Fluorescent Nanocable as a Biomedical Tool: Intracellular Self-Assembly Formed by a Natural Product Interconnects and Synchronizes Mitochondria

Self-assembly processes commonly occur in various biological contexts to form functional biological structures. However, the self-assembly of nanofibers within cells by heterologous molecules showing a biological function is rare. In this work, we reported the intracellular formation of fluorescent nanofibers by a natural small molecule, lycobetaine (LBT), which facilitated the direct physical connection between mitochondria and synchronized their membrane potential oscillations. The luminescent properties of LBT enabled the real-time observation of nanofiber formation, while the semiconductive nature of the LBT nanofiber facilitated electrical signal transduction among the connected mitochondria. This study introduces an approach to modulate mitochondrial connectivity within cells using “nano-cables” which facilitate studies on synchronized mitochondrial operations and the underlying mechanisms of drug action.

Mitochondria, being principal energy-generating organelles in eukaryotic cells, perform crucial roles in apoptosis, lipid metabolism, biosynthesis, and respiratory ATP production. 1 In the process of respiration, mitochondrial membrane potential (ΔΨ m ) 2 is a dynamic parameter that can be evaluated by membrane potential-sensitive fluorescent dyes. 3,4Although fluctuates from time to time, the ΔΨ m holds a stable homeostasis with the steady state of about −180 mV tolerating variations from −160 to −220 mV. 5,6Nonetheless, subtle deviations in the stable levels of the ΔΨ m can significantly impact mitochondrial functions, potentially leading to mitochondrial dysfunction. 7The decline in ΔΨ m levels serves as an indicator of cell disorders, which can subsequently trigger autophagy, 8 apoptosis, 9 and necrosis. 10The loss of ΔΨ m has been linked to various diseases, including keratitis, 11 diabetes, 12 neurodegenerative diseases, 13 and cancer. 14On the other hand, abnormally elevated levels of ΔΨ m can contribute to the development of severe conditions, including glaucoma and frontotemporal dementia. 15Therefore, it holds great significance to study ΔΨ m abnormality in both biological studies and the diagnosis of associated diseases.Much effort has been focused on monitoring and regulation at the level of individual mitochondria.However, the propagation of ion signals with ΔΨ m oscillations among interconnected mitochondria, particularly how individual mitochondria communicate with one another, remains obscure in cell-based studies.The linkage of the individual mitochondria by a conductive "nano-cable" may synchronize the ΔΨ m oscillations throughout the entire mitochondrial network and provide us with an opportunity to study the wave propagation at a cellular level. 16−19 One possible method to construct such a "nano-cable" is through self-assembly.The self-assembly of monomers is commonly found in nature and living beings. 20The cytoskeleton is formed from protein components that create a dynamic network of filaments within the cell for metabolism, proliferation, and intracellular transport. 21,22−41 However, intracellular selfassembly processes pose an inherent challenge due to the highly dynamic and crowded cellular environment, which makes achieving optimal scales and defined intracellular targets difficult.
−44 However, its intrinsic optical properties, similar to other natural products, 45−50 are often overlooked.In this work, we systematically investigate the rich photophysical properties of LBT in detail, including its pH-responsive fluorescence behavior and emission mechanism in both solution and aggregate states.The real-time monitoring of the formation of LBT nanofibers in situ is also illustrated.Assisted by a ΔΨ m probe, we observed synchronized ΔΨ m oscillations of LBT-nanofiber-connected mitochondria in live cells.Our research indicates that the oscillations in mitochondrial membrane potential are con- tingent on the K + concentration gradient.Consequently, LBT nanofibers, which facilitate interconnection between mitochondria, offer a tool for advancing mitochondrial research.

RESULTS AND DISCUSSION
Photophysical Properties of LBT.LBT is a π-conjugated molecule featuring a hydroxyl group, rendering it a prospective pH-responsive species. 51−53 Thus, we initially examined the photophysical characteristics of LBT in an aqueous solution with the addition of hydrochloric acid (HCl) or sodium hydroxide (NaOH).The ultraviolet−visible (UV−vis) spectra of LBT were subsequently recorded (Figure 1a).With increasing concentrations of HCl, the absorption band spanning 250 to 300 nm exhibited heightened intensity and sharper features, while the absorption band ranging from 320 to 420 nm remained relatively unchanged.Under excitation at 365 nm, a pronounced enhancement of the emission peak at 450 nm was observed (Figure 1b), accompanied by a gradual decrease in fluorescence at 545 nm upon the addition of HCl.In comparison to the solution without HCl, the fluorescence intensity of LBT at 450 nm exhibited an ∼21-fold increase, accompanied by a color transition from yellow to blue in the presence of 2.74 M HCl (Figure 1c).This indicates a pronounced protonation of LBT in highly acidic conditions.Conversely, the introduction of an aqueous NaOH solution induced a gradual emergence of the absorption band spanning 350 to 450 nm (Figure 1d).Simultaneously, the emission peak at 545 nm displayed a linear increase upon excitation at 420 nm (Figures 1e,f), indicating the deprotonation of LBT under alkaline conditions.Specifically, its emission intensity at a wavelength of 545 nm displayed an inverse trend when excited at different wavelengths in Britton−Robinson (B−R) buffer solutions within the pH range from 2.0 to 11.0 (Figure S1).To further investigate this phenomenon, we examined the excitation spectra.As shown in Figure S2, the excitation maxima were determined to be 368 and 403 nm, corresponding to the emission maxima of 450 and 545 nm, respectively.The corresponding emission lifetimes were measured to be 5.17 and 1.14 ns, indicating the nature of fluorescence emission (Figure 1g).These findings suggest that the emission arises from two distinct excited states.These results support the assertion that the protonation (cationic form) and deprotonation (zwitterionic form) of LBT occur in response to pH changes.
To further verify that the distinct emission properties of the probes at different pH originate from the cationic and zwitterionic forms, we conducted calculations and obtained density functional theory (DFT)-molecular electrostatic potential maps.By comparing the electron density distributions of the two forms, we sought to establish their correlations with the observed photoluminescence (PL) characteristics (Figure 1h). 54The N-cationic regions contain the lowest electron density and dominate the charges of the entire cationic form of the molecule.Meanwhile, the O-anionic regions, characterized by their high charge density, are prominent in the zwitterionic form.Overall, the charge density of the zwitterionic form is significantly higher than that of the cationic form.The proton nuclear magnetic resonance ( 1 H NMR) spectrum of LBT was obtained under conditions of both protonation (treated with HCl) and deprotonation (treated with NaOH) as shown in Figure S3.Upon deprotonation, the proton signals of LBT exhibited a highfield shift relative to those in the protonated state.This observation is in agreement with the theoretically calculated electronic environment.Next, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) plots were determined by DFT calculations (Figure S4).The corresponding energy gaps are calculated to be 5.60 and 4.36 eV for cationic and zwitterionic forms, respectively, which is consistent with the measurement of absorption spectra.Therefore, these results confirm that cationic and zwitterionic forms of LBT are in charge of the diverse luminescence in the solution state. 55BT Self-Assembles to Form Nanofibers.Aggregates often display distinct characteristics compared with their molecular counterparts, making them a subject of significant interest for further structure−property investigations.In addition to studying LBT at the molecular level, we also investigated the properties of LBT in its aggregate state.As illustrated in Figure 2a, the needlelike single crystals of LBT in their cationic form suitable for single-crystal X-ray diffraction (SXRD) analyses were obtained by slow solvent evaporation of acidified LBT solutions (Table S1).The SXRD results revealed that the LBT molecule adopted an almost planar conformation (Figure 2b).Additionally, stacked structures were observed, characterized by strong intermolecular π•••π interactions with distances below 3.5 Å.These interactions likely contribute to the nonemissive nature of LBT in its crystalline state (Figure 2c).In contrast, the alkalization of saturated LBT in aqueous solutions induced gelation, as illustrated in Figure 2d.Upon the addition of NaOH, the solution rapidly transformed into a gel state.In addition, the gel-to-solution transitions can be induced by the addition of HCl.The LBT hydrogel showed reversible responsiveness to varied pH values (Figure 2e).Its emission intensity showed an over 20-fold increase after gelation (Figure 2f).Interestingly, when the water was evaporated, the self-assembly of LBT into nanofibers exhibiting a yellow emission was observed under a fluorescence microscope (Figures 2g and S5).The imaging of LBT nanofibers using scanning electron microscopy (SEM) revealed that the nanofibers possessed a diameter below 100 nm, which was consistent with the observations made using a fluorescence microscope (Figure 2h,i).To gain a better understanding of the assembly process, positive electrospray ionization mass spectrometry (ESI-MS) 56 was employed to elucidate the components of LBT fibers.Monomers (m/z 266.3), dimers (m/z 531.3), and trimers (m/z 796.4) of LBT were obtained (Figure S6), which indicates the favorable formation of cationic and zwitterionic complexes.
Intracellular Nanofilaments Formation.We then explored the application of LBT in live-cell imaging.Before the cell imaging experiment, we used the Cell Counting Kit-8 (CCK-8) assay to assess the cytotoxicity of LBT in live cells. 57,58As illustrated in Figure S7, after incubation with 10 μM LBT for 24 h, the cell survival rates of HeLa and NIH/3T3 cells were about 70%.Live-cell fluorescence imaging of HeLa cells at 37 °C showed that only weak staining of cells was observed after 2 h of incubation with LBT (Figure S8).However, sporadic filaments were observed in some of the HeLa cells after 5 h of incubation.Similar findings were observed in NIH/3T3 cells, as illustrated in Figure S9.
In our experiments, we unexpectedly found that the cold environment greatly facilitates the formation of LBT nanofibers inside the cells.While HeLa cells stained with LBT at 37 °C did not show obvious nanofiber formation, a cold treatment (5 min, 15 °C) of the LBT-stained cells could significantly induce LBT fiber assembly.After cold treatment and a subsequent 1-h incubation, robust intracellular LBT networks were observed (Figure S10), and the same method yielded similar results in the other cell lines (NIH/3T3, and MDA-MB-231) tested (Figure 3).In particular, the formation of LBT filament networks in the cellular environment encompasses three stages: (i) enrichment of LBT molecules with adequate concentrations; (ii) induction of intracellular LBT filament formation via a temporary 5 min cold treatment; and (iii) extension of LBT filaments in the cellular environment upon further incubation.Intracellular LBT nanofibers were completely confined inside the cells with some fibers over 100 μm long or even with a bending angle greater than 90°.The LBT fibers seem to be physically harmless to the cells, as the cells with more fibers (with short cold treatment) demonstrated higher cell viability than the cells with fewer fibers (without short cold treatment; Figure S11).
LBT Nanofibers Physically Associate with Mitochondria.To further investigate the mechanism of intracellular LBT assembly, the relationship between LBT nanofibers and endogenous tubule-shaped networks, including microtubules and the endoplasmic reticulum (ER), was examined.The inhibition of tubulin polymerization using two microtubule destabilizers, 59 colcemid and nocodazole, did not affect the formation of LBT nanofibers in HeLa cells (Figure 4a).Similarly, treatment with tunicamycin, which induces ER stress, 60 did not significantly impact the formation of LBT nanofibers.These observations suggest that LBT fibers can form independently of the networks of microtubules or the ER.We proceeded to investigate whether LBT nanofiber formation is correlated to the tubular network of mitochondria.To explore the relationship, we costained the cells with LBT and TPE-Ph-In, 61−63 a mitochondria-specific probe.We confirmed that there is no fluorescence crosstalk between TPE-Ph-In and LBT (Figure S12).The resulting images revealed no clear spatial relationship between the signal of LBT and TPE-Ph-In (Figure 4b).Line scan analysis also supported such funding.However, after subjecting the cells to cold treatment, we observed that the LBT signal overlaid with TPE-Ph-In signals in mitochondria.Following a 1 h incubation period, LBT fiber networks were formed in HeLa cells, resulting in less overlapping with mitochondria.These observations were consistent with the results obtained using another mitochondrial probe, tetramethylrhodamine methyl ester (TMRM) (Figure S13).minute.In contrast, cells stained only with TPE-Ph-In did not exhibit any noticeable signal fluctuation (Figures 5c,d and  S15).TPE-Ph-In was reported to be sensitive to ΔΨ m fluctuation, and we then hypothesized that the spikes and oscillations in fluorescence intensity were caused by changes in ΔΨ m .To further verify the changes of ΔΨ m , a well-established commercial fluorescent probe for ΔΨ m , TMRM was used.The fluorescence intensity fluctuations of HeLa cells stained with LBT and TMRM were then recorded.Only longer oscillations were observed, with the entire event lasting approximately 60 s (n = 19 from three independent experiments).The TMRM signal displayed a decrease in fluorescence intensity within about 1.6 s, followed by a plateau and a slow recovery back to the baseline level, representing synchronized depolarization and repolarization processes (Figures 5e,f and S16).The time course oscillations of the traditional ΔΨ m probe TMRM were similar to the reported phenomena of mitochondrial metabolism oscillation. 64,65However, no fluorescence signal fluctuation was observed in cells stained only with TMRM (Figures 5g,h and S17).Unlike TPE-Ph-In, the transient spike signals were not recorded by TMRM, which suggested a low temporal sensitivity of TMRM.
To evaluate the frequency and amplitude of ΔΨ m signal flashing induced by LBT fibers, we analyzed the spatialtemporal relationship of the flashing events in individual mitochondria of the same cell.The cell shown in Figure 6a, selected from the representative fluorescence image in Figure S18, is given as an example.In this cell, distant mitochondria 3, 10, and 17, which were connected to the LBT fibers, exhibited in-phase ΔΨ m signal flashing (Figure 6b−e).In comparison, other mitochondria that were not connected to the LBT networks, such as mitochondria number 2, 14, and 15, displayed uncoupled ΔΨ m signal flashing.Subsequently, individual mitochondria within the same cell were selected as ROIs.Over 50% of the randomly selected mitochondria demonstrated synchronized depolarization and repolarization during the recording period (Figure S19).These results suggest that synchronized ΔΨ m oscillations in these LBTnanofiber-presented cells are observed.
The statistical study of flashing events was also conducted, as shown in Figure 6f,g.With an increase in LBT concentration from 0 to 10 μM, there was a corresponding rise in the percentage of cells exhibiting fluorescence flashing.After additional cold treatment, 74.8% of the 10 μM LBT stained cells showed flashing events.Since the cold treatment greatly facilitates intracellular LBT nanofiber formation, these fibers appear to contribute to the ΔΨ m oscillations.To further investigate the nature of these fibers, the electrical conductivity of the LBT nanofiber film was measured, showing a nonlinear behavior that was similar to semiconductors (Figure S20).This confirms that LBT fibers are capable of transmitting electronic signals. 66Based on these findings, it is postulated that the semiconductive LBT nanofibers establish physical connections between mitochondria, allowing for the conduction of electrical signals and the amplification of spontaneous spikes.
Consequently, the flashing events were eventually detected in situ.
K + Relevant Mitochondrial Flashing.To further investigate the physiological relevance to the synchronous oscillation of ΔΨ m , we treated the entire cell as the region of interest (ROI) and studied the influencing factors by observing the fluorescence changes in the cell before and after pharmacological treatments.We first treated the TPE-Ph-Instained LBT-nanofiber-presented cells with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-specific ionophore that dissipates ΔΨ m across the mitochondrial inner membrane.The TPE-Ph-In signals substantially increased shortly after CCCP was added to the medium.This increase was followed by a continuous decline in the TPE-Ph-In signals without any spontaneous spikes, confirming the dependence of the flash on the ΔΨ m value (Figure 7a).Treatment with sodium azide (NaN 3 ), which is a potent inhibitor of cellular respiration, particularly of the electron transport chain in mitochondria, did not affect the flashing of the TPE-Ph-In signals (Figure 7b).When oligomycin, a mitochondrial ATPase inhibitor used to increase ΔΨ m , was introduced into the medium, the TPE-Ph-In signals across the entire field of view significantly elevated, while the spontaneous spikes persisted.This indicated that although LBT-nanofiber-induced TPE-Ph-In flashing relies on ΔΨ m it is unrelated to the transient change of proton gradient across the inner mitochondrial membrane (Figure 7c).Furthermore, the addition of nigericin, an ionophore that facilitates selective exchange of K + and H + , resulted in the abolition of TPE-Ph-In signal spikes (Figure 7d).This observation suggests that ΔΨ m oscillation is influenced by the concentration gradient of these two ions across the inner mitochondrial membrane.In contrast, treatment with monensin, which promotes Na + /H + exchange and decreases the mitochondrial matrix pH, did not affect the occurrence of spontaneous spikes (Figure 7e).Finally, the addition of valinomycin, commonly used to eliminate the transmembrane potential by collapsing the K + concentration gradient, completely abolished the ΔΨ m oscillation as indicated by the TPE-Ph-In signal (Figure 7f).All control trials were included in Figure S21, which describes the intensity changes in TPE-Ph-In fluorescence.These results demonstrated that there was no fluorescence oscillation before or after the drug was added when living HeLa cells were treated solely with TPE-Ph-In.This further confirmed the crucial role of LBT nanofibers in the synchronized ΔΨ m oscillation.The frequency of ΔΨ m fluctuations in HeLa cells before and after treatment with specific drugs was statistically analyzed (Figure S22).The data represent the average of at least three independent experiments, demonstrating solid evidence of K + -dependent mitochondrial flashing.Valinomycin treatment has yielded similar results in the Huh-7 cell line (Figure S23), corroborating that it is a common phenomenon.
Collectively, our findings reveal the existence of a fast, frequent, low amplitude, and K + concentration gradientdependent ΔΨ m fluctuation, which is distinct from previously reported mitochondrial permeability transition pore (mPTP)dependent ΔΨ m oscillations.Based on these observations, we speculate that the rapid opening and closing of voltage-gated K + ion channels may be the primary cause of this ΔΨ m signal flashing.Although the mitochondrial-located voltage-gated potassium channel Kv1.3 has been associated with abnormal mitochondrial function, apoptosis, and abnormal steady ΔΨ m through function inhibition or gene silencing, 67,68 the regulation of ΔΨ m through this channel has not been investigated, not to mention the mechanism of the regulation.Our study provides an easy but powerful tool to observe transient mitochondrial membrane potential oscillation, which could explore the emerging field of regulation and maintenance of transient mitochondrial membrane potential homeostasis.

CONCLUSION
To summarize, a comprehensive investigation was conducted on the photophysical properties of a small-molecule LBT, encompassing its fluorescence property and emission mechanism at the molecular and aggregate levels.LBT could form crystals, gels, or nanofiber filaments in aqueous solutions with different pH values.Intracellularly, LBT was found to develop filament networks in live cells that were induced at low temperatures with proximity to mitochondria.Given the localization and electrical conductivity in mitochondria, the LBT filament network is capable of operating as an intracellular "nano-cable", enabling synchronization and amplification of ΔΨ m oscillations.As a result, metabolic oscillation of ΔΨ m or mitochondrial K + flux with spontaneous ΔΨ m flashing at a cellular level can be fluorescently observed in real-time through the ΔΨ m -sensitive probe TMRM or TPE-Ph-In.This work not only demonstrates the possibility of manipulating the connectivity of mitochondria but also introduces a platform facilitating the analysis of mitochondria within individual cells, highlighting synchronized cellular activities and enhanced ΔΨ m signal detection.

METHODS AND MATERIALS
Materials and General Instruments.All chemicals and reagents were commercially available and used as received without further purification.LBT was purchased from Psaitong (China) and used without further purification.The purity has been confirmed by 1 H NMR, high-performance liquid chromatography (HPLC), and high-resolution mass spectrometry (HRMS) analysis (Figures S24−S26).CCK-8 kits were purchased from Beyotime.Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphate buffered saline (DPBS), fetal bovine serum (FBS), trypsin-EDTA, and penicillin-streptomycin were obtained from Gibco.Tunicamycin, colcemid, nocodazole, and propidium iodide (PI) were purchased from Thermo Fisher Scientific.Milli-Q water was supplied by a Milli-Q Plus System (Millipore Corporation, United States).All of the solvents for optical spectroscopic studies were of spectroscopic grade.UV−vis absorption spectra were performed using a PerkinElmer Lambda 365 spectrophotometer.Fluorescence spectra were obtained by using a Horiba Duetta spectrofluorometer with a 10 mm quartz cuvette.Fluorescence images were collected on an Olympus IX71 inverted fluorescence microscope or Zeiss LSM 880 confocal laser scanning microscope.Scanning electron microscopy (SEM) micrographs were collected on a field emission scanning electron microscope (model: JSM-6700F).ChatGPT was utilized to assist in correcting grammar mistakes and providing language suggestions for this manuscript.
Single Crystal Cultivation.The single crystals of LBT in its cationic form were grown using the slow evaporation method at room temperature.An initial stock solution of LBT was prepared by dissolving the compound in a minimal volume of acetonitrile to achieve a supersaturated state.This solution was then filtered by using a 0.22 μm syringe filter to remove any particulate matter.A small volume of the HCl solution was carefully added.The formed needlelike crystals were examined for quality and size under a polarizing microscope before being selected for X-ray diffraction analysis.
Computational Details.LBT in its cationic form and zwitterionic form were fully optimized with the density functional theory (DFT) method by using the M06-2X density functional and 6-31+G(d,p) basis set.Analytical frequency calculations were also performed at the same level of theory to confirm that the optimized structures were at a minimum point.The frontier molecular orbitals (FMO) were displayed by using the IQmol molecular viewer package.All of the quantum chemical calculations were carried out using the Gaussian 09 program.
Cell Culture.HeLa cells, NIH/3T3 cells, MDA-MB-231, and Huh-7 cells were cultured in DMEM containing 10% FBS in a 5% CO 2 humidified incubator at 37 °C.Once the cells reached 80−90% confluence, they were dissociated into single cells with 0.05% trypsin-EDTA at 37 °C for 5 min and passaged at a ratio of 1:4−1:10 in one cell culture dish.
Cell Fixation.HeLa cells were fixed with 4% PFA for 10− 20 min at room temperature.Fixed cells were then washed with DPBS three times to remove the fixative solution.
CCK-8 Assay for the Determination of Cell Cytotoxicity.The cytotoxicity on cells was determined by the standard WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) (CCK-8) assay.HeLa cells and NIH/3T3 cells were seeded at a density of 7 × 10 3 cells and 1.4 × 10 4 cells per well, respectively, in standard 96-well clear microplates with 100 μL of culture medium and cultured overnight to reach 70−80% confluence.After that, the medium was replaced with 100 μL of fresh medium containing different concentrations of LBT (0, 2.5, 5, 10, 20, 30, and 40 μM), and water was used as vehicle control.After 24 h of incubation, 10 μL of 12 mM CCK-8 stock solution mixed with 90 μL of the abovementioned LBT containing fresh medium was added to each well for an additional 1 h of incubation.For cold treatment, cells were stained with 10 μM LBT for 1 h in the incubator followed by 5 min cold treatment (15 °C) and then another 1 h in the incubator.Control cells without LBT staining were incubated for 2 h in the incubator.After incubation, cells were changed to CCK-8 containing fresh medium and incubated for an additional 1 h in the incubator.The absorbance at 450 nm (OD 450 ) was measured using the SpectraMax M2 microplate reader (Molecular Devices).Cell viability (%) was calculated: (OD 450 sample/OD 450 control) × 100%.
Fluorescence Imaging of LBT.All fluorescence imaging was conducted by a fluorescence microscope or confocal laser scanning microscope.DMEM without phenol red was used as a staining and imaging medium.Cells were sequentially incubated with 10 μM LBT at 37 °C for 1 h, 15 °C for 5 min, and 37 °C for another 1 h.After the incubation, the cells were directly imaged without washing to reduce the perturbation of the LBT filaments.Confocal imaging was performed using the Zeiss LSM 880 confocal laser scanning microscope equipped with a Plan-Apochromat 63 × /1.4 NA oil objective lens, a photomultiplier tube, and a Gallium arsenide phosphide detector driven by the ZEN software (Carl Zeiss).For confocal imaging of LBT, the 488 nm laser was used and the emission between 500 and 700 nm were detected.For fluorescent imaging of LBT with inverted microscope, λ ex = 465−495 nm, λ em = 512−558 nm except in Figure 2 (λ ex = 450−490 nm, λ em = LP 515 nm); for fluorescent imaging of TPE-Ph-In or TMRM with inverted microscope, λ ex = 509− 519 nm; emission was collected using a long-pass filter with a cutoff wavelength of 590 nm.All filters used for excitation and emission were as indicated in the figure captions.All videos of TPE-Ph-In and TMRM were taken at a speed of 5 frames per second.Digital images were captured and processed by ZEN software (ZEN 2.5 lite) in grayscale and pseudo color.
Image Processing.Images were processed by Fiji (ImageJ 1.53s) software (NIH).Z-stacked images acquired on the Zeiss LSM confocal were processed on ImageJ.Images for colocalization studies were first processed with the background subtract function using Fiji and then analyzed with the colocalization function on Fiji.
Statistics and Reproducibility.All microscopy experiments were repeated with similar results.The details are listed in Table S2 (Supporting Information).

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.4c06186.CCDC 2202095 contains the supplementary crystallographic data for this paper.These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: + 44 1223 336 033.All data used to support the findings in the paper and Supporting Information are available from the corresponding authors upon reasonable request.

Figure 1 .
Figure 1.Photophysical properties of LBT in aqueous solutions.(a) Absorption spectra and (b) emission spectra (λ ex = 365 nm) of LBT at different HCl concentrations.The inset shows the chemical structure of the LBT.(c) Relative PL intensity changes of LBT at 450 nm.Inserts show the corresponding PL images.I 0 is the initial intensity at 450 nm.λ ex = 365 nm.(d) Absorption spectra and (e) emission spectra (λ ex = 420 nm) of LBT at different NaOH concentrations.(f) Relative PL intensity changes in LBT at 545 nm.The inset shows the linear relationship between PL intensity changes and NaOH concentrations.I 0 is the initial intensity at 545 nm.λ ex = 420 nm.(g) Timeresolved PL decay curves of LBT at room temperature.(h) The structures of cation and zwitterion forms of LBT and their corresponding electrostatic potential maps calculated by using the M06-2X density functional and 6-31+G (d, p) basis set.

Figure 2 .
Figure 2. Photophysical properties of LBT in different aggregate forms.(a) Illustration of crystal growth of cationic LBT with its (b) single crystal structure and (c) packing mode.(d) Illustration of the gelation process of zwitterionic LBT with its (e) corresponding images and (f) PL spectra (λ ex = 420 nm) before and after gelation.(g) A schematic diagram showing the self-assembly process of LBT and its (h) fluorescence (λ ex = 450−490 nm; emission was collected using a long-pass filter with a cutoff wavelength of 515 nm) and (i) SEM images.
LBT-Nanofiber-Mediated Synchronization of ΔΨ m Oscillations.During fluorescence imaging of HeLa cells stained with LBT and TPE-Ph-In, it was observed that the TPE-Ph-In channel exhibited spontaneous flashings at a cellular level, which indicated fluctuation in mitochondrial membrane potential. 4,61To further investigate the flashing signal of ΔΨ m , specific cells were selected as a region of interest (ROI) and the red fluorescence intensity was recorded over a period of 100 s.The blank region (No. 16) represented the background fluorescence intensity.Interestingly, two types of ΔΨ m oscillation patterns were observed (Figures 5a,b and S14).The first type consisted of transient spikes in fluorescence intensity, with a full width at half-maximum (fwhm) of approximately 0.5 s (n = 211 from three independent experiments), indicated by the blue arrows.The second type comprised oscillations lasting for more than half a

Figure 4 .
Figure 4. (a) Fluorescence images of live LBT-stained HeLa cells pretreated with colcemid, nocodazole, or cotreated with tunicamycin.All cells were imaged after the cold treatment.Scale bars: 20 μm.(b) Line scan analysis of LBT and TPE-Ph-In in live HeLa cells with the corresponding fluorescence (FL) intensity along the solid pink line.Cells at the three indicated time points were imaged.Green fluorescence represents LBT (λ ex = 465−495 nm, λ em = 512−558 nm); red fluorescence represents TPE-Ph-In (λ ex = 509−519 nm; emission was collected using a long-pass filter with a cutoff wavelength of 590 nm).Scale bars: 20 μm.

Figure 5 .
Figure 5. (a) Fluorescence image of living HeLa cells.Cells were incubated with LBT (10 μM) and TPE-Ph-In (5 μM) for 2 h followed by the cold treatment.(b) Time course analysis of TPE-Ph-In fluorescence intensity of cell No. 2 in (a), where F and F 0 represent the real-time and initial fluorescence intensities, respectively.(c) The fluorescence image of living HeLa cells.Cells were incubated with TPE-Ph-In (5 μM) only for 2 h followed by the cold treatment.(d) Time course analysis of TPE-Ph-In fluorescence intensity of cell No. 7 in (c).(e) The fluorescence image of living HeLa cells.Cells were incubated with LBT (10 μM) and TMRM (20 nM) for 2 h, followed by cold treatment.TMRM was used in nonquenching mode.(f) Time course analysis of TMRM fluorescence intensity of cell No. 10 in (e).(g) The fluorescence image of HeLa cells.Cells were incubated with TMRM (20 nM) only for 2 h, followed by the cold treatment.TMRM was used in nonquenching mode.(h) Time course analysis of TMRM fluorescence intensity of cell No. 12 in (g).Scale bar: 20 μm. Green fluorescence represents LBT (λ ex = 465−495 nm, λ em = 512−558 nm); red fluorescence represents TPE-Ph-In (a and c) or TMRM (e and g) (λ ex = 509− 519 nm; emission was collected using a long-pass filter with a cutoff wavelength of 590 nm).

Figure 6 .
Figure 6.(a) Fluorescence images of living HeLa cells.Cells were incubated with LBT (10 μM) and TPE-Ph-In (5 μM) for 2 h, followed by cold treatment.Scale bar: 5 μm.(b−e) Time course analysis of TPE-Ph-In fluorescence intensity of individual mitochondria in (a).Mitochondria were randomly picked and labeled as a fluorescence region of interest (ROI) and their fluorescence intensity.F and F 0 represent the real-time and initial fluorescence intensities, respectively.(f−g) Percentage of cells with ΔΨ m signal oscillations incubated with TPE-Ph-In (5 μM) and different concentrations of LBT with or without cold treatment.

Figure 7 .
Figure 7. Relative fluorescence intensity changes of TPE-Ph-In in live HeLa cells stained with LBT and TPE-Ph-In before and after the treatment of CCCP (a), NaN 3 (b), oligomycin (c), nigericin (d), monensin (e), and valinomycin (f), respectively.Arrows indicate the time points (100 s) of adding drugs.F and F 0 represent the real-time and initial fluorescence intensities, respectively.

Figure
Figure S1−S26: emission spectra of LBT; normalized excitation spectra of LBT; 1 H NMR spectra of LBT treated with HCl or NaOH; HOMO and LUMO plots and corresponding energy gaps from DFT calculations;