Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes

Abstract

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here, we demonstrate that, from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image four and with the help of self-labeling protein tags up to eight different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.

Genetically encoded fluorescence lifetime biosensors: overview, advances, and opportunities

“…Moreover, the phasor analysis allows the identification of different metabolic states of cells during differentiation even with small changes in their redox states, as well as rapid screen of sensors for their lifetime differences in fluorescence lifetime multiplexing. 58,59 Combined with the phasor analysis, the pH-sensitive GFP variant E 2 GFP was employed to monitor intracellular pH under different physiological conditions (Fig. 5a-f).…”

HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing

“…Together, the different variants enable multiplexed FLIM of up to three targets with a single fluorophore ligand, and up to eight targets when combined with other fluorophore labels. [91] The multiplexing capability was further exploited to design single wavelength cell-cycle Fucci biosensors, using HaloTag7 and HaloTag9 attached to human Cdt1 and Geminin respectively. [90] Advantageously, further multiplexing is possible, as demonstrated by simultaneous imaging of the HaloTag-Fucci labelled with MaP618 and a GFP-based RhoA GTPase activity biosensor.…”

“…The other two variants reduce quantum yield, and hence fluorescence lifetime, due to PeT quenching from a proximate tryptophan. Together, the different variants enable multiplexed FLIM of up to three targets with a single fluorophore ligand, and up to eight targets when combined with other fluorophore labels [91] . The multiplexing capability was further exploited to design single wavelength cell‐cycle Fucci biosensors, using HaloTag7 and HaloTag9 attached to human Cdt1 and Geminin respectively [90] .…”

Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging

“…However, it is difficult to perform highly multiplexed fluorescence imaging because fluorescence peaks are intrinsically broad, and so the number of simultaneously resolvable targets is limited. Consequently, there have been only a few successful demonstrations of multiplexed fluorescence imaging. − …”