Two-color, two-photon, and excited-state absorption microscopy

We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption mi- croscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the me- dium. We can easily measure 10 6 absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption TPA and the relatively long-lived excited-state absorption ESA of melanin are carried out in solution, and imaging capability is demon- strated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes one at 3p s, one lasting hun- dreds of nanoseconds when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy TPFM. This new technique could potentially provide valuable infor- mation in diagnosing melanoma. © 2007 Society of Photo-Optical Instrumentation