Abstract
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.
Highlights
Cellular processes govern and are the basis for life, where proteins play a key role
The highly complex cellular mechanics are governed by protein-protein interactions, which allow orchestrated action of chemical processes related to metabolism, cell growth, differentiation, and nutrient uptake, to name a few [1]
Imaging is performed in a simple single detector microscope setup coupled to time-correlated single-photon counting (TCSPC) electronics [22] (see Fig. 1(b))
Summary
Cellular processes govern and are the basis for life, where proteins play a key role. Protein-protein interactions often influence chemical reactions in the direct environment and the protein molecular complexes might change their tertiary conformation or intracellular location enabling trafficking, ion/proton transfer or the dislocation of molecules, e.g. along filament strands. These interactions mostly involve multiple constituents and to observe the effect of one protein action in sync with the reacting protein species is not trivial but could potentially enable a deeper understanding of the relevant interactions that govern the basic building blocks of living systems. Contrast between different cellular constituents is created via intensity, lifetime, spectra or labeling [2,3,4,5,6] and optical techniques are perfectly suited to solve the challenge of tracking protein-protein interactions in real time
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