Molecular Afterglow of Lophine-Based Luminophore and Its Imaging Applications

Abstract

Lophine, the first chemiluminescent compound discovered in history, has rarely been applied for in vivo imaging since its discovery in 1877. In this report, we demonstrate that lophine’s chemiluminescence emission could significantly be enhanced by caging the imidazole moiety via molecular afterglow mechanism. Notably, our study revealed a rare superoxide anion-mediated luminescence. We showed that caged lophine derivatives JIMI-11 and JIMI-12 could be used for in vivo mouse imaging. Compared to its uncaged form JIMI-6, JIMI-11 exhibited a significant enhancement (126-fold) in vitro and a 190-fold higher emission signal in vivo. We discovered that JIMI-11 selectively accumulates in white adipose tissues (WAT) and can be used to monitor changes in WAT mass in a mouse model of type-1 diabetes. Additionally, it can assess the therapeutic effects of Semaglutide in a mouse model of diet-induced obesity. Lastly, we designed JIMI-12 with a reactive oxygen/nitrogen species (ROS/RNS) responsive moiety as the caging group and demonstrated its utility for in vivo imaging of ROS in a lipopolysaccharide (LPS)-induced inflammatory mouse model. Our studies suggest that re-designing lophine-based probes could unlock their potential for both in vitro and in vivo applications. The ability to switch from chemiluminescence to molecular afterglow introduces a novel approach to designing imaging probes.

Graphical Abstract

We discovered that lophine—the first historically documented chemiluminescent compound—can be converted from weak chemiluminescence to bright molecular afterglow by caging its imidazole moiety. This switch greatly enhances its emission intensity, enabling, for the first time, the use of lophine derivatives for in vivo imaging applications, including reactive oxygen species (ROS) detection and monitoring adipose tissue changes induced by the GLP-1 agonist Semaglutide.

Introduction:

Imaging with molecularly produced light (“molecular light”), such as bioluminescence, chemiluminescence, and molecular afterglow, enjoys several advantages over fluorescence imaging (Figure 1A). The most important one is the high signal-to-noise ratio (SNR), due to the collected signals free from auto-fluorescence and scattering of the excitation light^1–8. However, the most significant disadvantage of “molecular light” is its low intensity, which often hampers its in vivo imaging applications^{2, 9, 10}. To overcome such limitations of “molecular light”, nano-platform is always used to boost the signals. However, nanoparticles have several limitations for in vivo imaging, such as limited penetration into target tissues and high accumulation in non-target organs (liver, spleen and kidneys)^11–14.

Figure 1.

Representative categories of molecularly produced light for imaging, including bioluminescence imaging via enzymatic reaction, chemiluminescence imaging via chemical reaction to generate high energy intermediate, and molecular afterglow imaging via photo-initiated [2+2] cycloaddition. (B) The switchable chemiluminescence/afterglow approach in this work. If G=H, the probe emits chemiluminescence, while if G is a caging group (G ≠ H), the probe JIMI-X could be switched to afterglow emission after light irradiation. The distinctive features of JIMI-X include: 1) it does not require an external photosensitizer, 2) its afterglow mechanism (type-I sensitization of O₂) is unique compared to other afterglow probes, 3) the afterglow can be activated using feeble visible light, and 4) JIMI-X enables in vivo imaging of fat and ROS without the need for skin removal or invasive surgery.

Molecular afterglow, an immerged direction for optical imaging, has been actively pursued over the past years. In 2017, Pu et al. discovered that poly(p-phenylenevinylene) (PPV)-based semiconducting polymers (SPs) could generate molecular afterglow light¹⁵ (Figure 1A). Since then, different types of molecular afterglow have been reported, such as light-induced afterglow, ultrasound-initiated afterglow, and recently, X-ray-initiated afterglow^{1, 16–18}. For the majority, afterglow imaging relies heavily on nano-platforms. To efficiently capture singlet oxygen generated by a photosensitizer and enhance emission intensity, nanoparticles are used to encapsulate both the photosensitizer and the afterglow substrate^{1, 16, –19–23}. In contrast, organic afterglow molecules that do not employ photosensitizers and nanoparticles are very rare, and existing small molecule afterglow luminescence probes are mostly confined to the scaffold of cyanine dyes^{24, 25}. In 2023, Song et al. reported that small-molecule fluorescence dyes could also generate afterglow^24–25. However, in many cases, both nanoparticle-based and small molecule-based afterglow probes remain unsatisfactory for in vivo imaging due to weak signals. In some instances, invasive laparotomy is necessary to observe the emitted signals¹⁰.

Lophine, chemically known as 2,4,5-triphenylimidazole, is a heterocyclic compound notable for its chemiluminescent properties^26–27. Lophine is the first chemiluminescence compound discovered in history, and it was discovered and reported in 1877²⁸. Lophine emits chemiluminescence in basic solutions in the presence of oxygen. Its chemiluminescence makes lophine valuable in various analytical applications, including as a sensitive detector in biochemical assays^{26, 29–31}. All the applications are in vitro tests; however, the in vivo chemiluminescence applications of lophine compounds have been rarely explored over the past 140 years, likely due to their weak chemiluminescence emissions.

Accidentally, we discovered that caged lophine lost its capacity to produce chemiluminescence; however, surprisingly, we observed that the caged lophines are capable of strongly emitting light via an afterglow mechanism (Figure 1B). Such switching from chemiluminescence to molecular afterglow could overcome the problem of weak intensity for in vivo applications. To the best of our knowledge, such a switch has not been reported in the literature. For the majority of molecular afterglow, the process needs photosensitizers to facilitate the generation of singlet oxygen, which reacts with -C=C- bond to form the high-energy intermediate 1,2-dioxetane^{1, 16–18}. In this report, we observed that the afterglow of caged-lophine could be initiated without photosensitizers, and our preliminary mechanism studies suggested that the reaction is not dependent on singlet oxygen. Instead, it involves superoxide species¹⁰. This unique property/mechanism sets it apart from most of other reported molecular afterglow, and it could open up a new avenue for searching molecular afterglow probes. In this work, we designed and synthesized a series of lophine derivatives, which we have designated as the JIMI-X probes (X =1–12).

During our in vivo studies, we found that caged-lophine could light up fat depots (or adipose tissue stores), which are critical components of the body that play diverse roles in energy storage, hormone production, and insulation³². Various imaging modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), and dual-energy X-ray absorptiometry (DEXA), have been applied for fat mass imaging to assess fat distribution and density^33–35. Fat mass imaging can offer insights into how fat deposits impact overall health and disease progression and aids in evaluating treatment efficacy and personalized treatment plans^32–33. Compared to MRI, CT and other imaging modalities, optical imaging offers low-cost options, particularly for preclinical animal studies^36–39.

Since the lophine probes have significant accumulations in fat depots, we speculated that this phenomenon could be utilized to monitor the changes in fat mass. Some type-I diabetic patients have lower subcutaneous fat compared to those without the condition^{40, 41}. In this report, we demonstrated that caged-lophine could be used to monitor the reduction of fat mass in streptozotocin (STZ)-induced type-I diabetic mouse models. Obesity is also highly associated with abnormal fat mass accumulations and distributions. Recently, anti-obesity (type-II diabetes) therapy has achieved a remarkable milestone with the agonists of glucagon-like peptide-1 (GLP-1), such as Semaglutide and Tirzepatide^{42, 43}. In this report, we demonstrated that JIMI-11 could be used to monitor the reduction of fat mass during Semaglutide therapy in diet induced obese (DIO)-obesity mouse models.

Based on our discovery of the molecular afterglow of caged-lophine, we proposed to harness this switch to monitor ROS changes in LPS-induced inflammatory mouse models. Indeed, we found that JIMI-12 was responsive to ROS and showed lower intensities in LPS-treated mice, compared to non-treated mice.

In this report, leveraging our discovery of the switchability between chemiluminescence and afterglow of lophine probes, we demonstrated that caged-lophine could be used for imaging fat depots, LPS-induced inflammation, and monitoring the progress of fat loss in STZ-induce type-I diabetes and therapy with the most popular anti-obesity/diabetes drug Semaglutide (Ozempic) with a mouse model. We believe that our work represents a new pathway for designing chemiluminescence and afterglow imaging probes.

Results and Discussion

1. Chemiluminescence of lophine derivatives

Our unexpected discovery stems from investigating the chemiluminescence of lophine-based derivatives. Lophine, the first known chemiluminescent compound, was discovered in 1877. The lophine compounds, derivatives of 2,4,5-triphenylimidazole, emit chemiluminescence under strongly basic conditions in the presence of O₂. The strong bases are essential because the initial step of the luminescent reaction involves the deprotonation of the N-H group in the imidazole ring²⁷. Despite their long history, in vivo applications have not been reported, likely due to the harsh conditions required for chemiluminescence, short wavelengths, and weak emission intensity. To address these issues, we proposed that conjugating different electron-rich auxiliary chromophores with the diphenyl imidazole core could enhance emission intensity. Additionally, by modifying the phenyl groups to modulate electron effects, we anticipated that the chemiluminescence-generation conditions could be milder, enabling the use of these probes for in vivo studies. In these regards, we first designed four lophine derivatives JIMI-X (X =1–4).

Among the four compounds, we aimed to achieve long emission via extending the π-π conjugation system. In this regard, we incorporated a dimethylamino-styrene group in JIMI-1 and a dimethylamino-benzothiazole moiety in JIMI-2 (Figure 2a). Previous studies indicated that benzofuran could enhance chemiluminescence emission by locking bond rotation^44–46. Based on this, we installed a diethylamino-benzofuran moiety in JIMI-3. To further extend the π-π conjugation of JIMI-3, we designed JIMI-4 by adding an additional double bond between the benzofuran and imidazole rings. The four designed compounds were synthesized by condensing aromatic aldehydes and benzil under the conditions of NH₄OAc and HOAc at 120 °C²⁷. The structures of these probes were confirmed by nuclear magnetic resonance (¹H-NMR and ¹³C-NMR) and high-resolution mass spectrometry (HRMS). These probes were then incubated with NaOH solutions, and their chemiluminescence (CL) was recorded using an IVIS imaging system. As we expected, JIMI-2 showed longer emission than JIMI-1 (580 nm) and lophine (540 nm), and JIMI-4 afforded the longest emission (660 nm) among the four probes (Figure 2b). Interestingly, the order of luminescence intensity was found to be JIMI-1 > JIMI-4 > JIMI-3 > JIMI-2 (Figure S1). JIMI-2 showed the weakest intensity, and this is probably due to the thiazole moiety⁴⁷. Notably, JIMI-3 exhibited weaker emission, which is different from our expectations. This is likely because the torsion of the bi-aryl connection of furan-imidazole reduces the overall planarity of the molecule.

Figure 2.

Synthetic route of the probes and chemiluminescence measurements under basic conditions. (a) Synthesis and the chemical structures of JIMI-X probes. (b) Chemiluminescence wavelength of JIMI-1, −2, −3, and −4 (250 μM) in NaOH solutions (2.5 mM). (c) Hammett plot. σ: substituent constant, ρ: reaction rate constant. (d) Chemiluminescence mechanism of JIMI-4 in basic solution. The well-established mechanism of lophine chemiluminescence involves two key steps: deprotonation (k₁) followed by oxidation (k₂). The Hammett curve in (c) indicates that R-substituent’s electronic properties significantly influence the rate-limiting steps-1 (k₁) and step-2 (k₂)

Considering that high emission efficiency benefits in vivo studies, we decided to further enhance the luminescence intensity of JIMI-4 by introducing electron-donating or electron-withdrawing groups at the para position of the phenyl ring. To this end, we synthesized a series of compounds, incorporating dimethylamino- (JIMI-5), methoxyl- (JIMI-6), fluoro- (JIMI-7), and cyano- group (JIMI-8), respectively. Under the same NaOH conditions, JIMI-6 exhibited the highest CL intensity among the compounds tested (Figure S2).

Notably, the CL intensity did not show a linear relationship with the electronic effects of the substituents. To further investigate the structure-activity (chemiluminescence) relationship, we utilized the results in Figure S2 to plot a Hammett curve (Figure 2c). The horizontal axis represents the σ value of each substituent, reflecting its electronic effect; more negative values indicate more electron-donating groups, while more positive values indicate more electron-withdrawing groups. The vertical axis shows the logarithm of the ratio of the chemiluminescence value of each compound to that of JIMI-4, which serves as the reference compound (R = H). The resulting Hammett plot is a downward opening, suggesting that altering the electronic effects of the substituents influences the rate-limiting step of the reaction⁴⁸, and consequentially impacts their CL emission intensities.

Mechanistically, several potential rate-limiting steps can be identified during the chemiluminescence generation reaction (Figure 2d). The first step involves deprotonating (k₁) the N-H group on the imidazole under basic conditions, producing a nitrogen anion. In the second step, the formed anion transfers a single electron to O₂, generating a superoxide anion (O₂^•─)⁴⁹ and forming a nitrogen radical on the imidazole moiety (k₂). In the third step, this radical quickly tautomerizes into an imidazole radical, which reacts with the superoxide to form the high-energy intermediate (HEI) 1,2-dioxetane. In the last step, the 1,2-dioxetane intermediate undergoes fragmentation, resulting in chemiluminescence. The Hammett curve suggests that the electronic properties of the R-substituent significantly influence the rate-limiting steps −1 and −2. For instance, if R = -N(CH₃)₂ (σ < 0) as in JIMI-5, the rate-determining step is likely step-1. Conversely, if R = -CN, as in JIMI-8, the rate-limiting step shifts to step-2 (Figure 2d). Collectively, in the case of electron-rich groups, deprotonation becomes more challenging while oxidation is facilitated. Conversely, electron-withdrawing groups favor the deprotonation step but make oxidation more difficult. As a result, both highly electron-rich and highly electron-deficient groups tend to decrease overall chemiluminescence.

2. Discovery of the switching from chemiluminescence to molecular afterglow with caged-lophines

Theoretically, if we install a caged group on the N-atom of the imidazole in JIMI-6, no chemiluminescence would be observed. To verify this, we synthesized JIMI-9 with a methyl group, JIMI-10 with a benzyl group, and JIMI-11 with an acetyl group on the imidazole ring (Figure 3a). The caged group was expected to block the deprotonation step, thereby inhibiting CL emission. Indeed, under strongly basic conditions, the CL of JIMI-9 and JIMI-10 was nearly completely abolished (Figure 3b). We found that JIMI-11 exhibited a weak CL intensity, which was 30% lower than JIMI-6, likely because the acetyl group could be removed under strongly basic conditions, releasing JIMI-6. Unexpectedly, we observed significant luminescence intensity from the Me and Bn-caged probes (JIMI-9 and JIMI-10) in DMSO solutions (no strong base was used), with intensities much stronger than JIMI-6 (G = H) and JIMI-11 (G = Ac) (Figure 3c, d). On the contrary, in PBS solutions (pH 7.4), JIMI-11 (G = Ac) displayed the highest luminous intensity among the four probes (Figure 3e), which was surprising because the acetyl group in JIMI-11 could not be removed at pH 7.4.

Figure 3.

Discovery of molecular afterglow luminescence of JIMI-Xs. (a) Synthesis and chemical structures of caged JIMI-X probes. (b) Chemiluminescence measurements of JIMI-6, −9, −10, and −11 (250 μM) in NaOH solutions (2.5 mM). (c) Afterglow luminescence imaging of JIMI-6, −9, −10, and −11 in DMSO solutions (50 μM). (d) Quantitative analysis of the afterglow images in (c) (e) Afterglow luminescence imaging of JIMI-6, −9, −10, and −11 in 20% DMSO/PBS solutions (50 μM). (f) Afterglow luminescence of JIMI-11 in 20% DMSO/PBS solution (50 μM) after light irradiation with iPhone 13. One column was covered with black paper to block the irradiation. (g) Afterglow luminescence of JIMI-6, −9, −10, and −11 in 20% DMSO/PBS solution (50 μM) before and after receiving blue LED light irradiation. (radiance unit: p/sec/cm²/sr). Error bar: mean ± SD, n = 3.

The observed striking luminescence phenomena in these caged molecules sparked our curiosity. We hypothesized that these probes were highly light-sensitive and that the emitted CL was actually molecular afterglow triggered by laboratory lighting. To validate our hypothesis, we added six JIMI-11 solutions to wells of two columns (three wells per column) on a 96-well plate. The right column was covered with black paper, and the left was irradiated with light from an iPhone-13 for 10 seconds. After the irradiation, the plate was subject to imaging with the IVIS imaging system. Clearly, the left column’s emission was much brighter than the right (Figure 3f). When we covered the left column and exposed the right column to the iPhone light, a reversed result was observed. These results strongly suggest that JIMI-11 is a molecular afterglow probe, and feeble light (iPhone light) is adequate to trigger the emission of molecular afterglow.

For convenience in subsequent experiments, we used a blue LED (470 nm LED Array, THORLABS (LIU470A); 24 V; 15 W) as a light source. As expected, JIMI-9 and JIMI-10 also showed strong molecular afterglow emission after LED irradiation (10 seconds). Interestingly, JIMI-6 (G = H) displayed afterglow capability as well, even though the intensity was weak. This suggests that the caged lophine structure is a new scaffold for generating molecular afterglow (Figure 3g). Among the tested probes, JIMI-11 showed the highest intensity of molecular afterglow (Figure 3g).

3. Characterization of caged-lophines as molecular afterglow probes

We measured the absorbance of JIMI-Xs (X =−6, −9, −10, and −11) (Figure 4a), and they exhibited two close absorption peaks. For instance, JIMI-11 showed peaks at λ = 420 nm and 440 nm. The fluorescence (FL) emission spectra revealed that JIMI-11 had a longer emission wavelength, while JIMI-10 had the highest intensity among the probes (Figure 4b). Furthermore, afterglow emissions showed that JIMI-6 and JIMI-11 had similar emission peaks (λ = 660 nm). Unlike the fluorescence spectra, JIMI-9 and JIMI-10 exhibited longer emissions (720 nm and 740 nm, respectively) than JIMI-11 (Figure 4c). To investigate the afterglow properties of the probes, we irradiated them with intermittent exposure (10 seconds) to LED light. As expected, the afterglow intensities of the three caged probes, JIMI-9, JIMI-10, and JIMI-11, displayed clear up-and-down cycles, whereas the uncaged probe JIMI-6 showed no significant changes between cycles (Figure 4d). Notably, no significant loss in afterglow intensity was observed for the three caged probes after five cycles. The afterglow intensity of JIMI-11 increased with concentration (Figure 4e); however, the intensities decreased with longer exposure times (Figure 4f). Additionally, we observed that JIMI-11 was consumed after prolonged LED exposure, as indicated by the reduction in absorbance at 440 nm (Figure 4g). This decrease is attributed to the afterglow reaction, where 1,2-dioxetane HEI is formed and subsequently breaks down to emit light. To determine if the light emission from JIMI-11 is a result of its reaction with various ROS species, we incubated JIMI-11 with ROS solutions. Unlike with LED irradiation, no significant signal increase was observed with the ROS solutions, indicating that JIMI-11 is indeed light-sensitive (Figure S3).

Figure 4.

Photophysical properties of JIMI-X probes and scanvenge experiment. (a-c) Normalized absorbance (Abs.) (10 μM), fluorescence emission (5 μM, unit: a.u.), and normalized afterglow luminescence (A.L.) (JIMI-6 and 11, 1 mg probe dissolved in DMSO/Cremophor/PBS (150/150/700 μL); JIMI-9 (5 mM in DMSO) and JIMI-10 (50 μM in DMSO)) spectra of JIMI-6, −9, −10, and −11. (d) Afterglow intensities of JIMI-Xs in 5 cycles after LED irradiation (50 μM). (e) Afterglow intensities of JIMI-11 at various concentrations after LED irradiation. (f) Afterglow intensities of JIMI-11 with different irradiation durations (50 μM). (g) Normalized absorbance of JIMI-11 with and without LED irradiation (10 μM). (h) Detection of superoxide anions with the fluorescence of DHE during JIMI-11 afterglow (JIMI-11 (20 μM) with DHE (50 μM)). (i) afterglow luminescence of JIMI-11 in PBS, argon (Ar) bubbled PBS, NAC and NaN₃ (JIMI-11 (50 μM) with scanvenger (500 μM)). (Afterglow radiance unit: p/sec/cm²/sr). Error bar: mean ± SD, n = 3. *p-value < 1.0, **p-value < 0.5 (t test).

4. Preliminary mechanism investigation of afterglow of caged-lophines

To investigate the mechanism of the afterglow reaction, JIMI-11 was irradiated with LED light overnight. HRMS detected a significant MS peak at 568.2430 ([M + H]), which was 32 more than JIMI-11 (536.2529, [M + H]) (Figure S4). This finding suggests that one molecular O₂ was attached to JIMI-11, indicating that intermediate dioxetane and subsequent fragmentation probably occur at the imidazole ring.

For the afterglow reaction, there are two potential pathways to form the 1,2-dioxetane HEI (Figure 5a). The most commonly reported Type-II pathway (energy transfer) involves the addition of singlet oxygen (¹O₂) to a -C=C- double bond via [2+2] cycloaddition. This ¹O₂ pathway is also widely utilized in Type-II photodynamic therapy. The second possible pathway involves the generation of the superoxide anion (O₂−) (Type-I, electron transfer). While this pathway is frequently employed in Type-I photodynamic therapy, it has been rarely reported in afterglow reactions¹⁰. To preliminarily investigate which pathway is the major mechanism of afterglow, we performed computations using density functional theory (DFT). The calculated energy gap ΔE_gap (E_LUMO−E_HOMO) for JIMI-6, JIMI-9, JIMI-10, and JIMI-11 was 7.8753, 7.8423, 7.8338, and 7.6807 eV, respectively (Figure 5b, Figure S5). Among these, JIMI-11 had the lowest HOMO-LUMO energy gap, giving insight that caging imidazole reduces HOMO-LUMO excitation, which endows compound high reactivity. Furthermore, we computed the energy gaps between the triplet state (T1) and the ground state (S0) (ΔE_T1-S0) of the four probes, all of which were lower than 0.98 eV (Figure 5b). This suggests that these probes are unable to convert triplet oxygen (³O₂) to singlet oxygen (¹O₂), as it is well-established that the energy required for this conversion must be higher than 0.98 eV^10,24. Thus, the Type-II energy transfer pathway is likely not favored in the afterglow reaction.

Figure 5.

Afterglow mechanism studies of JIMI-11. (a) Jablonski energy diagram for afterglow generation via two different pathways (Type-I: electron transfer, and Type-II: energy transfer). (b) Frontier orbital energies of four JIMI-X probes (X = −6, and −11) by DFT calculations. (c) Proposed reaction mechanism of the afterglow luminescence of JIMI-Xs.

To further investigate whether superoxide anion or singlet oxygen is produced and involved in the afterglow reaction, we incubated JIMI-11 with dihydroethidium (DHE), an indicator of superoxide anion, and anthracene-9,10-dipropionic acid disodium (ADPA), an indicator of singlet oxygen, under LED irradiation. Significant fluorescence changes were detected with DHE only in the presence of JIMI-11 and LED light (Figure 4h), indicating the generation of superoxide anion. However, the absorbance of ADPA showed only a slight decrease under the same conditions (Figure S6A), suggesting that singlet oxygen was not significantly involved in the afterglow reaction. This result was further confirmed using Singlet Oxygen Sensor Green (SOSG), another indicator of ¹O₂, which showed only a slight increase in fluorescence after LED irradiation of the SOSG and JIMI-11 mixture (Figure S6B). To confirm that oxygen is involved in the reaction, we bubbled argon into PBS buffer solutions containing JIMI-11 and then exposed them to LED light. This led to a significant decrease in afterglow luminescence from the argon-purged solutions (Figure 4i, Figure S7)⁴⁹. Additionally, the introduction of N-acetylcysteine (NAC), a superoxide anion scavenger⁵⁰, resulted in a significant reduction in afterglow intensity, whereas the singlet oxygen scavenger NaN₃ had a negligible impact on afterglow luminescence (Figure 4i). Collectively, these results confirm that superoxide anion, rather than singlet oxygen, is the major species in the afterglow reaction. The involvement of superoxide anion in the afterglow reaction is particularly notable, as most reported afterglow mechanisms involve the singlet oxygen pathway¹⁰.

Accordingly, based on the above experimental and DFT results, we proposed a possible mechanism for generating afterglow luminescence (Figure 5c). For caged probes (G ≠ H), the molecule can be excited by external light irradiation. The excited intermediate IN1 undergoes oxidation by oxygen through single electron transfer (SET), forming the radical cation intermediate IN2⁵¹. In this process, oxygen receives an electron to become a reactive superoxide anion, which further reacts with IN2 to form the 1,2-dioxetane HEI IN3. The radical cation intermediate IN2 and superoxide are reactive species. The reaction rate of two species should be faster than superoxide anion with probe itself, explaining why probe only responds to LED but not to superoxide. The unstable cyclic four-membered dioxetane IN3 then undergoes fragmentation, emitting light synchronously.

5. In vivo imaging of adipose tissue with JIMI-11

Before in vivo study, we evaluated the cytotoxicity of JIMI-11 in HEK293 cells using a standardized assay. The results indicated no significant cytotoxicity at the tested concentrations (Figure S8). To investigate whether the probes can be used for in vivo imaging, we first administered JIMI-6, −9, −10, and −11 into Balb/c mice through intravenous tail vein injection. We imaged the mice from both dorsal and ventral sides, and no significant signals were observed, suggesting that chemiluminescence from all the probes was weak and it was not feasible to use them for in vivo imaging.

Given that our in vitro studies suggested molecular afterglow was much stronger than the chemiluminescence of each probe, especially JIMI-11, we irradiated the mice from the ventricular side with LED light for 20 seconds after the probe injection to initiate molecular afterglow. The irradiated mice were immediately subjected to imaging acquisition on an IVIS imaging system. We were delighted to observe strong afterglow signals from JIMI-11 in the lower abdominal areas (Figure S9). Remarkably, the highest intensity reached approximately 3.5*10^7 photon/sec/cm^2/sr (radiance) in these areas (Figure S9b), which is roughly 10 times brighter than other reported small molecule afterglow probes^24,25. However, the other probes did not show strong afterglow signals like JIMI-11. The afterglow intensity from JIMI-11 was 190-fold higher than that from JIMI-6. Similarly, JIMI-11’s afterglow intensity was 185-fold and 146-fold higher than those from JIMI-9 and JIMI-10, respectively (Figure S9b). To investigate whether repeated irradiations would cause a diminishing of the signals, we turned the LED light on and off five times. We found no significant signal loss, suggesting that JIMI-11 can resist photo-bleaching in vivo (Figure S10). We also measured in vivo and in vitro half-life time of JIMI-11 (Figure S11). It suggested that in vivo half-life time (18 s) is 4.7-fold longer than in vitro (3.8 s).

After carefully surveying the abdominal areas with strong afterglow signals, we reasoned that JIMI-11 accumulated in fat depots. To verify this, we irradiated the mice from the dorsal side and observed very strong afterglow signals from the interscapular white adipose tissue (iWAT), indicating JIMI-11’s accumulation in fat tissues (Figure S12a). Next, we monitored the signals from iWAT daily. Surprisingly, even one month after the iv injection of JIMI-11, strong afterglow signals were still observable. Interestingly, inguinal white adipose tissue (ingWAT) showed stronger luminescence than iWAT, suggesting a faster metabolism of JIMI-11 in iWAT compared to ingWAT (Figure S12b).

To further confirm JIMI-11’s selectivity towards white adipose tissue (WAT), we conducted an ex-vivo experiment. One day after iv injection of JIMI-11, the mouse was sacrificed, and the main organs were dissected, including gonadal white adipose (gWAT), iWAT, ingWAT, and brown adipose tissue (BAT). These tissues were then irradiated by LED light. As expected, all the WAT exhibited strong luminescence, whereas the brain, lungs, liver, stomach, spleen, kidneys, heart, and intestines showed no apparent afterglow signals, and BAT also showed low intensity (Figure S13a). We also performed ex vivo fluorescence imaging to further support these results (Figure S13c). Additionally, we found that afterglow intensities had a good linear relationship with WAT weight (Figure S13b). Collectively, these experiments clearly demonstrate that JIMI-11 specifically accumulates in WAT.

6. In vivo imaging of STZ-induced Type-1 diabetes model with JIMI-11

STZ-induced type 1 diabetes (T1D) is a widely used experimental model to study diabetes and its associated complications^52–53. In this animal model, STZ administration leads to a significant reduction in overall fat mass due to insulin deficiency and increased lipolysis⁵⁴. Insulin is crucial for the storage of glucose and fats, and its deficiency results in the breakdown of fats in adipose tissue, reducing fat stores^{53, 54}. Given that JIMI-11 has demonstrated excellent capability for imaging WAT, we hypothesized that JIMI-11 could be used to monitor changes in WAT mass in STZ-induced T1D mouse models.

Since our preliminary study showed a slow metabolism rate of JIMI-11 in vivo, it suggested that signals could be monitored without multiple injections of the probe. In this study, we injected JIMI-11 twice over a 20-day period. Before the STZ treatment, two groups of mice (control and STZ-treated) were intravenously administered JIMI-11 through the tail vein on day −8 (8 days before the end of STZ-induction and 8 days before the second JIMI-11 injection). The dorsal signals, including iWAT and ingWAT, were monitored every two days. To establish the diabetes model, one day after the first JIMI-11 injection, STZ was administered through intraperitoneal (ip) injection for seven consecutive days, while saline was ip injected into the control group (Figure 6a). During the STZ treatment days, no significant difference between the two groups was observed for the first five days. However, a significant difference (1.5-fold, Figure S14) emerged on day −3. This difference disappeared on day 0 after the first JIMI-11 injection. On this day, we re-injected JIMI-11 and continued monitoring the signals every two days. A significant difference was observed on day 7 after stopping STZ treatment and persisted on days 9 and 12 (Figure 6c). Additionally, the STZ group exhibited significant weight loss (Figure 6d), and the measured blood glucose levels (> 600 mg/dL) in all five mice confirmed the successful establishment of the T1D model (Figure 6e).

Figure 6.

In vivo imaging studies of JIMI-11 in the STZ-induced diabetes model. (a) The treatment and imaging scheme for the control group (saline) and the experimental group (STZ). (b) Representative images of afterglow luminescence with JIMI-11 for the saline and STZ-treated groups. (c) Quantitative analysis of the afterglow luminescence signals from iWAT of the saline and STZ-treated groups. (d) Body weight of the saline and STZ groups at day 12. (e) Blood glucose level of the saline and STZ groups at day 12. (f) The afterglow intensities from iWAT at day 12. (g, h) The afterglow intensities from ingWAT (left side and right side) at day 12. (radiance unit: p/sec/cm²/sr). Error bar: mean ± SD. **p-value < 0.01 (t test).

We compared the afterglow signals in iWAT and ingWAT between the STZ group and the control group and found that the total afterglow signals in the STZ group were significantly lower. Specifically, at day 12, the intensity in iWAT was 2.6-fold lower (Figure S15). However, one mouse in the STZ group displayed an abnormally higher signal, despite its blood glucose level indicating diabetes. Excluding this outlier, the difference between the two groups increased to 12.2-fold (Figure 6f). Additionally, we observed that the left ingWAT was 20.6-fold higher (Figure 6g), and the right ingWAT was 15.1-fold lower (Figure 6h) in the STZ group at day 12. These results indicate that JIMI-11 can be used to monitor changes in WAT in vivo.

7. In vivo imaging monitoring with JIMI-11 for fat mass changes under Semaglutide therapy.

Semaglutide, a GLP-1 receptor agonist, has garnered significant attention for its effectiveness in treating type II diabetes and long-term weight management^{42, 43}. Given that weight loss is closely associated with fat reduction and that our probe JIMI-11, accumulates strongly in fat tissue, we utilized JIMI-11 to monitor afterglow luminescence in saline- and Semaglutide-treated mouse models (Figure 7). Before starting the drug treatment, JIMI-11 was injected intravenously into the mice, and the afterglow intensities in the iWAT were recorded at the following day. The obese mice were then treated with subcutaneous injections of semaglutide, while the control healthy mice received saline every three days. After one month of treatment, JIMI-11 was re-administered via tail vein injection to both groups, and the afterglow signal was recorded the next day (Figure 7a).

Figure 7.

Monitoring changes in fat mass with JIMI-11 for the Semaglutide-treated obese mouse model. (a) The treatment and imaging scheme for the saline and Semaglutide groups. (b) Monitoring body weight of the saline and semaglutide groups. (c) Representative images of afterglow luminescence with JIMI-11 before and after saline/Semaglutide treatment. (c) iWAT afterglow luminescence images of the saline/Semaglutide treatment groups. (d) Quantitative analysis of the afterglow intensities from iWAT at day 1 before the treatment. (e) Quantitative analysis of the afterglow intensities from iWAT at day 34 after the treatment. (radiance unit: p/sec/cm²/sr). Error bar: mean ± SD.

During this period, the mice’s weight was also tracked after each treatment. As expected, the Semaglutide-treated mice exhibited significant weight loss, and visibly reduced fat accumulation (Figure 7b, 7c). In contrast, mice in the saline-treated group experienced a slight weight increase, suggesting negligible acute toxicity of our probe to mice (Figure 7b). Before the drug treatment, the iWAT afterglow intensity in obese mice was 4.9-fold higher than that of healthy mice (Figure 7d). After one month of the Semaglutide treatment, this ratio decreased to 1.8-fold (Figure 7e). The average afterglow intensity in obese mice before treatment was 1 × 10^8 (radiance), which dropped to 4 × 10^7 after treatment, indicating a positive correlation between the afterglow intensity and the fat content.

8. In vivo imaging of ROS/RNS with JIMI-12

Our in vitro studies demonstrated that caging lophine with acetyl, benzyl, and methyl groups could switch chemiluminescence to molecular afterglow, leading to increased emission intensity upon LED irradiation. Leveraging this switching phenomenon, we designed JIMI-12 as an afterglow probe for detecting ROS/RNS. In this probe, a (pinacolato)boryl carbamate group, the most widely used responsive unit for ROS/RNS, was introduced to cage the imidazole of JIMI-6 (Figure 8a). The synthesis of JIMI-12 was straightforward. Upon completion, we characterized the probe and found it to be resistant to photo-bleaching, as evidenced by no apparent intensity decrease after five consecutive LED irradiation cycles (Figure S16a). We also recorded its emission spectrum, which peaked around 640 nm (Figure S16b). As the probe concentration increases, the afterglow intensity also increases (Figure S16c). As we expected, when incubated with H₂O₂ (Figure S16d) and SIN-1 (a precursor for ONOO- generation, Figure 8b), the immolative (pinacolato)boryl carbamate was removed, producing JIMI-6 and resulting in decreased luminescence emission intensities.

Figure 8.

The design and application of JIMI-12 as a switchable probe upon reacting with ROS. (a) The design of JIMI-12 with an immolative linker that is responsive to ROS. Upon reacting with ROS species, the (pinacolato)boryl carbamate linker could be cleaved, which could lead to JIMI-12 being uncaged. (b) Sin-1 response of JIMI-12. Error bar: mean ± SD, n=3. (c) Schematic monitoring abnormal afterglow luminescence signal of the LPS model using JIMI-12. (d) Afterglow signal intensity in saline- and LPS-treated mice model. (d) Representative images of afterglow luminescence with JIMI-12 in saline and LPS mice model. Error bar: mean ± SEM, n=3. (radiance unit: p/sec/cm²/sr).

To investigate whether this probe can be employed for in vivo studies, we used lipopolysaccharide (LPS) to induce whole-body inflammation in mice⁵⁶. We simultaneously injected LPS and JIMI-12 intraperitoneally and monitored the afterglow signals for 13 days (Figure 8c). Compared to the control group (saline injection), significant differences were observed, with the LPS group showing lower intensities (Figure 8d), which revealed that signal reduction in vivo is more contributed to LPS induced ROS production rather than probe metabolism.

In this study, we unexpectedly discovered that caging the imidazole moiety of lophine significantly enhances its light emission through a molecular afterglow mechanism. We attribute this notable enhancement to the following factors: A) The chemiluminescence of uncaged lophine involves multiple rate-limiting steps, such as deprotonation by a strong base and subsequent single-electron transfer to O₂ to generate superoxide anions (Figure 2d). Since these steps can be slow, the overall chemiluminescence efficiency is significantly reduced. This explains why uncaged lophine typically exhibits very weak chemiluminescence. B) In contrast, the molecular afterglow pathway begins with faster steps, including molecular excitation followed by electron capture by O₂ in the excited state (Figure 5c). Both of these steps can occur rapidly, likely contributing to the higher emission efficiency observed in caged lophine. This distinction in reaction kinetics between the two pathways provides a plausible explanation for the enhanced light emission in caged lophine systems.

Interestingly, JIMI-11 exhibited approximately 150-fold higher afterglow intensity in vivo compared to JIM-9 and JIMI-10 (Fig. S8). In PBS buffer, JIMI-11 also demonstrated brighter afterglow (Fig. 3e and Fig. 4d), whereas in DMSO solutions, the trend was reversed. Based on the mechanism proposed in Fig. 5c, we offer the following tentative explanation: compared to the Me- and Bn-groups, the intermediate IN2 with the Ac-group has a greater capacity to stabilize the radical cation in polar environments, such as PBS buffer and physiologically relevant conditions in vivo. This enhanced stability of IN2 allows more JIMI-11 to react with superoxide anions, forming intermediate IN3 and resulting in stronger light emission. Notably, an inverse relationship was observed between the emission peaks of fluorescence and afterglow for JIMI-9, −10, and −11 (Figure 4b and Figure 4c). While the fluorescence emission spectra align with the ΔE_gap (E_LUMO−E_HOMO), the afterglow emissions deviate from this pattern. This discrepancy is likely due to differences in electron distribution between the chemi-excited states and the LUMO states of these probes.

In this work, we demonstrated the potential of JIMI-11 for in vivo imaging of fat mass. However, the mechanism by which the probe accumulates in fat depots remains unclear, though it appears to be related to the hydrophobic nature of JIMI-11. Further investigation into the uptake mechanism will be the focus of our future work.

While this study demonstrates the successful application of caged lophines for adipose tissue and ROS imaging, their potential extends far beyond these targets. By conjugating targeting warheads, these probes could be adapted for tumor-specific imaging through binding to cancer-associated proteins or enzymes. Additionally, strategically designed caged lophine derivatives could serve as ligands for imaging pathological protein aggregates, including amyloid beta plaques and tau tangles in neurodegenerative diseases. Another promising direction involves exploiting the afterglow signal to monitor prodrug activation, where caged lophines would function as both therapeutic agents and imaging reporters. Our team is actively pursuing these exciting applications to expand the utility of this versatile platform.

Beyond in vivo imaging, lophine derivatives have been explored for potential therapeutics^57–59. For example, lophine analogue SB431542 has been comprehensively investigated as a TGF- β1 inhibitor^{58, 60}. Interestingly, Taomoki et al. recently reported that the caged form of SB431542 could be photo-activated to release its pharmacologically active form⁶⁰. Mutoh recently reported that hexaarylbiimidazole compounds, dimeric forms of lophine derivatives, could change their absorption from visible into NIR-II via sequential photochromic reactions⁶². In addition, lophine derivatives have been developed as in vitro analytical tools/reagents²⁶.

Conclusions:

In this study, we discovered that caging the imidazole moiety of lophine derivatives enabled a switch from chemiluminescence to molecular afterglow, resulting in a remarkable enhancement of luminescence emission (~190-fold). Such an enhancement could have great potential to expand the applications of lophine probes for in vivo imaging. In addition, we revealed that the molecular afterglow mechanism via type-I activation is different from most reported afterglow molecules. We believe our findings could greatly broaden the potential applications of lophines for in vivo imaging and beyond.