SyncRGB-FLIM: synchronous fluorescence imaging of red, green and blue dyes enabled by ultra-broadband few-cycle laser excitation and fluorescence lifetime detection

• Published in: Biomedical optics express 2019-04, Vol.10 (4), p.1891-1904
• Main Authors: Maibohm, Christian, Silva, Francisco, Figueiras, Edite, Guerreiro, Paulo T, Brito, Marina, Romero, Rosa, Crespo, Helder, Nieder, Jana B
• Format: Article
• Language: English
• ISSN: 2156-7085; 2156-7085
• DOI: 10.1364/BOE.10.001891

We demonstrate for the first time that an ultra-broadband 7 femtosecond (fs) few-cycle laser can be used for multicolor nonlinear imaging in a single channel detection geometry, when employing a time-resolved fluorescence detection scheme. On a multi-chromophore-labelled cell sample we show that the few-cycle laser can efficiently excite the multiple chromophores over a >400 nm two-photon absorption range. By combining the few-cycle laser excitation with time-correlated single-photon counting (TCSPC) detection to record two-photon fluorescence lifetime imaging microscopy (FLIM) images, the localization of different chromophores in the cell can be identified based on their fluorescence decay properties. The novel SyncRGB-FLIM multi-color bioimaging technique opens the possibility of real-time protein-protein interaction studies, where its single-scan operation translates into reduced laser exposure of the sample, resulting in more photoprotective conditions for biological specimens.