Luminescent Terbium Protein Labels for Time-Resolved Microscopy and Screening

摘要

Lanthanide luminescence offers several advantages for fluorescence-based biological assays: 1) large Stoke's shifts (>150 nm) and multiple narrow emission bands (< 10 nm at half-maximum) allow efficient spectral separation of emission signals; 2) long luminescence lifetimes (microsecond to millisecond) enable time-resolved detection methods to remove scattering and autofluorescence background; and 3) relative insensitivity to photobleaching allows for prolonged detection. Terbium and europium probes typically incorporate the metal ion into an organic chelating ligand that contains a sensitizing chromophore. When excited with near-UV light in the absorption band, the chromophore transfers energy by intersystem crossing to the triplet excited state and intramolecular transfer to the emissive level of the chelated metal. Direct conjugation of lanthanide probes to antibodies, oligonucleotides, and proteins has enabled the development of sensitive, time-resolved fluorescence resonance energy transfer (TR-FRET) assays of biomolecular interactions in purified biochemical preparations, cellular extracts, and on cell surfaces. Recent efforts have sought to develop lanthanide probes for live-cell imaging applications using time-resolved microscopy with pulsed, near-UV single photon excitation or two-photon excitation.