Quantitative Time-Gated Lanthanide Microscopy to Study Protein Dynamics in Live- Cells

In order to deduce the molecular mechanisms of biological function, it is necessary to monitor changes in the subcellular location, activation, and interaction of proteins within living cells in real time. Förster resonance energy-transfer (FRET)- based biosensors that incorporate genetically encoded, fluorescent proteins permit high spatial resolution imaging of protein−protein interactions or protein conformational dynamics. However, a nonspecific fluorescence background often obscures small FRET signal changes, and intensity-based biosensor measurements require careful interpretation and several control experiments. Moreover, the broad excitation and emission spectra of fluorescent proteins makes it difficult to image multiple FRET events in a single cell. These problems can be overcome by using lanthanide [Tb(III) or Eu(III)] complexes as donors and green fluorescent protein (GFP) or other conventional fluorophores as acceptors. Essential features of this approach are the long-lifetime (approximately milliseconds) luminescence of Tb(III) complexes and time-gated luminescence microscopy. This allows pulsed excitation, followed by a brief delay, which eliminates nonspecific fluorescence before the detection of Tb(III)-to-GFP emission. While time-gating increases signal-to-background ratio, the inherently low photon emission rates of long-lived lanthanide probes may yield unacceptably low signal-to-noise ratios (S:N) because S:N depends on the total number of photons acquired in an image. This study quantitatively evaluated the performance of lanthanide-based FRET microscopy with respect to such performance measures as photon collection efficiency, temporal resolution, signal-to-noise ratio (S:N) and dynamic range. Images of Tb(III) luminescence and Tb(III)-to-GFP FRET were acquired in living mammalian cells under experimental conditions that approximated those used when imaging fluorescent protein biosensors to study their application in lanthanide-based biosensors for cellular imaging. Analyses showed that Tb(III) and Tb(III)-mediated FRET signals could be quantified with high precision (S:N > 5) despite relatively low numbers of photons acquired (<30 per pixel) and that temporal resolution and dynamic range were similar to those seen with fluorescent protein FRET. Additional experiments highlight the potential of multiplexed biosensor imaging using a single Tb(III) donor and two or more differently colored fluorescent protein acceptors.