Fluorescence detection is the dominant technology for cellular imaging, clinical diagnostics, DNA analysis and drug discovery. In fluorescence microscopy, the fluorophores are subject to photobliching and detectability is limited by cellular autofluorescence. We describe new approach to develop fluorescent probes that display long emission wavelength, long decay times, and high quantum yield and high fluorescence brightness. These luminophores are covalently linked pairs of long-lifetime fluorophores (like metal-ligand complexes) and a short-fluorescence-lifetime and high quantum yield dyes. Using resonance energy transfer (RET) it is possible of obtaining desirable spectral properties and long fluorescence lifetime in covalently linked pairs. The long-lifetime donor results in a long-lived fluorescence component in the acceptor decay. Importantly the emission spectrum of the luminophore is that of the acceptor and quantum yield of the luminophore approaches that of the higher quantum yield acceptor. Such luminophores are suitable for fluorescence measurements in biological samples with the use of real time background suppression to eliminate autofluorescence. We discuss experimental examples based on long lived metal-ligand-complexes and long wavelength acceptors like Texas Red. The emission maxima (spectra) and decay time of such RET tandems can be readily adjusted by selection of the donor, acceptor and distance between them. Such luminophores with long-wavelength emission and adjustable long lifetime can have numerous applications in one-photon and multi-photon cellular and tissue imaging with the use of off-gating the excitation pulse and sample autofluorescence.
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