Two-color excited-state absorption imaging of melanins

Abstract

We have demonstrated a new method for imaging melanin with two-color excited state absorption microscopy. 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 place in the medium. We can easily measure 10-6 absorption changes caused by either instantaneous two-photon absorption or relatively long lived excited state absorption with a RF lock-in amplifier. Eumelanin and pheomelanin exhibit similar excited state dynamics. However, their difference in excited state absorption and ground state absorption leads to change in the phase of the transient absorption signal. Scanning microscopic imaging is performed with B16 cells, melanoma tissue to demonstrate the 3D high resolution imaging capability. Different melanosome samples are also imaged to illustrate the differences between eumelanin and pheomelanin signals. These differences could enable us to image their respective distribution in tissue samples and provide us with valuable information in diagnosing malignant transformation of melanocytes.