Open AccessSyncRGB-FLIM: synchronous fluorescence imaging of red, green and blue dyes enabled by ultra-broadband few-cycle laser excitation and fluorescence lifetime detection
Author(s) -
Christian Maibohm,
Francisco Silva,
Edite Figueiras,
Paulo T. Guerreiro,
Marina Brito,
Rosa Romero,
Helder Crespo,
Jana B. Nieder
Publication year - 2019
Publication title -
biomedical optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.10.001891
Subject(s) - femtosecond , fluorescence , fluorescence lifetime imaging microscopy , laser , chromophore , optics , materials science , laser induced fluorescence , photon counting , microscopy , excitation , absorption (acoustics) , optoelectronics , photon , chemistry , photochemistry , physics , quantum mechanics
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.