Figure 1.

Comparison of one-and two-photon excitation. (A) Cartoon illustrating the fundamental differences between normal (i.e., one-photon) absorption and two-photon absorption. As blue light is focused by a lens during one-photon excitation of a chromophore the concentration of excited molecules increases but the same number of molecules are excited in each z section. Whereas, during two-photon irradiation with NIR the total flux in each z section is constant (red), but excitation only occurs at the focus (green). Hence, for fluorescence emission can be seen throughout the light path for one-photon, but is pin-point for two-photon. Adapted from Ellis-Davies (2011b), by permission of the American Chemical Society. (B) Simple Jablonski diagram illustrating the absorption of light from the ground state (S₀) to excited state (S₁) occurs directly with one-photon absorption (hu₁), and goes via a “virtual state” K in the case of two-photon excitation (hu₂).