Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein-protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of proteinprotein interactions. A luminescent terbium complex, TMP-Lumi4, was introduced into cultured cells using two methods: (i) osmotic lysis of pinocytic vesicles; and (ii) reversible membrane permeabilization with streptolysin O. Upon intracellular delivery, the complex was observed to bind specifically and stably to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals from cellular autofluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 μs) between excitation and detection. Background elimination made it possible to detect interactions between the first PDZ domain of ZO-1 (fused to eDHFR) and the C-terminal YV motif of claudin-1 (fused to GFP) in single microscope images at subsecond time scales. We observed a highly significant (P < 10 −6 ), six-fold difference between the mean, donornormalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing noninteracting mutants. The results show that time-resolved LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications. cellular imaging | dihydrofolate reductase | Forster resonance energy transfer | lanthanide luminescence | protein labeling P rotein-protein interactions, often mediated by modular interaction domains, play a fundamental role in the dynamic organization of cells (1). Various experimental techniques such as immunoprecipitation, affinity chromatography, and yeast twohybrid analysis have been used to identify putatively interacting proteins and deduce the biomolecular mechanisms of cell function (2, 3). However, cell-free studies and screening assays do not provide information about the spatio-temporal organization of protein networks in the natural environment of the living cell or organism. A variety of optical methods are available for monitoring protein interactions in cells, including fluorescence cross correlation spectroscopy (FCCS) (4, 5), bimolecular fluorescence complementation (6), translocation-based assays (7-9), and methods that detect intermolecular Förster resonance energy transfer (FRET). Among these methods, only FRET allows dynamic and reversible imaging of protein-protein interactions while simultaneously preserving information about their subcellular distribut...

Brilliance of terbium: Heterodimeric conjugates of trimethoprim covalently linked to sensitized terbium chelates bind to Escherichia coli dihydrofolate reductase fusion proteins with nanomolar affinity (see picture). Terbium luminescence enables sensitive and time‐resolved detection of labeled proteins in vitro and on the surface of living mammalian cells.

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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 (micro-to millisecond) enable time-resolved detection methods to remove scattering and autofluorescence background; and 3) relative insensitivity to photobleaching allows for prolonged detection. [1] 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 via intersystem crossing to the triplet excited state and intramolecular transfer to the emissive level of the chelated metal. [1,2] 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. [3][4][5][6][7][8] Recent efforts have sought to develop lanthanide probes for live cell imaging applications using timeresolved microscopy with pulsed, near-UV single photon excitation or two-photon excitation. [9][10][11][12][13][14][15]

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