A versatile bioluminescent probe with tunable color

Abstract

Bioluminescence is a powerful method for imaging in vivo, but applications at the microscale are far from routine. This is due, in part, to a lack of versatile tools for visualizing dynamic events. To address this void, we developed a new platform-Bioluminescence Resonance Energy mAKe over with a Fluorescence-Activating absorption-Shifting Tag (BREAKFAST). BREAKFAST features a bright luciferase combined with a chemogenetic tag (pFAST) for rapid color switching. In the presence of luciferin and a discrete fluorogenic ligand, signal is observed via resonance energy transfer. We evaluated spectral outputs with various fluorogens and established the utility of BREAKFAST for combined fluorescence and bioluminescence imaging. Dynamic, four-color visualization was achieved with sequential ligand addition and spectral phasor analysis. We further showed selective signal quenching with a dark fluorogen. Collectively, this work establishes a new method for bioluminescence imaging at the cellular scale and sets the stage for continued probe development.

Fig. 1. BREAKFAST components and overall design. (a) NanoLuc luciferase catalyzes the oxidation of furimazine (Fz) to produce furimamide and light. (b) Fluorogenic ligands that reversibly bind to pFAST. (c) Tuning BREAKFAST emission via fluorogen addition.

Fig. 2. Bioluminescence emission spectra with BREAKFAST probes. (a) Structures of candidate fluorogens. Spectra were acquired with BREAKFAST in the presence of (b) Fz only, (c) Fz and HMBR, (d) Fz and HBR-3,5DM or (e) Fz and HBR-3,5DOM. For all measurements, Fz and fluorogen were used at equimolar ratios (50 μM or 1 : 50 dilution of commercial stocks). B.E. = BRET efficiency.

Fig. 3. BREAKFAST can be resolved with both fluorescence and bioluminescence imaging using phasor analysis. HEK293 cells expressing BREAKFAST were incubated with fluorogens. (a) Fluorescence images of cells treated with HMBR, HBR-3,5DM, or HBR-3,5DOM and the associated phasor plots. (b) Bioluminescence images of cells treated with Fz and either HMBR, HBR-3,5DM, or HBR-3,5DOM. The associated phasor plots are also shown. For (a) and (b), scale bars = 10 μm.

Fig. 4. Color tuning with BREAKFAST probes. A549 cells were engineered to express NanoLuc-pFAST in the nucleus. Luciferin (Fz) and fluorogens were then flowed in as depicted, and images were acquired between each addition. Distinct spectral outputs were observed depending on the ligand present. Some cells detached in the imaging chamber under flow. For all images, scale bars = 10 μm.

Fig. 5. Reversible bioluminescence quenching. (a) Cartoon representation of the quench and rescue experiment. (b) HeLa cells were transiently co-transfected with ER-localized YeNL (KDEL-YeNL) and nuclear localized BREAKFAST reporters. Cells were treated with Fz only, followed by HBIR-3M (quencher), and finally HMBR (fluorogen). Vertical scale bars for the whole cell images represent pixel intensity (in a.u.). Images are representative of n = 3 independent experiments. Scale bars = 10 μm. (c) Quantification of bioluminescence signal quenching for BREAKFAST and YeNL in (b).