Although Förster resonance energy transfer is well suited to the investigation of protein interactions in vitro and within live cells, its efficacy can be diminished by a variety of factors during studies, including photobleaching, crosstalk and autofluorescence. In addition, FRET efficiency can be limited by the structure, size, geometry and separation of the donor and acceptor probes used in the studies. For example, the chromophores within cyan and yellow fluorescent protein reside deep within the protein matrix.Researchers in Gerard Marriott’s laboratory at the University of Wisconsin-Madison are interested in mapping changes in the distribution of protein complexes during cell motility. FRET is the most widely used technique for monitoring protein interactions in live cells, but there are limitations of the technique for such applications. Researchers have reported a technique that improves FRET sensitivity. GFP serves as the donor probe in a GFP-AGT protein complex. GFP does not engage in FRET with the spiro (SP) state. However, the absorption of the merocyanine (MC) state, formed on UV or two-photon excitation of SP, exhibits a strong spectral overlap with GFP emission that results in FRET and a decrease in GFP emission. Excitation of MC with 543-nm light generates the SP state (no FRET) to complete a single optical switch cycle.“It is highly possible that only a few percent of a donor-labeled protein will engage in FRET with the acceptor probe in a cell, and so meaningful results, especially in studies requiring multiple or long-term measurements of protein interactions, can only be obtained by greatly improving the accuracy of the determination of FRET efficiency,” Marriott said. Thus, the researchers started to think about a different way to measure fluorescence emission for conventional and FRET imaging and proposed a new “optical lock-in detection” (OLID) approach that would allow accurate imaging of protein complexes, even when less than 10 percent of the donor probe engages in FRET.One of the OLID imaging techniques is detailed in the Feb. 15 online issue of Biophysical Journal. In the paper, the Marriott group, in collaboration with colleagues at Vanderbilt and Stanford universities, reported a method that significantly improves the sensitivity of the FRET technique. It is based on an optical switch acceptor probe — a GFP-alkylguaninetransferase fusion protein labeled with benzylguanine-NitroBIPS, generally referred to as, simply, NitroBIPS — that allows the investigators to turn FRET on and off over many cycles of optical switching, generating a highly modulated FRET waveform.NitroBIPS and related optical switch probes exist in two distinct structural states that can be efficiently interconverted using two wavelengths of light. The absorption of the merocyanine structural state strongly overlaps with the emission of donor probes such as GFP, acting as an efficient acceptor probe in FRET with GFP, whereas the spiro structural state does not. Previous studies by the Marriott laboratory have established that it is possible to generate the merocyanine state by exciting the spiro state at 365 or 720 nm (two-photon excitation) and the spiro state by exciting the merocyanine state at 543 nm. A bonus property of the NitroBIPS is that the excited state of merocyanine can sometimes return to its ground state with the emission of a red photon, and this emission can be used to read out the state of the switch in complex environments such as within a living cell.The researchers took advantage of these properties when developing the new technique. By applying a defined sequence of two-photon and 543-nm scans of a sample containing interacting GFP and NitroBIPS-labeled proteins, they could modulate the amount of the merocyanine state in the system, thereby modulating the efficiency of FRET. Because the intensity of GFP fluorescence in a GFP-NitroBIPS complex varies with the waveform of optical switching, this fluorescence could be isolated from background signals and from GFP probes not engaged in FRET using an analysis based on a simple lock-in and phase-sensitive detection of the modulated GFP signal.Conceptually, the technique is much like combining photoactivation and photobleaching of the acceptor, both of which facilitate detection of FRET between labeled proteins and, therefore, can serve as indicators of the formation of a complex in cells. Photoactivation and photobleaching, however, are irreversible and require control experiments to ensure that they have not destroyed local protein activity or triggered a stress response in the cell. Furthermore, these techniques usually allow only a single measurement of a relative measure of FRET because of time-dependent change in the donor population. With the OLID-FRET technique, investigators can make multiple, absolute determinations of FRET efficiency by reversibly switching the acceptor probe completely on and off during each optical switching cycle and without the release of photoproducts.The investigators demonstrated the new method using in vitro and in vivo samples. They performed OLID-FRET by using two-photon excitation of spiro (720 nm) to trigger a transition to merocyanine, and by using excitation at 543 nm to trigger the transition from merocyanine to spiro. They performed the experiments on a Zeiss microscope equipped with a 40×, 1.2-NA objective. A Coherent Ti:sapphire laser oscillator provided the 720-nm excitation, and a helium-neon laser resident in the Zeiss microscope provided the 543-nm excitation.In addition, the investigators developed an in vivo labeling method to deliver NitroBIPS probes to specific proteins, enabling the method to be used with proteins in living cells. This involved labeling a commercially available genetically encoded Snap-tag reagent from Covalys Biosciences AG of Witterswil, Switzerland, with a suicide substrate linked to NitroBIPS. The in vivo labeling reaction between the two proved very efficient in vitro and in vivo.The experiments showed that FRET efficiencies as low as 1 percent can be measured using OLID-FRET between GFP and NitroBIPS, recommending the technique for a range of applications. “This property should greatly improve the accuracy and the types of protein interactions that can be imaged in cells, especially in the majority of cases where endogenous — that is, unlabeled — proteins dilute the probability of FRET between the donor and acceptor probes,” Marriott said.He noted that fluorescence lifetime imaging microscopy (FLIM) also can be used to image protein interactions in cells and offers the added advantage of not requiring knowledge of the number of donor probes that engage in FRET with an acceptor. “However, FLIM is still limited by how well the lifetimes … of donor-acceptor population can be resolved from the uncomplexed donor probes. Generally, this is no better than 10 percent.”The researchers in the Marriott laboratory are working to extend the technique in several ways. First, they have made optical switches whose merocyanine state acts as an efficient acceptor for redshifted dyes and fluorescent proteins; using this in conjunction with NitroBIPS could enable imaging of two different protein complexes in cells. Also, Marriott explained that, given the ease of synthesis, there is no reason why they cannot extend the wavelength of the merocyanine state beyond 800 nm, which would create opportunities for true multiplexed imaging of protein interactions in cells and in tissue.Finally, the researchers have developed an OLID technique that employs particular genetically encoded proteins as optically switchable acceptor probes. These will enable them to sidestep the need for in vivo labeling, allowing the improvement of imaging of specific proteins and their interactions in the tissue of living organisms. Also on the horizon is a new class of synthetic OLID probe that will make it possible to isolate signals from specific labeled proteins within a large background at the level of single molecules.