Luminescent proteins for high-speed single-cell and whole-body imaging

Abstract

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca(2+), cyclic adenosine monophosphate and adenosine 5'-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.

Figure 1. Development of the bright luminescent protein Nano-lantern.

(a) Schematic of the domain structure of Nano-lantern. Numbers represent the relative amino-acid position in the original protein. (b) Emission spectra of equimolar amounts of the luminescent proteins Nano-lantern, VRL10.3, eBAF-Y⁹, RLuc8_S257G, RLuc8 and RLuc. Emission spectra measurements were performed at least in triplicate, and the averaged data are shown. (c) Luminescence (left) and fluorescence (right) imaging of HeLa cells expressing Nano-lantern targeted to cytoplasm, mitochondria and histone H2B. The exposure times for the luminescence images were 3 s, 3 s and 1 s, respectively. The exposure time for all fluorescence images was 1 s. The reference fluorescence signal was captured by exciting Venus with light at 490 nm. Scale bars, 50 μm.

Figure 2. Luminescence imaging of mice with Nano-lantern-expressing tumours.

(a) Position of colon26 cells expressing Nano-lantern (green circles) or RLuc8 (red circles) in the transplanted mice (dorsal view). (b,d) Representative luminescence images of the indicated numbers of injected cells at 600 s (b) and 1 s exposures (d). (c) Comparison in relative light units (RLU, pixel-integrated counts per s) of 1 × 10⁷ cells of colon26/RLuc8 and Nano-lantern. Measurements were performed in triplicate, and the averaged data and s.d. are shown. (e) Consecutive frames of video-rate images of Nano-lantern-expressing tumour cells in an unshaved mouse. The luminescent signal every 60 ms is shown in green in the still images and as a series of pseudo colours (blue, cyan, green, yellow, red and magenta) in the merged image. Scale bars, 1 cm.

Figure 3. Development of a Nano-lantern-based luminescent Ca²⁺ indicator.

(a) Schematic representation of the domain structures of CaM-M13@91_Nano-lantern and CaM-M13@228_Nano-lantern (Nano-lantern (Ca²⁺)). (b) Relative brightness of recombinant Nano-lantern, CaM-M13@91_Nano-lantern and CaM-M13@228_Nano-lantern (Nano-lantern (Ca²⁺)), with or without Ca²⁺. Measurements were performed at least in triplicate, and the averaged data and s.d. are shown. (c) A series of pseudo-coloured ratio images of HeLa cells expressing Nano-lantern (Ca²⁺) taken at video rate, following histamine stimulation. Scale bar, 10 μm. (d) Time course of the L/L₀ ratio change at an ROI (white box in (c)) in a HeLa cell expressing Nano-lantern (Ca²⁺). Number indicates the time point of each image in (c). Representative data from at least five experiments are shown in (d) and (e). (e) A typical time course of the spatial L/L₀ ratio change in ROIs (cyan, magenta and yellow boxes in (c)) in another HeLa cell. (f) Time course of the L/L₀ ratio change in a rat hippocampal neuron co-expressing Nano-lantern (Ca²⁺) with the ChR2. ChR2 was repeatedly photo-activated in illumination sessions (~2 s), consisting of 15 cycles of camera exposure (100 ms) and pulses of blue light (0.8 ms) that were delivered during periods (30 ms) when camera exposure was off. Numbers indicate the time points of each image in (g). Representative data from five measurements are shown. (g) A series of pseudo-coloured ratio images of a rat hippocampal neuron expressing Nano-lantern (Ca²⁺). Scale bar, 10 μm.

Figure 4. Development of a Nano-lantern-based luminescent cAMP indicator.

(a) Schematic of the domain structure of Nano-lantern (cAMP). (b) Relative brightness of recombinant Nano-lantern and Nano-lantern (cAMP) with or without cAMP. These measurements were performed at least in triplicate, and the averaged data and s.d. are shown. (c) A cAMP image of ~100,000 D. discoideum cells expressing Nano-lantern (cAMP1.6) during aggregative morphogenesis. Scale bar, 1 mm. (d) Space and time plot along the yellow lines shown in (c) for 60 min. (e) Time course of the luminescence intensity change at the ROIs indicated in (c). These are typical data from five measurements.

Figure 5. Development of a Nano-lantern-based luminescent ATP indicator.

(a) Schematic of the domain structure of Nano-lantern (ATP1). (b) Relative brightness of recombinant Nano-lantern and Nano-lantern (ATP1) with or without ATP. These measurements were performed at least in triplicate, and the averaged data and s.d. are shown. (c) Time course of the luminescence intensity change in a plant leaf expressing Nano-lantern (ATP1) with (dashed line) or without (solid line) the mitochondrial ATP synthesis inhibitor oligomycin A. Representative data from five measurements are shown. (d) Plant leaf luminescence images of CT-Nano-lantern (ATP1), chloroplast autofluorescence and merge. Scale bar, 20 μm. (e) A typical pattern of the time course of the luminescence intensity change in a plant leaf expressing CT-Nano-lantern (ATP1) with (dashed line) or without (solid line) the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea with weak light irradiation (0.3 mW cm⁻²). Representative data from five measurements are shown. (f) A typical pattern of the time course of the luminescence intensity change in a plant leaf expressing CT-Nano-lantern (ATP1) (solid line) or in a wild-type plant (dashed line) with pulsed light (8.2 mW cm⁻²) indicated as a white rectangle in the scheme above the graph. Representative data from five measurements are shown.