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...

By imaging the release of a GFP-based viral content marker produced upon virus maturation, we have previously found that HIV-1 fuses with endosomes. In contrast, fusion at the cell surface did not progress beyond a lipid mixing stage (hemifusion). However, recent evidence suggesting that free GFP can be trapped within the mature HIV-1 capsid raises concerns that this content marker may not be released immediately after the formation of a fusion pore. To determine whether a significant portion of GFP is trapped in the mature capsid, we first permeabilized the viral membrane with saponin. The overwhelming majority of pseudoviruses fully released GFP while the remaining particles exhibited partial loss or no loss of content. The extent of GFP release correlated with HIV-1 maturation, implying that incomplete Gag processing, but not GFP entrapment by mature capsids, causes partial content release. Next, we designed a complementary assay for visualizing pore formation by monitoring the intraviral pH with an additional pH-sensitive fluorescent marker. The loss of GFP through saponin-mediated pores was associated with a concomitant increase in the intraviral pH due to equilibration with the pH of an external buffer. We next imaged single HIV-cell fusion and found that these events were manifested in a highly correlated loss of content and increase in the intraviral pH, as it equilibrated with the cytosolic pH. Fused or saponin-permeabilized pseudoviruses that partially lost GFP did not release the remaining content marker under conditions expected to promote the capsid dissociation. We were thus unable to detect significant entrapment of GFP by the mature HIV-1 capsid. Together, our results validate the use of the GFP-based content marker for imaging single virus fusion and inferring the sites of HIV-1 entry.

Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (\100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, k em 5 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Europium chelate-impregnated microspheres were used to evaluate instrument performance in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast, and signal-to-noise ratio (SNR). About 200 nm microspheres were imaged within the time resolution limit of the ICCD (66.7 ms) with complete autofluorescence suppression. About 40 nm microspheres containing $400 chelate molecules were detected within $1-s acquisition times. A luminescent terbium complex, Lumi4-Tb 1 , was introduced into the cytoplasm of cultured cells at an estimated concentration of 300 nM by the method of osmotic lysis of pinocytic vesicles. Time-resolved images of the living, terbium complex-loaded cells were acquired within acquisition times as short as 333 ms, and the effects of increased exposure time and frame summing on image contrast and SNR were evaluated. The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions. ' 2010 International Society for Advancement of Cytometry Key terms fluorescence microscopy; lanthanide; time-resolved; signal-to-noise ratio THE sensitivity and precision of fluorescence-based bioassays and microscopy is often diminished by autofluorescence background emitted from samples and containers or coverslips. Time-resolved luminescence (TRL) measurements can effectively eliminate autofluorescence when the detected analyte has an emission lifetime exceeding that of endogenous fluorophores (\100 ns) (1). TRL instrumentation uses a finite pulse of light to excite a sample (Fig. 1). Then, the detector is electronically switched on, or unshuttered after a short interval (the gate delay) during which sample autofluorescence has diminished. The detector remains on for a finite interval (the gate width), and the output from multiple excitation/emission cycles can be integrated to increase signal.The most common emissive probes for TRL-based bioassays are ligand-sensitized, coordination complexes of lanthanide cations. Lanthanide complexes (LCs) have long lifetimes (ls-ms) that facilitate TRL detection as well as large Stokes shifts ([150 nm) and multiple, narrow emission bands that make ...

Retinal vascular diseases, including diabetic retinopathy, neovascular age related macular degeneration, and retinal vein occlusion, are leading causes of blindness in the Western world. These diseases share several common disease mechanisms, including vascular endothelial growth factor (VEGF) signaling, hypoxia, and inflammation, which provide opportunities for common therapeutic strategies. Treatment of these diseases using laser therapy, anti-VEGF injections, and/or steroids has significantly improved clinical outcomes. However, these strategies do not address the underlying root causes of pathology, and may have deleterious side effects. Furthermore, many patients continue to progress toward legal blindness despite receiving regular therapy. Nanomedicine, the engineering of therapeutics at the 1-100 nm scale, is a promising approach for improving clinical management of retinal vascular diseases. Nanomedicine-based technologies have the potential to revolutionize the treatment of ophthalmology, through enabling sustained release of drugs over several months, reducing side effects due to specific targeting of dysfunctional cells, and interfacing with currently “undruggable” targets. We will discuss emerging nanomedicine-based applications for treatment of complications associated with retinal vascular diseases, including angiogenesis and inflammation.