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 Ca2+, 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. SIMILAR CONTENT BEING VIEWED BY OTHERS

MULTIPLEXED BIOLUMINESCENCE MICROSCOPY VIA PHASOR ANALYSIS Article 23 June 2022 BRIGHT AND PHOTOSTABLE YELLOW FLUORESCENT PROTEINS FOR EXTENDED IMAGING Article Open access 04 April 2025 A

GENERAL METHOD FOR THE DEVELOPMENT OF MULTICOLOR BIOSENSORS WITH LARGE DYNAMIC RANGES Article Open access 08 June 2023 INTRODUCTION Although fluorescence imaging is widely used, its

dependence on external illumination prevents its universal application. For example, fluorescence imaging cannot be easily used to study light-dependent biological processes, such as visual

photoreception or photosynthesis. Although optical recording of the light-sensitive retina has been successfully performed using 2-photon excitation with a 930-nm femtosecond laser1, this

method is not versatile because many biological molecules have significant absorption at both visible and infrared wavelengths2. Therefore, methods based on fluorescence with both 1-photon

and 2-photon excitation cannot always be used to study light-dependent biological processes. Moreover, fluorescence is incompatible with non-invasive deep tissue imaging of whole organisms

and other applications where the cellular substrate is autofluorescent (for example, chloroplasts of photosynthetic plants), saturated with photopigments (porphyric hepatocytes, melanocytes

or retinal pigment epithelia) or extremely photosensitive. Experimental problems also arise when external illumination is required, as for biological technologies such as optogenetics,

chromophore-assisted light inactivation and photolysis of caged compounds, which prevents simultaneous use of fluorescence imaging. Finally, the general power density of external

illumination for live-cell microscopy with fluorescence (sub W cm−2) sometimes causes phototoxic effects in visualized substrates, which alters cellular behaviour and ultimately leads to

cell death. In contrast, chemiluminescence generates a visible light signal through a localized chemical reaction without the need for external illumination. Because the _k_cat values of

conventional luciferases range from 0.1 for firefly luciferase (quantum yield=0.5)3 to approximately 4.4 for _Renilla_ luciferase (RLuc)8 (quantum yield=0.053)4, transient expression of

micromolar concentrations of luciferases, for example, RLuc8, generates a power density of emitted light that is 1/103 that of the general power density required for fluorescence emission in

live-cell imaging (approximately 0.1 μW cm−2). Therefore, although chemiluminescent proteins, including aequorin and luciferases, have been used to image living cells and organisms5,6, the

light output from these proteins is insufficient to provide temporal and/or spatial resolution equivalent to fluorescence. In the case of luminous organisms, such as the sea pansy _Renilla

reniformis_, the problem of low brightness has been solved in nature by the phenomenon of bioluminescence resonance energy transfer (BRET), in which the excited energy of a luminescent

substrate, coelenterazine, bound to RLuc is efficiently transferred intermolecularly to the acceptor _Renilla_ green fluorescent protein (quantum yield=0.3) by a Förster resonance energy

transfer (FRET) mechanism, thereby increasing the emitted photon number approximately six-fold7. Based on this natural intermolecular BRET, intramolecular BRET probes, such as aequorin-GFP8

and BAF-Y9, have been developed. Although these probes allow for live-cell imaging with improved resolution in space and time, they still underperform compared with fluorescent protein-based

probes because of low brightness. To address this problem, we obtained a brighter RLuc by random mutagenesis and fusion to a yellow fluorescent protein (YFP) with high BRET efficiency. The

fusion protein showed much brighter luminescence than BAF-Y, enabling not only real-time imaging of intracellular structures in living cells but also sensitive tumour detection in freely

moving mouse. Moreover, we developed Ca2+, cyclic adenosine monophosphate (cAMP) and adenosine 5′-triphosphate (ATP) indicators based on this bright luminescent protein. These luminescent

indicators will allow visualization after the optical control of cellular or enzymatic activity at the single-cell, organ and whole-body level in animals and plants. RESULTS DESIGN AND

APPLICATION OF THE BRIGHT LUMINESCENT PROTEIN To improve brightness, we designed a chimeric protein based on eBAF-Y9 (Supplementary Note 1), which is a fusion of enhanced YFP and an enhanced

RLuc, RLuc8 (4). Because BRET efficiency, and thus brightness, depends on the photochemical and physical properties of the donor and acceptor, we honed these parameters by improving donor

brightness, maximizing spectral overlap between the donor emission and acceptor absorbance using Venus10 and optimizing the spatial arrangement of the donor and acceptor in the fusion

construct (Supplementary Note 1, Supplementary Fig. S1 and Supplementary Table S1). The resulting protein, which we called Nano-lantern, reminiscent of a light source with nanometre scale

(Fig. 1a), exhibited 5.3 and 2.9 times greater luminescence than RLuc8 and eBAF-Y, respectively, over the entire emission range (Fig. 1b). The improved brightness of Nano-lantern should

generate power densities in the range of 1 μW cm−2 (versus 0.1 μW cm−2 for RLuc8) following transient overexpression in the micromolar range in human cells, and thus increase imaging

potential. Indeed, when Nano-lantern was expressed in HeLa cells, a luminescence image with quality almost comparable to that of fluorescence images was obtained. Nano-lantern and fusions

with defined localization tags enabled visualization of cell compartments and organelles, such as the cytoplasm, mitochondria and nucleus (histone H2B), in living cells by using low

magnification lens (X20 dry objective) with brief, 1–3-s exposures (Fig. 1c). Nano-lantern also allowed for visualization of finer structures, including microfilaments, microtubules and

their tips (EB3) with a high magnification lens (X60 oil-immersion objective) and longer, 3–60 s, exposures (Supplementary Fig. S2). Therefore, the enhanced luminescent signal (μW cm−2) from

Nano-lantern enables imaging at high spatial resolution until at least the subcellular level, with a quality approaching that of fluorescent protein (FP)-based imaging. This is because

although the power density of the emitted light of Nano-lantern is still ~1/102 smaller than that of general fluorescence emissions (at ~100 μW cm−2), image quality is defined by the

signal-to-noise ratio (S/N ratio). In fluorescence imaging, this is compromized by high noise levels from stray excitation light and autofluorescence from both the sample and the medium. In

chemiluminescence imaging, quality does not suffer from low signal levels because of the very low noise level. Because external illumination is not needed, an adequate S/N ratio is

preserved. Currently, chemiluminescence is commonly used for non-invasive tumour detection in living mice for tumour metastasis research and anti-cancer-drug screening11. Brighter

luminescence should enable detection of smaller tumours. Indeed, when adenocarcinoma colon26 cell lines that stably express Nano-lantern or RLuc8 (colon26/Nano-lantern and colon26/RLuc8) was

subcutaneously implanted into BALB/c nude mice (Fig. 2a), and we detected as few as 103 Nano-lantern-expressing cells 1 day after transplantation (Fig. 2b), which is 1 order of magnitude

better than RLuc8-expressing tumours (Fig. 2c). The increased brightness of Nano-lantern allowed for imaging of 105 and 106 cells at 0.1 and 1 Hz sampling rates, respectively (Fig. 2d).

Surprisingly, we could even obtain video rate (30 Hz) imaging of tumours 17 day after implantation of 106 of tumour cells in freely moving unshaved mice (Fig. 2e and Supplementary Movie 1).

In previous reports of luminescent tumour cell imaging in non-anaesthetized mice12, successful images were only obtained with nude mice, larger tumour samples and prolonged exposure times

(160 or 200 versus 33 ms in this report). Increased sensitivity and faster imaging without the need for anaesthesia using Nano-lantern greatly facilitates the analysis of tumour growth and

metastasis in living animals. FUNCTIONAL INDICATORS BASED ON NANO-LANTERN Having demonstrated the benefit of the improved luminescent properties of Nano-lantern, micrometre spatial

resolution in living cells and improved tumour resolution in whole animals, we sought to develop functional indicators for bioactive molecules such as Ca2+, cAMP and ATP based on

Nano-lantern. Thus far, two types of chemiluminescence-based functional indictors have been reported; one uses the complementation of split luciferase (CSL), in which the luciferase molecule

is split, with one portion fused to the sensor domain of a bioactive molecule and the other to a peptide that binds the sensor domain. This type of indicator emits a signal upon bioactive

molecule-binding-mediated reconstitution of the split luciferase13. The other uses a BRET-based indicator composed of the sensor domain of a bioactive molecule flanked by an intact

chemiluminescent donor and a fluorescent acceptor, in this case, a cellular response is detected as a change in BRET efficiency14. Although CSL-type indicators have a larger signal change,

their absolute intensity is often too low to be imaged. Conversely, BRET-based type indicators have sufficient signal intensity for imaging; however, its signal change upon target molecule

binding is generally small. To overcome these drawbacks, we sought to insert a sensor domain into Nano-lantern to create a hybrid CSL/BRET indicator that would exhibit a signal change with

sufficient intensity for imaging (Supplementary Fig. S3a). A CA2+ INDICATOR BASED ON NANO-LANTERN Given the novel hybrid approach, we first sought to develop a Nano-lantern-based indicator

for Ca2+, one of the most important second messengers in living cells. We cloned the Ca2+-sensing domain (CaM-M13) of yellow cameleon, an established FRET-based Ca2+-indicator15, and

searched for the optimal insertion site in Nano-lantern that would yield a large signal change and preserve high intensity emission. Other important properties, including Ca2+ affinity and

reversibility of the catalytic activity of luciferase, were also examined. Ultimately, of the sites tested, we found that insertions into the non-structural Gly228/Gly229 loop in RLuc8 that

links the hydrolase and cap domains were well tolerated (Supplementary Fig. S3b and Supplementary Note 2). The resulting construct, named Nano-lantern (Ca2+), showed a 300% signal change at

35% brightness of the parental protein at saturating Ca2+ concentrations (Fig. 3a). The Ca2+ affinities of Nano-lantern (Ca2+) and its derivatives (Supplementary Fig. S4 and Supplementary

Table S2) were almost identical to that of the parental yellow cameleon series, suggesting that reconstitution of split-Rluc8 in Nano-lantern (Ca2+) did not interfere with the Ca2+-induced

conformational change in the CaM-M13 domain (Supplementary Note 2). To compare the performance of this Ca2+ indicator to a known standard, we expressed Nano-lantern (Ca2+) in HeLa cells.

Following stimulation with 10 μM histamine, an acute Ca2+ spike (S/N=70) followed by Ca2+ oscillations with smaller amplitudes (S/N=29) were detected with a sampling rate as fast as 30 Hz

(Fig. 3c). We could even observe the propagation of a cytoplasmic Ca2+ wave from one end of the cell to the other, as was previously observed using yellow cameleon15 (Fig. 3e and

Supplementary Movie 2). Histamine-induced Ca2+ oscillations were abolished by the addition of the histamine receptor antagonist cyproheptadine, confirming that the luminescent signal change

reflects changes in cytoplasmic [Ca2+] (Supplementary Fig. S5). Previous CSL-based Ca2+ indicators13 were not fully reversible in response to physiological or stimulated Ca2+ release. We

confirmed by co-imaging Nano-lantern (Ca2+) with R-GECO116 that the kinetics of Nano-lantern (Ca2+) parallel those of fluorescence-based probes (Supplementary Fig. S6). Encouragingly,

Nano-lantern (Ca2+), when expressed in dissociated rat hippocampal neurons (Supplementary Fig. S7), and imaged at 10 Hz, could report spontaneous cytoplasmic [Ca2+] spikes and transient

[Ca2+] changes due to reversible stimulation (50 mM KCl). To realize the theoretical advantage of Ca2+ imaging using luminescence probes over fluorescence-based indicators, we performed Ca2+

imaging combined with the control of neuronal activity using optogenetic tools. Ectopic expression of photoreceptor proteins, such as halorhodopsin and channelrhodopsin2 (ChR2), has enabled

the functional analysis of neuronal cells and their networks with ms time resolution17. Yet, these tools are spectrally incompatible with fluorescent Ca2+ indicators. Excitation light can

be used either to control the photoreceptor or to measure the Ca2+ transient, but not for both in the same cell at the same time. Because luminescence imaging can be performed in the absence

of external light, it can be partnered with an optogenetic strategy without undesired interference due to the activity of the photoreceptor. To demonstrate this, we co-expressed

Nano-lantern (Ca2+) and ChR2 in dissociated rat hippocampal neurons. The luminescent signal was imaged every 100 ms, and ChR2 was activated with a 0.8-ms pulse of blue light (438 nm) during

the dead time of the CCD chip for data transfer18. In response to 15 cycles of activation light, a rapid increase was observed in the luminescent signal (Fig. 3f and Supplementary Movie 3).

We also detected a gradual decrease in the signal to the basal level when the illumination was terminated. Catalytic consumption of substrate (coelenterazine-h) causes a gradual decrease in

both the baseline and the amplitude of transients. These results suggest that these synergistic neurobiology tools can now be combined into a single experimental approach. In the future, it

may also be possible to combine other optical control systems, such as chromophore-assisted light inactivation of target molecules19 and photolysis of caged compounds20, with luminescent

functional indicators. IMPROVED CAMP INDICATORS BASED ON NANO-LANTERN Imaging intracellular cAMP, another important second messenger, using fluorescent indicators has been challenging

because of the small signal change (1–15%) with current FP-based indicators21,22,23. To examine whether the CSL/BRET hybrid strategy could be applied to construct a high-performance cAMP

indicator, we cloned a series of cAMP-binding motifs from the PKA regulatory subunit or EPAC1, into Nano-lantern at the 228/229 position (similar to in Nano-lantern (Ca2+)) (Supplementary

Fig. S8a). Recombinant proteins were purified and screened for intensity changes in response to cAMP. Among the nine constructs tested, three cAMP-recognition motifs gave a large signal

change with distinct cAMP affinities (_K_d=0.4, 1.6 and 3.3 μM), and one of these three, Nano-lantern containing a fragment of EPAC1 (G170 to A327 with Q270E), which we named Nano-lantern

(cAMP1.6) (from its _K_d), showed the largest signal increase (130%) upon cAMP stimulation (Fig. 4a, Supplementary Fig. S8b–d and Supplementary Note 3). In contrast, FRET-based indicators

with an identical cAMP-recognition domain showed only a small signal change (11.7% for CFP/YFP-based constructs; Supplementary Fig. S8e and Supplementary Note 3). To examine the ability of

Nano-lantern (cAMP1.6) to detect intracellular cAMP concentration changes, we used _Dictyostelium discoideum_ cells because cytoplasmic cAMP concentration (hereafter [cAMP]) in

_Dictyostelium_ cells increases in response to extracellular cAMP stimulation24,25, which indirectly increases [cAMP] through the action of the cell-surface receptor and cAMP synthase

(adenylate cyclase A). Cells stably expressing Nano-lantern (cAMP1.6) whose _K_d was best fitted to the estimated range of oscillating cellular [cAMP] (Supplementary Fig. S8d and

Supplementary Table S3)26 showed a rapid increase in luminescent signal (40% increase in initial intensity), followed by a gradual decay in response to 100 μM cAMP stimulation (Supplementary

Fig. S9). These luminescence signal changes do indeed reflect cytoplasmic cAMP dynamics, as we confirmed that this transient cAMP-stimulated signal increase was lost in adenylate cyclase A

mutant cells (Supplementary Fig. S10). During morphogenesis, populations of _Dictyostelium_ cells form circular and spiral aggregation patterns27. This is achieved by the repeated

intracellular synthesis and intercellular relay of cAMP (at a 6–10-min interval), which functions as a chemoattractant molecule. Direct imaging of this with fluorescence reporters has never

been previously possible because of their limited dynamic range. When cells stably expressing Nano-lantern (cAMP1.6) were cultured on an agar plate (106 cells cm−2), we observed a

propagating spiral wave of cAMP with a 6-min periodicity that had a larger signal change (Fig. 4c, and Supplementary Movie 4) than that detected with a currently available FRET-based

indicator (Supplementary Fig. S11 and Supplementary Movie 5). We were able to successfully detect cAMP at the single-cell level in _Dictyostelium_ amoebae (cell volume, approximately 1/20

that of HeLa cells) with FRET-based indicators (Supplementary Fig. S11b); however, this was not possible with Nano-lantern (cAMP). In fact, approximately 20 single cells were required to

obtain a luminescence image at high magnification (Supplementary Fig. S9) due to the low absolute intensity in this organism because of its lower expression compared with that in mammalian

cells. However, these results still demonstrate that luminescent indicators have a clear performance advantage over fluorescence-based indicators when performing macroscopic analysis of

populations of living cells for prolonged periods without obvious cell injury. VISUALIZING ATP PRODUCTION IN PLANT CHLOROPLASTS Luminescence imaging can be applied in systems where

fluorescence observation is impossible. A typical example is the chloroplast of photosynthetic plants, which converts the energy of sunlight into chemical energy in the form of NADPH and

ATP. Fluorescence-based imaging cannot provide the needed spatiotemporal resolution for this process due to the strong autofluorescence of chlorophyll and the intrinsic photosensitivity of

photosynthesis. Therefore, as another demonstration of luminescence-based functional imaging, we sought to visualize the dynamics of ATP production and utilization in chloroplasts. To this

end, we developed a Nano-lantern-based ATP indicator using the ε subunit of the bacterial FoF1-ATP synthase similar to that found in the fluorescent ATP indicator ATeam28. This was also

inserted into the 228–229 position in a modified Nano-lantern (VRL10.0_S257G) (Supplementary Note 4). The generated construct, which we named Nano-lantern (ATP1), exhibited a 200% increase

in light output upon the addition of ATP with a _K_d of 0.3 mM (Fig. 5a, Supplementary Fig. S12 and Supplementary Table S4). When expressed in a live plant leaf, Nano-lantern (ATP1) was able

to detect the decrease in cytoplasmic ATP levels resulting from the addition of a mitochondrial ATP synthase inhibitor, oligomycin A (Fig. 5c). To visualize ATP production in chloroplasts

during photosynthesis, transgenic _Arabidopsis_ plants were constructed that could target Nano-lantern (ATP1) to the chloroplast stroma by using a transit peptide fusion29 (CT-Nano-lantern

(ATP1)). The luminescent signal for CT-Nano-lantern (ATP1) in the mesophyll of leaf cells overlapped with the red autofluorescence signal of the chloroplasts (Fig. 5d), and an increase in

ATP levels was detected after weak light irradiation. This response was effectively suppressed by treatment with the photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea30 (Fig.

5e), demonstrating that this response in ATP levels was photosynthesis dependent. Further monitoring of the regulation of chloroplast ATP levels was possible by pulsing strong

photo-irradiation (Fig. 5f and Supplementary Movie 6). During photo-irradiation, ATP levels increased rapidly in the first few seconds, and then continued to increase at a moderate rate. We

speculate that this slower increase is due to downregulation of the photosynthesis rate by energy-dependent quenching (qE quenching)31, a rapid mechanism used to modulate the amount of light

accepted and to prevent the harmful effects of excess light irradiation. To our knowledge, this is the first visualization of qE quenching-mediated downregulation of ATP synthesis in live

plant cells. Taken together, these results demonstrate the potential of Nano-lantern-based indicators as powerful tools for functional observation in biological materials with strong

autofluorescence and photosensitivity. Other cells with these properties include porphyric hepatocytes and erythrocytes, which accumulate the photo-sensitizing substance porphyrin, and

retinal ganglion cells, which absorb light in animal retinas. Nano-lantern-based indicators may be useful for analysing the native properties of these cells, which cannot always be

determined with existing FP (because of the need for external illumination) or chemiluminescent indicators (because of their poor indicator performance). DISCUSSION By optimizing the BRET

efficiency between Venus and improved RLuc8, we developed Nano-lantern, the brightest luminescent protein reported to date. This enhanced brightness enables luminescent imaging on a

micrometre scale in single cells, and tumour imaging in freely moving, unshaved animals. We expect that the increased brightness of Nano-lantern will make more advanced applications

possible, such as high-throughput drug screening and single-cell tracking in living animals and plants. In addition, Nano-lantern can help realize the other advantages of low-intensity light

imaging, including diminished photobleaching and phototoxicity, which allow for prolonged observation without harming the cellular substrate. However, rapid consumption of the luminescent

substrate by Nano-lantern hampers prolonged observation of biological events, especially, in the case of whole-body imaging. Therefore, to facilitate indefinite observation, a method that is

applicable to the entire organism and provides a continuous luminescent substrate must be developed. An important aspect of this study is the development of a simple method for the

construction of high-performance luminescence-based indicators by inserting the sensor domain of bioactive molecules into the non-structural Gly228/Gly229 loop of the RLuc8 moiety in

Nano-lantern. However, saturation screening to determine better insertion sites might also improve the functional indicator performance in terms of the rate constants for the association and

dissociation of the ligand with the indicator, both of which are important properties for reliable reporting of bioactive molecule dynamics. Finally, a further increase in brightness is

clearly desirable. The efficiency of intermolecular BRET between RLuc and _Renilla_ green fluorescent protein was reported to be 1.0 (ref. 7), suggesting that Nano-lantern, the BRET

efficiency of which is estimated to be approximately 0.2, could be improved. If this was achieved, luminescent proteins might allow imaging on even smaller spatial or temporal scales,

possibly even at the single-molecule level. METHODS GENERAL The detailed experimental conditions are described in the Supplementary Methods and Supplementary Tables S5–S7. SCREENING OF

BRIGHT RLUC8 GENE LIBRARIES RLuc8 cDNA was generated by introducing eight mutations, A55T/C124A/S130A/K136R/A143M/M185V/M253L/S287L, into pGL4.70 [hRLuc] (Promega) by site-directed

mutagenesis4. To obtain the cDNA libraries of mutated RLuc8 (mut-RLuc8), 30 rounds of error-prone PCR were performed under conditions to achieve a mutation rate of 5-bp/kb. _Escherichia

coli_ JM109 (DE3) cells were transformed with pRSETB_mut-RLuc8 and were spread on LB/ampicillin agar plates. Following a 20-h incubation at 37 °C, the _E. coli_ colonies were sprayed with a

phosphate-buffered saline (PBS) solution containing 5 μg ml−1 coelenterazine-h and were examined with an LAS-1000 luminescence imaging system (GE Healthcare). The brightest colonies were

picked, and their brightness was tested. CONSTRUCTION OF NANO-LANTERN To obtain eBAF-Y9 cDNA, the enhanced YFP and RLuc8 regions were amplified by PCR. The two resulting fragments were

ligated into the pRSETB vector (Life Technologies) to generate pRSETB_eBAF-Y. Venus, the circularly permutated Venus series (cp50, 157, 173, 195 and 229Venus)15, and the C-terminally 10

amino-acid-truncated Venus (VenusΔC10) were amplified by PCR. RLuc8 and the N-terminally 1–6-truncated RLuc8 (ΔN1–6) were amplified by PCR. The resulting fragments of Venus and RLuc were

ligated into the _BamH_I/_EcoR_I site of the pRSETB vector to yield a series of pRSETB_Venus-RLuc8 fusion proteins. CONSTRUCTION OF NANO-LANTERN (CA2+) To insert a Ca2+-sensing domain into

the RLuc8 moiety of Nano-lantern, a cDNA fragment encoding the CaM-M13 peptide of YC3.6015 was amplified by PCR to add _Nco_I and _Sac_I sites to its 5′ and 3′ ends by using the fCaM

(_Nco_I) and rM13 (_Sac_I) primers, respectively. These restriction sites were also introduced at the 91/92 or 228/229 position of RLuc8_S257G in pRSETB_RLuc8_S257G and pRSETB_Nano-lantern

by PCR. These fragments were then ligated to yield pRSETB_CaM-M13@91_RLuc8_S257G, pRSETB_CaM-M13@91_Nano-lantern and pRSETB_CaM-M13@228_Nano-lantern. CONSTRUCTION OF NANO-LANTERN (CAMP) The

cDNA clone encoding the human PKA regulatory subunit Iα was purchased from DNAFORM (Japan), and the cDNA fragments corresponding to the cAMP-binding domain and its flanking region and

Nano-lantern region were amplified by PCR. The obtained fragments were ligated to yield pRSETB_Nano-lantern (cAMP). A Q270E mutation was introduced into EPAC1 by site-directed mutagenesis. A

fragment encoding the 12-amino-acid linker PWNWDSGCSREG was attached to the junction between 228 of Nano-lantern and the N-terminus of the cAMP-binding motif by inserting an oligonucleotide

cassette. CONSTRUCTION OF NANO-LANTERN (ATP) The cDNA fragment encoding the ATP-recognition domain (the ε subunit of the bacterial FoF1-ATP synthase) was PCR-amplified using ATeam1.03 cDNA

as a template28. The _Nco_I_CaM-M13__Sac_I fragment of Nano-lantern (Ca2+) was replaced with the ATP-recognition motif to yield pRSETB_Nano-lantern (ATP0). RLuc8_S257G (ΔN3) of

pRSETB_Nano-lantern (ATP0) was replaced with RLuc8_S257G (ΔN0) to generate pRSETB_Nano-lantern (ATP1). PROTEIN PURIFICATION AND _IN VITRO_ SPECTROSCOPY _E. coli_ strain JM109 (DE3) was

transformed with a pRSETB vector carrying Nano-lantern and its derivative cDNA and was grown at 23 °C. Recombinant proteins were purified using Ni-NTA agarose (Qiagen) and then diluted to 20

 nM in 50 mM HEPES (pH 7.2). The emission spectra of the purified proteins were measured using a micro-plate reader (SH-9000; Corona Electric) or a spectrofluorometer (F-4500; Hitachi). A

final concentration of 1–10 μM coelenterazine-h was used as the luminescent substrate for these measurements. Experiments were performed at least in triplicate, and the averaged data were

used for further analysis. For Nano-lantern (cAMP), × 6 His-tagged recombinant Nano-lantern (cAMP) proteins were expressed in _Dictyostelium_ cells and purified using Ni-NTA agarose, because

synthetic genes containing cAMP-recognition motifs were not efficiently expressed in _E. coli_. The emission spectrum of 100 nM Nano-lantern (cAMP) was measured in the presence of 6 μM

coelenterazine-h using a spectrophotometer (F-4500; Hitachi). For the FRET constructs, 200 μM recombinant protein was used in the spectral analysis. The emission spectrum upon excitation

with 425 nm light was measured in the presence or absence of 100 μM cAMP. PREPARATION AND IMAGING OF MICE All animal experiments were carried out in accordance with the Principles of

Laboratory Animal Care, as adopted and promulgated by the Guidelines for Animal Experiments at Kyoto University and Hokkaido University. For tumour cell imaging, 5-week-old male nude BALB/c

mice (_nu/nu_) were anesthetized. At six different sites on the back, 1 × 101–1 × 107 colon26 cells expressing RLuc8 or Nano-lantern were subcutaneously transplanted, as illustrated in Fig.

2a. On the next day, the mice were anesthetized again, and 80 μg of coelenterazine-h/mouse in 20% ethanol: 80% PBS (in a final volume of 200 μl) was intravenously administered. For the

video-rate imaging of freely moving mice, 1 × 106 colon26 cells stably expressing Nano-lantern were subcutaneously implanted into the back of anesthetized 5-week-old male BALB/c mice. On the

17th day after implantation, 8 μg of coelenterazine-h/mouse in 20% ethanol: 80% PBS (in a final volume of 60 μl) was injected into the tumour. Detailed imaging conditions are described in

Supplementary Methods and Supplementary Table S6. PREPARATION AND IMAGING OF RAT HIPPOCAMPAL NEURON CULTURES On day _in vitro_ 7, dissociated rat hippocampal neurons cells were transfected

with pcDNA3_ Nano-lantern (Ca2+) and pcDNA3.1_hChR2 by calcium phosphate precipitation. The neuronal cells were imaged on day _in vitro_ 9–11 by using an inverted wide-field epifluorescence

microscope as described in Supplementary Methods. Just before observation, 20 μM coelenterazine-h was added to the culture medium. To image the transient Ca2+ induced by high-potassium

stimulation, cells were treated with 50 mM KCl. Luminescence images were collected at 10 Hz. PREPARATION AND IMAGING OF _D. DISCOIDEUM_ Strain Ax-2 was cultured in HL-5 medium as previously

described27. Expression plasmids were electroporated into cells, and transformants were selected in the presence of 10 μg ml−1 G418 (Life Technologies). For cAMP imaging, the cells were

starved in development buffer (10 mM K/Na2 phosphate buffer, 1 mM CaCl2 and 2 mM MgCl2; pH 6.5) for at least 6 h and were plated in 35-mm glass-bottom dishes or on 1% agar plates. The

detailed imaging conditions are described in the Supplementary Methods and Supplementary Table S6. PREPARATION AND IMAGING OF PLANTS Wild-type and transformed _Arabidopsis thaliana_ plants

(ecotype Columbia, Col-0) were grown at 22 °C under 16 h light/dark. The _Arabidopsis_ plants were transformed with pH2GW7_CT- Nano-lantern (ATP1) using _Agrobacterium tumefaciens_ (strain

GV3101) to generate stable transgenic plants. Transformants were selected on MS medium containing 20 μg ml−1 hygromycin B, and the T2 plants were used for imaging. Wild-type _Nicotiana

benthamiana_ plants were grown at 26 °C under 16-h light/dark. _N. benthamiana_ plants were transfected with pH2GW7_Nano-lantern (ATP1) for transient expression by the agroinfiltration

method and were imaged 1 day after agroinfiltration. To observe the effect of photosynthesis on ATP dynamics, we applied 438/24 nm blue light (LightEngine SPECTRA; Lumencor). Because visible

light excitation causes strong chloroplast autofluorescence, we used the same instrumental and optical setup as described above for the Nano-lantern (Ca2+) imaging induced by ChR2

stimulation to avoid perturbation of CT-Nano-lantern (ATP1) signals. The detailed imaging conditions are described in Supplementary Methods and Supplementary Table S6. ADDITIONAL INFORMATION

Accession codes: Sequences have been deposited in GenBank under the accession codes JN859492 (Nano-lantern), JN859493 (Nano-lantern (Ca2+) CaM-2G), JN859494 (Nano-lantern (Ca2+) CaM-2GS),

JN859495 (Nano-lantern (Ca2+) CaM-4GS), JN859496 (Nano-lantern (Ca2+) CaM(E104Q)-2G), JN859497 (Nano-lantern (Ca2+) CaM(E104Q)-3GS), JN859498 (Nano-lantern (Ca2+) CaM(E104Q)-4GS), JN859499

(Nano-lantern (cAMP 0.4)), JN859500 (Nano-lantern (cAMP1.6)), JN859501 (Nano-lantern (cAMP 3.3), JN859502 (Nano-lantern (ATP1)), JN859503 (CT-Nano-lantern (ATP1)) and JN974906 (Nano-lantern

(ATP0)). HOW TO CITE THIS ARTICLE: Saito, K. _et al._. Luminescent proteins for high-speed single-cell and whole-body imaging. _Nat. Commun._ 3:1262 doi: 10.1038/ncomms2248 (2012). ACCESSION

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Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Jin Zhan, Michael W Davidson, Feng Zhang and Hiromi Imamura for providing the plasmids; the Nikon Imaging

Center at Hokkaido University for technical support; Ryouichi Tanaka, Atsushi Takabayashi and Matthew Daniels for their valuable discussions. This work was supported by Grant-in-Aid for

Scientific Research on Innovative Areas ‘Spying minority in biological phenomena (No.3306)’ from MEXT (23115003, T.N.); Grant-in-Aid for Scientific Research on Innovative Areas ‘Fluorescence

Live imaging’ from MEXT (23113501, K.S.); Grants-in-Aid for Young Scientists (B) from MEXT (22770182, K.H.); Management Expenses Grants from NIG (K.H); FIRST from JSPS (T.N.); and Grant

from the Cooperative Research Programme of ‘Network Joint Research Centre for Medical Devices’ and PRESTO from JST (Y.H. and T.N.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Institute of

Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan Kenta Saito, Y-F Chang, Noriyuki Hatsugai, Tomoki Matsuda, Yoshiyuki Arai & Takeharu

Nagai * Research Institute for Electronic Science, Hokkaido University, N20, W10 Kita-ku, Sapporo, Hokkaido, 001-0020, Japan Kenta Saito, Yu Yoshida, Tomoki Matsuda, Yoshiyuki Arai & 

Takeharu Nagai * Centre for Frontier Research, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan Kazuki Horikawa * Research Centre for Cooperative Projects,

Hokkaido University, N15, W7 Kita-ku, Sapporo, Hokkaido, 060-8638, Japan Noriyuki Hatsugai & Takeharu Nagai * Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29

Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan Yuriko Higuchi & Mitsuru Hashida * PRESTO, Japan Science and Technology Agency, Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan

Yuriko Higuchi & Takeharu Nagai Authors * Kenta Saito View author publications You can also search for this author inPubMed Google Scholar * Y-F Chang View author publications You can

also search for this author inPubMed Google Scholar * Kazuki Horikawa View author publications You can also search for this author inPubMed Google Scholar * Noriyuki Hatsugai View author

publications You can also search for this author inPubMed Google Scholar * Yuriko Higuchi View author publications You can also search for this author inPubMed Google Scholar * Mitsuru

Hashida View author publications You can also search for this author inPubMed Google Scholar * Yu Yoshida View author publications You can also search for this author inPubMed Google Scholar

* Tomoki Matsuda View author publications You can also search for this author inPubMed Google Scholar * Yoshiyuki Arai View author publications You can also search for this author inPubMed 

Google Scholar * Takeharu Nagai View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.N. designed all the experiments. K.S. performed the

experiments for Nano-lantern (Fig. 1 and Supplementary Figs. S1,2). Y.H. performed luminescence imaging of mice (Fig. 2) with the support of M.H. Y-F.C. performed the experiments for

Nano-lantern (Ca2+) (Fig. 3 and Supplementary Figs. S4–7). K.H. performed the experiments for Nano-lantern (cAMP) (Fig. 4 and Supplementary Figs S8–11). N.H. performed the experiment for

Nano-lantern (ATP) with the help of Y.Y (Fig. 5 and Supplementary Fig. S12). T.M. performed preparation of primary culture neurons (Fig. 3f and Supplementary Fig. S7) and crystal structure

analysis (Supplementary Fig. S3b). Y.A. constructed light illumination system during luminescence imaging (Figs 2e, 3f). T.N., K.H., K.S., Y-F.C. and N.H. wrote the manuscript, and all the

authors participated in editing it. K.S., Y-F.C., K.H. and N.H. equally contributed to this study. CORRESPONDING AUTHOR Correspondence to Takeharu Nagai. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figures S1-S12, Supplementary Tables S1-S6, Supplementary

Methods, Supplementary Notes 1-4 and Supplementary References (PDF 6578 kb) SUPPLEMENTARY MOVIE 1 Nano-lantern-expressing tumor cells in an unshaved, freely moving mouse. The luminescent

signal recorded every 60 ms is green in the movie. (MOV 1364 kb) SUPPLEMENTARY MOVIE 2 Pseudo-coloured ratio images of HeLa cells expressing Nano-lantern (Ca2+) recorded at 30Hz. (MOV 1857

kb) SUPPLEMENTARY MOVIE 3 Pseudo-coloured ratio images of a rat hippocampal neuron expressing Nano-lantern (Ca2+) recorded at 10Hz. (MOV 1638 kb) SUPPLEMENTARY MOVIE 4 A propagating cAMP

spiral wave of _Dictyostelium discoideum_ cells expressing Nano-lantern (cAMP1.6) during aggregative morphogenesis (MOV 1134 kb) SUPPLEMENTARY MOVIE 5 A propagating cAMP spiral wave of

_Dictyostelium discoideum_ cells expressing FRET-based cAMP indicator during aggregative morphogenesis. (MOV 1017 kb) SUPPLEMENTARY MOVIE 6 ATP-synthesis and consumption in a plant leaf

expressing CT-Nano-lantern (ATP1) in repeated light and dark condition, respectively. (MOV 1974 kb) RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons

Attribution-NonCommercial-No Derivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Reprints and permissions ABOUT THIS ARTICLE

CITE THIS ARTICLE Saito, K., Chang, YF., Horikawa, K. _et al._ Luminescent proteins for high-speed single-cell and whole-body imaging. _Nat Commun_ 3, 1262 (2012).

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