Figure 2. Energy diagrams of Two-Photon Autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS). A: Energy diagram of TPAF, in which an autofluorescent molecule simultaneously absorbs two optical infrared photons (E_2p). After internal-crossing (IC), in which some energy is lost, the fluorescent molecule will emit a fluorescence photon (E_em). B: Energy diagram of CARS, in which two optical photons with the photon energy difference (E_pump - E_Stokes) equaling to the vibrational energy of a molecule (E_Ω) is used to excite the vibrational motion of the molecule. A third photon (E_probe) is subsequently used to interact with the vibtational motion of the molecule, resulting in the emision of an energy-upshifted photon (E_CARS). C: A schematic diagram illustrating the CARS process. The pump and the Stokes photons are simultaneously exciting the lipid molecule, with the energy difference between the two photons equal to the vibrational energy of the molecule bond (E_Ω). Subsequent interaction of the probe photon coherently interacts with the vibrational motion of the molecule to generate a release of the CARS photon.