Multiplexed fluorescence lifetime imaging by concentration-dependent quenching.

This study sought to use the undesirable concentration-dependent quenching to propose a simple multiplexed imaging analysis for histopathological identification of different stained tissues. To verify this point, the relationship between the fluorescence lifetime and eosin concentration was obtained. At low concentrations, the fluorescence lifetimes of eosin were independent of the concentration (<0.25 μg ml). At moderate concentrations (0.25-1 μg ml), eosin was quenched and its fluorescence lifetime was shortened gradually. Interestingly, the fluorescence of eosin was still quenched when the concentration exceeded 1 μg ml, but its corresponding fluorescence lifetimes increase with increased concentration (>100 μg ml). To further verify that multiplexed imaging of different tissues could be achieved only by eosin, we used fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence lifetimes from hematoxylin and eosin (H&E) stained sections. Working directly on an average fluorescence lifetime (τ) histogram for lifetime-based separation easily achieved multiplexed imaging in situ. H&E stained erythrocytes, smooth muscles, collagen and artificial structures on a prepared microscopic slide could be identified without the need of alternating laser excitation, using hyperspectral systems and special staining or multi-labeled immunofluorescence. Using only eosin, different types of tissues could be distinguished by eosin concentration-dependent quenching. Hence, eosin fluorescence lifetimes potentially simplify multiplexed imaging and may have potential applications for pathological diagnosis.