Wang, KeLiu, Tzu-MingWu, JuwellHorton, Nicholas G.Lin, CharlesXu, Chris2013-03-192012Wang, Ke, Tzu-Ming Liu, Juwell Wu, Nicholas G. Horton, Charles P. Lin, and Chris Xu. 2012. Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy. Biomedical Optics Express 3(9): 1972-1977.2156-7085http://nrs.harvard.edu/urn-3:HUL.InstRepos:10448706We demonstrate a fiber-based, three-color femtosecond source for simultaneous imaging of three fluorescent proteins (FPs) using two-photon fluorescence microscopy (2PM). The three excitation wavelengths at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation (SHG) of the 1550-nm pump laser and the 1728-nm and 1900-nm solitons generated through soliton self-frequency shift (SSFS) in a large-mode-area (LMA) fiber. These energetic pulses are well matched to the two-photon excitation peaks of red, cyan and yellow fluorescent proteins (TagRFPs, TagCFPs, and TagYFPs) for efficient excitation. We demonstrate simultaneous 2PM of human melanoma cells expressing a “rainbow” combination of these three fluorescent proteins.en-USJournal Article2013-03-1910.1364/BOE.3.001972