5.3.2　Principles of two-photon fluorescence imagin...

Two-photon fluorescence is the process in which a fluorophore is excited via near simultaneous absorption of two photons，each having half the energy（twice the wavelength）required for the transition from the ground to the first singlet excited state.The prerequisite for near-simultaneous absorption and the timescale of molecular light absorption（10-16 s）dictates the use of specialized excitation sources.In current instruments，a mode-locked Tisapphire laser delivers infrared light pulses of femtosecond duration at high repetition rates.Two-photon excited fluorescence has a characteristic dependence on the square of the excitation light intensity.Namely，doubling the excitation intensity quadruples the fluorescence signal.In contrast，fluorescence derived from conventional one-photon absorption exhibits linear dependence on excitation light intensity.When irradiated by a laser of a suitable wavelength，some molecules in intravital tissues can emit stable fluorescence，and they are therefore called endogenous fluorophores.Endogenous fluorophores are widely distributed in intravital tissues，such as reduced nicotinamide adenine dinucleotide phosphate（NADPH），porphyrin derivatives，and melanin.Usually，the morphology and boundary of melanocytes in the area of melanoma will change，which can be used for early diagnosis of melanoma.Besides，the percentage of NADPH in skin tissue is related to the level of intracellular redox and metabolism，which can serve as molecular markers for skin aging detection.Therefore，two-photon fluorescence imaging technique has a wide range of applications in the early diagnosis of dermatosis and detection of skin aging.(https://www.daowen.com)