energy levels and dashed lines represent virtual energy levels