Imaging mitochondrial membrane potential via concentration-dependent fluorescence lifetime changes

Domain: biology
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
Mitochondria are central to cellular metabolism. Various fluorescence tools have been developed for imaging the mitochondrial environment. Yet, new reporters and imaging methods for directly reading the mitochondrial status are needed for high spatial-temporal resolution imaging. Here, we introduce PKMitoDeepRed (PKMDR), a low-phototoxicity mitochondrial probe for time-lapse imaging, whose fluorescence lifetime serves as a sensitive indicator of mitochondrial membrane potential (Δψm). The positively charged PKMDR accumulates within mitochondria under a higher Δψm, leading to concentration-induced quenching and a measurable decrease in fluorescence lifetime. Since mitochondrial respiration primarily regulates Δψm, PKMDR’s fluorescence lifetime effectively reports on the status of oxidative phosphorylation. Using PKMDR with fluorescence lifetime imaging microscopy (FLIM), we visualize heterogeneous Δψm across individual cells, organoids, and tissues over time. This method reliably reveals the heterogeneity between metabolically active peripheral mitochondria and relatively inactive perinuclear mitochondria in various cell types. Overall, PKMDR-FLIM is a robust tool for directly visualizing Δψm with high spatiotemporal resolution.

Introduction
Mitochondria are essential organelles that serve as powerhouses of the cell and act as transition hubs for multiple cellular signaling pathways, such as autophagy and apoptosis. Mitochondrial dynamics are closely correlated with cellular activities. Thus, regulating their plasticity and dynamic features is crucial for controlling cellular respiration and maintaining homeostasis in organisms1,2. In oxidative phosphorylation (OXPHOS), ATP synthase and the electron transport chain (ETC) generate ATP, which relies on mitochondrial membrane potential (MMP, or Δψm) for the proton-motive force. High-energy electron carriers (NADH and FADH2) produced in the Krebs cycle sequentially transfer their electrons to oxygen along the chain through ETC complexes, while protons are pumped out of the matrix by harnessing the released energy, establishing MMP between the intermembrane space and the matrix3.

Alterations in Δψm can disrupt proton gradient formation, leading to metabolic disturbances4. Therefore, monitoring the membrane potential across the inner membrane is crucial for assessing the metabolic activity of mitochondria and cell viability. Classical MMP probes are cationic small-molecule dyes, such as Rhodamine 1235, TMRM (tetramethyl rhodamine methyl ester), TMRE6, and JC-17, whose accumulation in the inner membrane is proportional to Δψm according to the Nernst equation. Among these, TMRM and TMRE are widely regarded as “gold standards” for measuring Δψm through fluorescence intensity readouts8,9. However, intensity readouts may produce misleading information due to inner filter effects—absorption and scattering events that modulate detected fluorescence intensity10. Additionally, their application is susceptible to concentration fluctuations, sample geometry, and photobleaching10. The J-aggregate-induced spectral shifts of the JC-1 molecule render it a ratiometric probe capable of reading absolute potential via an intensive parameter rather than an extensive one. However, the JC-1 protocol suffers from weak signals and cumbersome calibrations8.

The fluorescence lifetime (τ) of a fluorophore is the average time it spends in the excited state before returning to the ground state. For a fluorescent probe, τ serves as a kinetic parameter that indicates the intensity decreases to 1/e of its initial value. Fluorescence lifetime imaging microscopy (FLIM) is gaining increasing attention in biological imaging. Tailored FLIM sensors have been developed for various parameters, such as pH11, viscosity12,13, temperature14 and calcium15. Compared to intensity imaging, FLIM has the advantage of measuring absolute values, as lifetime is an intensive parameter whose value is not affected by fluorescence signal strength16. Therefore, FLIM can bypass the drawback of intensity imaging to a certain extent17.

Recently, scientists have developed several strategies to study mitochondria using FLIM. For instance, MitoFlipper has been reported to respond to osmotic shocks and provide membrane tension readouts18. MitoPB Red indicates polarity changes under oxidative stress19. Classic dyes such as TMRM and SYTO are also responsive under FLIM, with their signals attributed to mitochondrial membrane potential20. Moreover, the fluorescence lifetime of Mitorotor-1 is affected by molecular free volume, which relates to lateral diffusion and is further subject to change in IMM fluidity21. Nonetheless, these probes have not yet established a clear-cut relationship between the metabolic state of mitochondria and the fluorescence lifetime of probes, and their versatility with advanced samples has not been fully demonstrated.

Here, we leverage PKMDR, a gentle far-red mitochondrial probe developed by our group, to precisely report mitochondrial metabolic heterogeneity from individual cells to tissues using fluorescence lifetime imaging technology. PKMDR offers high brightness and very low phototoxicity, making it ideal for long-term time-lapse recordings22. Although it is commonly accepted that fluorescence lifetime is independent of the concentration of fluorescent molecules, we demonstrate that the fluorescence lifetime of PKMDR decreases at high concentrations due to concentration-induced quenching. Since the mitochondrial accumulation of PKMDR depends on mitochondrial membrane potential, the fluorescence lifetime of PKMDR can directly reflect the membrane potential of mitochondria. We showcase advanced applications under different metabolic conditions, including senescence, knockout cells, oocytes at various stages, and T cells before and after activation. Additionally, PKMDR reveals metabolic heterogeneity within individual cells, across different cell cycles, between individual cells of embryos, and in organoid and tumor tissues.

Results
Cationic dyes generally exhibit concentration-dependent fluorescence lifetime due to quenching

In our earlier study, we devised PKMito dyes, which were red and far-red probes specifically designed for mitochondrial imaging22,23. Thanks to the alleviated phototoxicity rendered by the intramolecular triplet state quenchers, PKMito dyes demonstrated minimal levels of phototoxicity, which was ideal for time-lapse imaging. Among them, PKMitoDeepRed (PKMDR) was based on the far-red chromophore Cy5, which was particularly prone to aggregation24,25. A previous study has shown that Cy5 on a heavily labeled secondary antibody, with a degree of labeling (DOL) greater than 3, exhibited a lower fluorescence lifetime compared to antibodies with a DOL of 126. This prior data led us to speculate that PKMDR may display a concentration-dependent fluorescence lifetime (Fig. 1a), especially in mitochondrial imaging.

To test this hypothesis, we measured the fluorescence intensity and lifetime of PKMDR at various concentrations of DMSO using a fluorescence spectrophotometer, whose cuvette-based setup offers accurate measurements. The relationship between fluorescence intensity and dye concentration deviated from linearity starting at 20 μM, indicating self-quenching behavior (Supplementary Fig. 1a)27. Accordingly, the fluorescence lifetime of PKMDR sharply declined above 20 μM (Fig. 1b), collectively supporting the emergence of concentration-induced quenching. Notably, while PKMDR was mainly in a dispersed state in solution below 20 μM, there is a known artifact of radiative energy transport (RET). Within the long light path of the cuvette, the re-absorption of the emitted fluorescence photon at another dye molecule would give rise to an apparent increase in lifetime, accounting for the fluorescence lifetime appeared to increase between 1 μM and 20 μM17,28. At concentrations above 20 μM, as the PKMDR molecules started to self-quench, the fluorescence lifetime decreased with increasing concentration, yet the total fluorescence intensity was still going upwards (Fig. 1c). At above 500 μM, the fluorescence intensity of the solution finally decreased due to a strong quenching effect17.

The counterintuitive variation in fluorescence lifetimes with concentration revealed alterations in the photophysical properties of the fluorophore at elevated concentrations, potentially indicative of behavior arising during the aggregation process of this fluorescent molecule. We speculated that such behavior should be general among fluorescent dyes29. Concentration-induced quenching can also be recorded with the DMSO solution of TMRM and TMRE, which gave a decreasing fluorescence lifetime at mM concentration ranges (Fig. 1d, Supplementary Fig. 1b). Notably, TMRM and TMRE were less hydrophobic than PKMDR, which translates into higher critical concentrations of fluorescence lifetime drop.

We further established the generality of concentration-caused lifetime changes in membranous environment, where the fluorescence lifetime of PKMDR in giant unilamellar vesicles (GUVs) at various concentrations was measured using FLIM imaging (Fig. 1e). GUVs were prepared using the electro-formation technique, where dyes were doped into the GUVs solution. Of note, the concentration of dyes referred to the apparent concentrations in the GUVs solution, as the absolute concentration of dyes in the 2D lipid bilayer was hard to quantify. In GUVs, PKMDR lifetime showed a consistent decrease as the apparent concentration of the dye increased (Fig. 1g). Mitorotor-1, whose fluorescence lifetime was suggested as a microviscosity sensor21, gave a concentration-dependent lifetime in GUVs as well (Fig. 1f, h). Such a trend was also generalizable to TMRM and TMRE in GUVs (Fig. 1i, j and Supplementary Fig. 2), although occurring at significantly higher dye concentrations. Notably, PKMDR exhibited the largest dynamic range of fluorescence lifetime variation among the tested dyes, spanning from 0.2 ns to 1.5 ns across the concentration range tested (Fig. 1g–j). Moreover, we also examined the impact of temperature and pH on the lifetime of PKMDR. The fluorescence lifetime of PKMDR was measured in phosphate buffer solutions across pH 5.8–7.8. The results demonstrated that the fluorescence lifetime remained stable and independent of environmental pH within this physiologically relevant range (Supplementary Fig. 3a). To evaluate temperature effects, PKMDR was incorporated into DOPC vesicles. The fluorescence lifetime exhibited a temperature-dependent change of 0.38 ns over the range of 25°C to 45°C (Supplementary Fig. 3b). Importantly, this variation is significantly smaller than the 1.24 ns lifetime shift induced by concentration-dependent aggregation of PKMDR (Fig. 1g). While temperature modulates the fluorescence lifetime to a measurable extent, its contribution is ~3.3-fold weaker compared to concentration-dependent effects. This indicates that PKMDR’s sensing performance is primarily governed by its concentration rather than thermal fluctuations under physiological conditions. Overall, these data suggested that the concentration-dependent fluorescence lifetime was a general phenomenon in cationic mitochondrial dyes, where PKMDR was a potential FLIM sensor due to its low critical concentration, high brightness, and large dynamic range in fluorescence lifetime (Supplementary Table 1).

PKMDR is a FLIM probe that reveals the mitochondrial inner membrane potential

We then exploited the concentration-based lifetime change of PKMDR in mitochondrial imaging. PKMDR was an established inner-membrane stain, as confirmed with two-color SIM imaging of U2OS cells using Tomm20-GFP as an outer-membrane counter-marker (Fig. 2a). PKMDR, like many cationic dyes, exhibits a high affinity to mitochondria due to the electrochemical drive of the negative potential of mitochondrial membrane potential (MMP). In contrast to TMRM mitochondrial dye, which mainly accumulates in the mitochondrial matrix, the hydrophobic nature of PKMDR renders its selective partition at the inner mitochondria membrane (IMM), giving a higher concentration and an amplified change-of-concentration in the 2D compartment. Therefore, we speculate that the concentration-induced lifetime change of PKMDR can be a sensitive readout for mitochondrial MMP. In the mitochondria with high MMP, the local concentration of PKMDR can be very high at the IMM, triggering dye quenching, resulting in a lower fluorescence lifetime. On the contrary, in mitochondria with low MMP, PKMDR was relatively dilute, giving a lower fluorescence intensity and a longer fluorescence lifetime (Fig. 2b).

To test our hypothesis, we treated HeLa cells with FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, mitochondrial oxidative phosphorylation uncoupler), antimycin A (ETC complex III inhibitor), rotenone (ETC complex I inhibitor) and oligomycin (ATP synthase inhibitor), resulting in a decrease (FCCP, antimycin A, rotenone) or in an increase (oligomycin) in MMP30 (Fig. 2c, Supplementary Fig. 4). Fluorescence lifetime imaging of PKMDR, referred to as PKMDR-FLIM, shows a clear increase after FCCP, antimycin A, and rotenone treatment (Δτ = 0.56 ns, 0.37 ns, 0.24 ns, respectively), which was in line with a decrease in MMP (Fig. 2d). Similarly, oligomycin treatment resulted in a reduced PKMDR-FLIM value (Δτ = −0.29 ns). We also measured the lifetime of PKMDR in cells treated with high and low osmolarity solutions, which resulted in mitochondrial swelling and crumpling. If the PKMDR lifetime is sensitive to changes in membrane tension, we would expect to observe opposite trends in the FLIM changes following hypo-osmotic versus hyper-osmotic shock. However, both treatments resulted in increases in the fluorescence lifetime of PKMDR, supporting the hypothesis that alterations in osmotic pressure led to mitochondrial dysfunction and a subsequent decrease in MMP (Supplementary Fig. 5). To rule out the potential influence of other membranous organelles during the FLIM measurements, we isolated the mitochondria from mouse cardiomyocytes and performed time-lapse imaging experiments using purified mitochondria. Consistent with the cell-based experiments, a gradual increase of fluorescence intensity, along with the concomitant decrease of fluorescence lifetime of PKMDR, was recorded in purified mitochondria upon the addition of succinate (substrate for complex II). Finally, upon the addition of the FCCP decoupler, which dissipated the MMP and dye accumulation, a sharp increase in PKMDR lifetimes was recorded, along with the change in fluorescence intensity in the opposite direction (Fig. 2e, f, and Supplementary Movie 1). The MMP changes during succinate and FCCP treatments were confirmed using TMRM stain and intensity imaging (Supplementary Fig. 6). The time-lapse imaging results using isolated mitochondria can rule out possible biochemical changes to a maximum extent, therefore strongly supporting that the fluorescence lifetime of PKMDR can directly report on MMP in live cells.

PKMDR-FLIM can report mitochondrial respiration

The MMP is primarily generated by the ETC in the IMM. Electrons move through the ETC, driving protons from the matrix into the intermembrane space, creating a proton gradient. This gradient establishes the MMP, essential for ATP synthesis via mitochondrial F1FO ATP synthase. MMP magnitude indicates mitochondrial health and metabolic state (OXPHOS); a higher membrane potential suggests more active metabolism, whereas a reduced potential indicates mitochondria respiration slowdown or mitochondrial dysfunction. We therefore tested PKMDR-FLIM as an indicator of mitochondrial activity. The increase of mitochondrial activity via nutrient starvation is a canonical cellular response31–33. Depriving key nutrients like pyruvate and glutamine increases the levels of OXPHOS, which has been used as a stimulus to activate mitochondria31–33. In our test, nutrient starvation caused a decrease in PKMDR lifetime (Fig. 3a, b, Supplementary Fig. 7). Mitochondrial respiration and ATP production increased during the first two hours of serum starvation34. In line with this, PKMDR lifetime also decreased in the FBS starvation experiment (Fig. 3a, b, Supplementary Fig. 7). In contrast to starvation, senescent cells undergo various alterations in terms of their mitochondria’s function, structure, and dynamics. Senescent cells have reduced mitochondrial membrane potential and lower ratios of ATP/ADP and NAD+/NADH35,36. We induced senescence in human umbilical vein endothelial cells (HUVEC) using the DNA-damaging agent doxorubicin37. Senescence was confirmed by senescence-associated β-galactosidase (SABG) staining and by measuring the expression of senescence-associated genes p16 and p21 by RT–qPCR (Supplementary Fig. 8). In senescent HUVECs, PKMDR-FLIM indicated increased fluorescence lifetimes, corroborating the reduced MMP (Fig. 3a, b).

Beyond the starvation and senescence models, the regulation of proteins in the mitochondrial respiratory chain also impacts the state of OXPHOS. We therefore tested if PKMDR-FLIM is responsive to OXPHOS deficiency, specifically a change in ETC complexes. NDUFS2, a core subunit of mitochondrial complex I, was essential for acute oxygen-sensing, development, and metabolism38. COX4I1, a common isoform of the COX4 subunit, was responsible for regulating COX and catalyzing the last electron transfer step in the respiratory chain of the mitochondria, which was the primary oxygen consumer (Fig. 3c)39. Genetically inactivating complex IV and complex I by knocking out its subunit COX4Il and NDUFS2 in 143B cells40 would both reduce respiration, as characterized by the elevation of PKMDR lifetimes (Fig. 3d, e, Supplementary Fig. 9).

The mitochondrial metabolism within a cell undergoes dynamic alterations across various stages of its lifecycle, manifesting as distinct metabolic profiles that are finely tuned to meet the specific energetic and biosynthetic demands of each phase. We next challenged whether PKMDR-FLIM can track mitochondrial metabolic dynamics throughout different stages. Mitochondria in early oocytes exhibit lower ETC activity compared to those of neighboring granulosa cells, and mitochondria metabolism increases during oocyte maturation41,42. Consistent with the fluorescence intensity imaging results of the TMRM (Supplementary Fig. 10), we found that PKMDR-FLIM can accurately map the mitochondria respiration of oocytes at different stages together with the granulosa cells (Fig. 3f–i). Meanwhile, it was well known that mitochondrial metabolism was accelerated after T cell activation. PKMDR-FLIM can also profile such a change in mitochondria, where the naïve T cells had a longer lifetime compared to activated T cells, which exhibited a shorter fluorescence lifetime (Fig. 3j, k). Overall, in starvation and senescence, ETC complex protein deficiency, oocyte development, and T cell activation models, PKMDR-FLIM was a robust protocol for direct visualization of mitochondrial metabolic status across various samples.

PKMDR-FLIM unveils mitochondrial metabolic heterogeneity in tissues and developing embryos

Within the complex architecture of tissues, differentiated cells exhibit diverse metabolic specialization. Thus, the study of mitochondria metabolic heterogeneity facilitates a deeper understanding of the development of various diseases. Previous membrane potential probes, such as TMRM, are subject to inaccuracies in fluorescence intensity imaging of tissues due to background fluorescence, light scattering, and variations in the path length of emitted light. Additionally, the fluorescence intensity signals can be compromised by uneven tissue sample thickness and defocusing, which may not accurately reflect the genuine changes in biological processes. Therefore, we explored whether PKMDR can label mitochondria in tissue samples and perform fluorescence lifetime imaging. As shown in Fig. 4a, high-quality images of mitochondria in brain organoids and tumor tissue can be recorded. The far-red fluorescence of PKMDR rendered compatibility for multiplexing with other fluorescent proteins or dyes. The strong fluorescence signal and the robust staining protocol of PKMDR allowed us to map the MMP heterogeneity at tissue level. At the same time, PKMDR-FLIM can clearly distinguish MMP differences between cells in brain organoids and tumor tissue (Fig. 4a).

We further investigated MMP in early-stage mouse embryos. In developing embryos, mitochondria play a crucial role, with their distribution, activity, and structure being closely tied to cellular functions43,44. From the two-cell to blastocyst stage, mouse embryos were approximately 50 to 150 μm. In these samples, intensity imaging of mitochondrial MMP using a TMRM probe was no longer accurate, as the light scattering, refraction, and de-focusing in thick samples will complicate the signal analysis (Fig. 4b). Yet, PKMDR-FLIM suggested that from the 4-cell stage onward, MMP began to exhibit a heterogeneous pattern across different cells (Fig. 4b, c). It has been reported that certain pluripotency regulators were highly heterogeneously expressed among blastomeres of the 4-cell embryo45–48, suggesting the presence of factors that modulate mitochondrial metabolic activity in individual blastomeres. Overall, PKMDR-FLIM offers direct visualization methods of MMP with spatial precision in tissues. The absolute-reading of FLIM is particularly advantages over intensity-based methods in imaging thick and scattering samples to avoid signal distortion.

Mitochondria in a single cell exhibit temporal and spatial heterogeneity

Mitochondrial metabolism was a dynamically changing process in both space and time. Yet, studying such heterogeneity heavily hinges on fluorescence imaging as it offers subcellular resolutions. Using PKMDR-FLIM, we investigated the distribution of mitochondrial membrane potential in various cell types. During early embryo development, the distribution and membrane potential of mitochondria were stage-specific and exhibited heterogeneity not only between cells but also within individual cells49–51. PKMDR-FLIM suggested that mitochondria with high MMP were distributed in the cell periphery, whereas low-MMP mitochondria were located in the central region of MII oocytes (Fig. 5a). Such a distribution pattern was also found in zygotes (Fig. 5b). Notably, the spatial distribution patterns of MMP in oocytes remain a subject of ongoing debate. Conflicting results have been reported between TMRM measurements (showing MMP gradient decreasing from the central region to the periphery) and those obtained through JC-1 imaging (demonstrating an inverse peripheral-to-central gradient)52–55. Our analysis aligns closely with the spatial patterns observed in JC-1, which providing support and insights for the study of spatial heterogeneity of MMP.

We also mapped the MMP in HUVEC and neurons. In these spreading cells, mitochondrial membrane potential increased from the nuclear region to the cell periphery (Fig. 5c, d). These results highlight how mitochondrial membrane potential varies spatially. Furthermore, the FLIM analysis of mitochondria in different regions in neurons showed decreased levels of MMP in the neuronal soma compared to terminals (Fig. 5e, f). This result was in line with a previous study, where the metabolic flux and cellular respiration analyses of isolated neuronal somata and synaptosomes evidence suggested that the neuron soma exhibit increased levels of aerobic glycolysis and decreased levels of OXPHOS compared to the terminals, both in their resting and activated states56. Therefore, using PKMDR-FLIM, the heterogeneity of MMP at the sub-cellular level can be unambiguously imaged, supplementing and supplanting other methods for studying metabolism with lower spatial and temporal resolutions.

Finally, we demonstrated that the extraordinarily low phototoxicity of PKMDR enables time-lapse FLIM recording, revealing the temporal regulation of mitochondrial MMP. The long stretch of video captures multiple mitochondrial events with sudden decreases in membrane potential followed by recoveries (Fig. 6a, b and Supplementary Movie 2). These events were reminiscent of the well-studied Mito-Flash events, which were optical readout systems that use frequency coding to represent free-radical generation and energy metabolism processes within individual mitochondria57,58. In primary neurons, PKMDR-FLIM can record the real-time transport of mitochondria between neurites and the soma, simultaneously revealing both their location and membrane potential (Fig. 6c, Supplementary Movie 3). The complexity of mitochondrial trafficking in neurons has remained difficult to study due to the limitations of traditional imaging techniques, and the relationship between mitochondrial transport and metabolism has long been a subject of debate59–62. PKMDR-FLIM holds the potential to address key questions regarding mitochondrial function and its role in neuronal health and disease.

Methods
Fluorescence microscopy
Confocal laser scanning microscopes (TCS SP8 or STELLARIS 8 FALCON; Leica) with a pulsed white light laser (WLL) fitted with an APO 100×/1.40 oil and an APO 40×/1.32 oil objective lens, as well as HyD detectors, were used for confocal and FLIM imaging. FLIM images were acquired under excitation at 633 nm (SP8, WLL, 80 MHz) or 638 nm (STELLARIS 8, WLL, 80 MHz) and the emission was collected in the range of 650‒700 nm. The pinhole was set to 1.0 AU. Data were collected using LAS X (Leica) at 512 × 512 or 1024 × 1024 pixel resolution and frame accumulations of 1–10 times, or collecting 500 photons per pixel (Supplementary Table 2). FLIM data were analyzed using the LAS X software (Leica Microsystems) by fitting a mono-, bi-, or triexponential decay model (n-exponential reconvolution, Eq. 1) to the decay (χ2 < 1.3, Eq. 2) (Supplementary Table 2). Intensity-weighted lifetime (Eq. 3) was used as mean fluorescence lifetime in each image. The instrument response function (IRF) used for deconvolution was measured under the same microscope settings. For time-lapse imaging, multiple images were acquired with a time delay of 11 s or 15 s interval between the starting point of capturing two successive images. Phasor plot analysis in Fig. 5 is performed in LAS X software (Leica Microsystems). After selecting a region of interest around the cell for analysis, the corresponding phasor plot is displayed, and pixels with similar lifetimes are grouped into clusters. Distinct clusters emerge when MMPs in an individual cell are different. Pixels forming each cluster can be identified in the lifetime image and thus separated. No threshholding is applied.

y(t) = IRF(t + Shift_IRF) * (Bkgr_IRF + sum_{i=1}^{n} A_i * e^{-t/τ_i}) + Bkgr_IRF (1)

n: Number of exponential components
A: Amplitudes – Exponential pre-factors
τ: Exponential Decay Times (e.g. lifetimes)
Bkgr: Tail Offset – Correction for background
Shift_IRF: IRF Shift – Correction for IRF displacement
Bkgr_IRF: Irf Background – Correction for IRF background

χ^2 = sum_{k=1}^{n} (N(t_k) - N_c(t_k))^2 / N(t_k) (2)

N(t_k): Measured fluorescence decay function
N_c(t_k): Calculated decay function
n: Evaluated across the number of data points

τ_AvInt = sum_{k=0}^{n-1} I_k * τ_k / sum I_k (3)

I: Intensities associated with each exponential component, normalized to the time resolution of the measured decay curve in order to be displayed in photon counts
I_Sum: of Fluorescence Intensity for all components
τ_AvInt: Mean Photon Arrival Time – Intensity weighted average lifetime

HIS-SIM imaging (Fig. 2a) was performed by following the previous report63. A commercial structured illumination microscope (HIS-SIM) was used to acquire and reconstruct the cell images. To further improve the resolution and contrast in reconstructed images, sparse deconvolution was used by following the previous report64.

Cell culture and treatment
HeLa, U2OS, MC-38, 143B and L929 cell lines were grown in DMEM medium with 10% v/v fetal bovine serum (FBS, Vistech), 1% v/v penicillin-streptomycin mixture (Gibco) and GlutaMax™ (Gibco) supplement addition. Regarding that COX4I1-KO 143B and NDUFS2-KO 143B cell lines had conspicuous attenuation in oxygen consumption rate, we added extra 3 mM pyruvate and 50 μg/mL uridine in aforementioned DMEM system to sustain the cell viability for these two gene knocked-out cell lines. HUVEC were cultured in endothelial cell medium (ScienCell) with 5% FBS (ScienCell), 1% ECGS (ScienCell) and 1% P/S (ScienCell). All cells were incubated at 37°C with 5% CO2. For regulating mitochondrial membrane potential (Fig. 2c-d), FCCP (2 μM), antimycin A (10 μM), rotenone (10 μM), oligomycin (20 μM) were used for modulation of mitochondrial membrane potential in live cells.

For induction of senescence (Fig. 3a, b), HUVEC cells were treated with 200 nM of doxorubicin for 48 h, and then the culture medium was refreshed. Cells were maintained in culture and imaged at days 2 after treatment.

Hypertonic and hypotonic treatment (Supplementary Fig. 5) were performed using a previously published protocol21. Hypotonic solutions was salt-free HANKS buffer (140 mM NaCl, 2.5 mM KCl, 0.5 mM MgCl2, 1.2 mM CaCl2, 5 mM glucose, 10 mM HEPES pH 7.5), and supplemented with 500 mM of NaCl was used to make hypertonic solutions. Cell starvation model was established using a previously published protocol with modifications21. For FBS starvation (Fig. 3a, b), cells were cultured in FBS-free media for 2 h before imaging. For nutrient starvation (Fig. 3a, b), cells were cultured in DMEM media without glucose, sodium-pyruvate and glutamine (Thermofisher Scientific) for 2 h before being taken for imaging.

PKMDR labeling for live-cell FLIM imaging
Cells were seeded into a glass-bottom dish (Cellvis) a day before imaging with the cell density at around 4 × 10^4 cells/mL. For labeling of mitochondria, cells were stained with DMEM supplemented with 250 nM PKMDR for 20 min at 37°C. Following the staining procedure, the cells were washed up to three times with pre-warmed PBS or culture medium to remove the unbound dye, followed by the addition of DMEM for live cell imaging. If necessary, further incubation at 37°C for 15 min before imaging can reduce unspecific ER labeling. For multicolor labeling, we recommend sequential staining if the used dyes require different incubation times.

Lentiviral production and generation of stable cell lines
pBOB-EF1-H2B-GFP plasmid, psPAX2 and pMD2.G were kindly offered by Dr. Jing Hu’s lab (Peking University). Lentivirus production was carried out in HEK293T cells cultured to 90–95% confluency. The cells were co-transfected using Lipo8000™ Transfection Reagent (Beyotime) with a mixture of lentiviral vectors (containing the cDNA of interest), along with the packaging plasmids psPAX2 and pMD2.G at a ratio of 10:7:3. Virus-containing supernatants were collected and filtered through 0.45 μm filters 48 h post-transfection, then employed to infect target cells for 24 h in the presence of 10 μg/ml polybrene (Beyotime). Infected cells were selected by 10 μg/ml puromycin (Beyotime). H2B-GFP expression was validated by confocal imaging (Fig. 4a).

Tumor tissue imaging
All animal procedures were approved by the Peking University Animal Use and Care Committee and complied with the standards of the Association for Assessment and Accreditation of Laboratory Animal Care (code number IMM-ChenZX-1). For cancer models (Fig. 4a), female C57BL/6J mice (8 weeks) (Beijing Vital River Laboratory Animal Technology) were inoculated subcutaneously with 1 × 10^6 MC38 H2B-GFP cells into the right flank. After tumor growth up to 100 mm^3, tumors were rapidly removed and placed in cold (4°C) HBSS for 1 min. Next, tumors were dissected for mounting to the cutting stage, and 100 μm thick slices were cut in ice-cold HBSS using a VT1200 vibratome (Leica). The slices were stained with DMEM supplemented with 500 nM PKMDR for 45 min at 37°C. After wash for 3 times, the slices were placed to glass-bottom dishes (Cellvis) and imaged using TCS SP8 FALCON confocal microscope (Leica).

Sen-β-Gal activity
Cells were fixed for 15 min in 2% PFA at room temperature, and then stained for 16 h at 37°C in the dark using the Senescence β-Galactosidase Staining Kit (Beyotime). After incubation, cells were imaged using a Zeiss Axiovert 200 microscope (Zeiss) and analyzed with ImageJ (Supplementary Fig. 8).

qPCR
Total RNA (1 μg) was extracted from cells with RNA simple Total RNA Kit (TIANGEN) and reverse-transcribed into cDNA by HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The cDNAs were amplificated by the TB Green® Premix Ex Taq™ II (Takara) using the CFX96™ Real-Time System (Bio-Rad). 2−ΔΔCt method was used to quantify the relative expression. The mRNA levels were normalized by GAPDH. The primer sequences are as follow: (1) 5’-GATCCAGGTGGGTAGAAGGTC-3’ as p16-F and 5’-CCCCTGCAAACTTCGTCCT-3’ as p16-R; (2) 5’-TGTCCGTCAGAACCCATGC-3’ as p21-F and 5’-AAAGTCGAAGTTCCATCGCTC-3’ as p21-R; (3) 5’-GGAGCGAGATCCCTCCAAAAT-3’ as GAPDH-F and 5’-GGCTGTTGTCATACTTCTCATGG-3’ as GAPDH-R (Supplementary Fig. 8).

Fluorescence intensity and lifetime measurement
PKMDR, TMRM, and TMRE were dissolved in DMSO for fluorescence intensity and fluorescence lifetime measurement (Fig. 1b–d, Supplementary Fig. 2). 10 mM stock solution was diluted with DMSO to different concentration ranging from 10 nM to 5 mM for each dye. Fluorescent intensity curve was acquired by TECAN Infinite® 200 Pro Multimode Microplate Readers, where 384-well microplate was used to hold diluted samples for fluorescence scanning at room temperature. Fluorescent lifetime measurement was conducted using FLS980 Spectrometer (Edinburgh Instruments) containing an integrating sphere at room temperature with in a 1 cm square quartz cuvette. Fluorescence lifetime data were analyzed using the Fluoracle software (Edinburgh Instruments) by fitting a mono-exponential decay model (n-exponential reconvolution) to the decay. The instrument response function (IRF) used for deconvolution was measured under the same instrument settings. Measurements were carried out at their excitation wavelength (PKMDR, 644 nm; TMRM and TMRE 515 nm).

Measurement of ROS generation of fluorophores
Singlet oxygen quantum yields of TMRM, JC-1, Mitorotor-1, SiRM, and PKMDR were measured with 1,3-Diphenylisobenzofuran (DPBF, Dibai), which can combine with singlet oxygen as a decaying fluorescent indicator. Absorbances at 525 nm of TMRM, JC-1, and Mitorotor-1 solution are modulated to 0.11 in air-saturated ACN where TMRM played as a reference. Absorbances at 625 nm of SiRM and PKMDR solution are modulated to 0.11 in air-saturated ACN in which SiRM played as a reference. Treated the system with DPBF (final concentration 8 × 10^-5 M), then irradiated TMRM, JC-1, and Mitorotor-1 with 520–530 nm LED lamp (50 mW/cm^2, SiRM and PKMDR with 620–630 nm LED lamp). The slope of linear decay of DPBF absorption at 415 nm is proportional to the absolute singlet oxygen quantum yield (Supplementary Table 1).

GUV preparation and imaging
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Fig. 1e−j) were used as lipids for the preparation of giant unilamellar vesicles (GUVs), which is dissolved in CHCl3 to a final concentration of 10 mM. GUVs were prepared using electro formation technique by Vesicle Prep Pro (Nanion Technologies). To immobilize lipid vesicles for confocal imaging, GUVs solution supplemented with different concentration of PKMDR was mixed with 0.5 wt% low gelling temperature agarose (Sigma) solution in 1:1 ratio. The mixture was dropped onto a glass slide and imaged with TCS SP8 FALCON confocal microscope (Leica).

Isolation and imaging of cardiac mitochondria
Mouse cardiac mitochondria (Fig. 2e, f) were isolated using a previously published protocol with modifications65. Glass-bottom dishes were coated with a poly-D-lysine solution (Gibco) and placed at 37°C overnight. Female C57BL/6J mice (8 weeks) (Beijing Vital River Laboratory Animal Technology) were anesthetized with an i.p. injection of tribromoethanol (500 mg/kg) and dissected. Heart was removed and washed with pre-cooled mitochondria isolation medium (300 mM sucrose, 10 mM HEPES (pH 7.2), 1 mM EGTA, and 0.5 mg/mL BSA, adjust pH to 7.2 with potassium hydroxide), minced, and homogenized on ice. Centrifuge the homogenate at 4°C for 10 min at 600 × g. Then collect the supernatant and further centrifuge at 4°C for 10 min at 6000 × g. Re-suspend the pellet and place them to poly-D-lysine coated glass-bottom dishes, then centrifuge at 4°C for 6 min at 3220 × g. Replace the isolation medium to respiration buffer (300 mM sucrose, 10 mM KCl, 10 mM Tris-HCl, 10 mM K2HPO4, adjust pH to 7.2 with potassium hydroxide) at room temperature. After staining with 500 nM PKMDR, 2.5 mM of succinate or 2 μM FCCP was added to mitochondria in respiration buffer at the time of imaging for modulation of mitochondrial membrane potential.

Oocyte and early embryo collection
To obtain oocytes or pre-implantation embryos (Figs. 3f−i, 4b, c, 5a, b), 3-week-old C57BL/6N (10–12 g) female mice were intraperitoneally injected with pregnant mare’s serum gonadotropin (PMSG, 10 IU) and human chorionic gonadotrophin (HCG, 10 IU). The zona pellucida was gently removed by treatment with Tyrode’s solution (Merck Millipore, MR-004-D). Oocytes at GV and MII stages were collected at 0, 12 h post HCG. To collect GV oocytes, cumulus–oocyte complexes (COCs) was obtained by manually rupturing antral ovarian follicles, and cumulus cells were removed by repeatedly pipetting. To collect MII oocytes, COCs were harvested from the ampullae of the oviduct, and cumulus masses were removed by incubation in a hyaluronidase medium (Sigma, H4272). Pre-implantation embryos were cultured in KSOM media (Merck Millipore, MR-121-D) at 37°C with 5% CO2 and collected at the following timepoints after HCG stimulation: Zygote (21–23 h), two-cell embryos (43–45 h), four-cell embryos (54–58 h), eight-cell embryos (68–70 h), morula-stage (78–80 h) and blastocysts (96–100 h).

Oocytes and preimplantation embryos (zygote to morula) were treated with 500 nM PKMDR in pre-warmed KSOM medium for 20 min and blastocyte-stage embryos were treated for 1 h at 37°C with 5% CO2 subsequently washed three times in fresh KSOM medium. Images were taken with the STELLARIS 8 FALCON confocal microscope system (Leica).

T cell isolation and culture
Murine CD8+ T cells (Fig. 3j, k) were isolated from the spleens of C57BL/6J mice (8 weeks) by EasySep™ Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies) activated with Dynabeads® Mouse T-Activator CD3/CD28 beads (Thermofisher) for 48 h and maintained in RPMI 1640 medium (Gibco) with 10% FBS, 50 μM β-mercaptoethanol (Sigma-Aldrich) and 60 U of IL-2 (PeproTech). For labeling of mitochondria, naïve or activated CD8+ T cell transferred to glass-bottom dishes coated with poly-D-lysine and stained with RPMI 1640 medium supplemented with 250 nM PKMDR for 30 min at 37°C, respectively.

Neuron isolation and culture
For primary rat hippocampus neuron culture (Figs. 5d−f, 6c), glass-bottom dishes were coated with a poly-D-lysine solution (Gibco) and placed at 37°C overnight. In order to isolate neurons, the skulls of neonatal Sprague-Dawley rats were cut off using scissors. Next, the brain was extracted from the skull and placed into a DMEM medium pre-cooled to 4°C. Subsequently, the hippocampi were isolated from the brains using a dissecting microscope, fragmented into small segments, and then exposed to Trypsin-EDTA (0.25%) for a duration of 15 min at a temperature of 37°C. Subsequently, the trypsin was carefully substituted with preheated (37°C) DMEM solution containing 10% FBS. Following a series of pipetting actions for a duration of 1 min and subsequent incubation on ice for 5 min, the tissue sections were then settled at the bottom of the centrifuge tube. The supernatant was collected and diluted with neural culture medium (Neurobasal™ medium supplemented with B-27™ supplement, GlutaMAX™ supplement, and penicillin-streptomycin) and then transferred to glass-bottom dishes coated with poly-D-lysine. Every four days, half of the neural culture medium was substituted with a new medium. For labeling of mitochondria or cell membrane and mitochondria, cells were stained with DMEM supplemented with 10 nM or 250 nM PKMDR for 20 min at 37°C, respectively.

Organoids
Cortical organoids (Fig. 4a) were generated using a previously published protocol with modifications66. In brief, human embryonic stem cells (hESCs) were cultured to 70%–80% confluent and incubated with Accutase (Invitrogen) at 37°C for 5 min and dissociated into single cells. About 9000 live cells in 150 μl PGM1 medium (Cellapy) supplemented with CEPT Cocktail (GLPBIO) and 0.0125% PF-127 (Sigma) were added to each well of a 96-well non-treated round-bottom microplate (Corning) to form embryoid bodies. Twenty-four hours following cell aggregation (day 0), 3D hESC spheroids were treated with KSR medium supplemented with 10 μM SB-431542 (MedChemExpress), 2.5 μM dorsomorphin (Selleck) and 1.25 μM XAV-939 (MedChemExpress). For imaging, cortical organoids were collected at day 16, treated with 300 nM PKMDR and 5 μg/mL Hoechst 33342 in pre-warmed culture medium for 30 min.

Mitochondrial segmentation and tracking
The image analysis platform was set up using a workstation with the following specifications was employed: a 3.70 GHz Intel i9-10900X CPU, an RTX TITAN NVIDIA GPU with 24 GB of memory, and 256 GB of RAM. The software platform used was based on Python 3.8. Additionally, certain algorithms relied on ImageJ67 and TrackMate68 for their implementation.

For the images acquired from the imaging system, we initially apply the median filtering and Rolling ball algorithms69 to denoise. Subsequently, the denoised images are binarized using the Otsu algorithm70 for mitochondrial segmentation. The morphological opening operations were introduced to eliminate small objects. Following this, label images were generated for each mitochondrial region. During mitochondrial tracking, the aforementioned mitochondrial image is input into TrackMate for Simple LAP Tracker analysis.

To measure the relationship between the fluorescence lifetime of each mitochondrion and its distance from the nucleus. We first manually labeled the position of the nucleus centroid for each cell. The average distance from the nucleus and the average fluorescence lifetime of all pixels within each mitochondrion were then calculated. Each mitochondrion constitutes a point on the scatter plot. Finally, the Spearman correlation coefficient was calculated (Fig. 5c, d).

Statistical analysis
Statistical analysis and drawing of graphs were performed using Prism version 10.0 (GraphPad Software). Normality tests were used where appropriate to select statistical tests. Each figure legend indicates the statistical tests used as well as the threshold below which p-values are plotted. Data are displayed as scatter plots, with single points either representing biological replicates (study participants) or single cells, overlaid on bars + standard error of mean. All t-tests were performed as two-tailed tests.

Ethics approval and consent to participate
All procedures were approved by the Peking University Animal Use and Care Committee and complied with the standards of the Association for Assessment and Accreditation of Laboratory Animal Care (code number IMM-ChenZX-1).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This article presents a new approach to monitor mitochondrial membrane potential (MMP) using the PKMDR probe in combination with fluorescence lifetime imaging microscopy (FLIM). The study demonstrates that PKMDR’s fluorescence lifetime serves as a sensitive readout for MMP, revealing mitochondrial metabolic heterogeneity in various cell types, organoids, and tissues. The authors provide a thorough literature review on existing mitochondrial imaging tools and their limitations, setting the stage for their new method. They explain the photophysical properties of PKMDR and how its fluorescence lifetime changes with concentration. The experimental validation, involving both in vitro and in vivo systems, is sufficient, demonstrating PKMDR-FLIM’s utility under various metabolic conditions and in different cell types. The findings regarding metabolic heterogeneity within and between cells, as well as the temporal dynamics of mitochondrial membrane potential, are partially novel and have good implications for mitochondrial biology and related diseases.\nHowever, some comments and suggestions for improvement could strengthen the manuscript:\n1. The fluorescence lifetime of a dye is generally considered to be independent of its concentration, which represents a significant advantage over fluorescence intensity measurements, as it is not affected by variations in dye concentration within cells. However, the authors present a unique probe, PKMDR, whose fluorescence lifetime specifically depends on concentration to characterize mitochondrial membrane potential. This raises an important question: How can the authors ensure that the parameter being characterized (such as ion concentration) is independent of the amount of dye taken up by the cells?\n2. While the authors propose concentration-induced quenching as the mechanism for the decrease in PKMDR’s fluorescence lifetime at high concentrations, more experimental evidence supporting this mechanism would be valuable. For example, control experiments with structurally similar but non-quenching dyes could help confirm that the observed effect is indeed due to quenching and not other factors.\n3. What is the typical imaging speed for FLIM using PKMDR in cells? Generally, TCSPC, integrated in the system TCS SP8 FALCON or STELLARIS 8 FALCON, often requires relatively long “photon counting” durations. For long-term time-lapse FLIM imaging as seen in Movie S1-3, are the lifetime values in each frame accurate, given that sufficient photon counts are essential for high goodness of fit? What are the the R-squared values or Chi-squared values used for estimation of goodness of fit throughout thr article?\n4. Some of the data presented are based on a relatively small number of biological replicates (e.g., n=6 cells in Fig. 1,2,3. Mostly less than 10). Performing statistical tests to confirm the significance of the observed differences and including error bars or confidence intervals in the figures would strengthen the conclusions.\n5. In Figure 4b, it is noticed that the TMRM-intensity and PKMDR-FLIM images do not depict 4-cell or 8-cell stages, whereas the 3D image does. Is the 3D view a representation of the fluorescence lifetime map? Additionally, the observation of fewer cells in the 2D view raises the question of whether this is due to limited depth of field, and if so, does this represent a limitation of the technique? The apparent discrepancy in the number of cells observed between the 2D and 3D views may be attributed to the limitations of depth of field in the 2D images. 2D images may only capture cells that are within a narrow focal plane, leading to an underestimate of the total cell count.\n6. How was the phasor plot in Figure 5 obtained? The authors did not mention the parameter settings in the Materials and Methods section. Given that the phasor plot is a powerful tool for distinguishing between different fluorescence lifetime components, it is surprising that the authors did not utilize it to differentiate and visualize between cellular components. For the sake of clarity and reproducibility, it would be beneficial for the authors to provide a detailed description of the methodology used to generate the phasor plot, including all relevant parameter settings, and to explain why it was not employed for cellular component differentiation in this study.\n7. I have noticed that in Supplementary Figures, there is a lack of spaces between some numbers and their units (e.g., “5μM” instead of “5 μM”, 2.25±0.13). This inconsistency in formatting may lead to confusion or misinterpretation of the data, as proper spacing is crucial for clarity and adherence to scientific conventions. It is recommended that the authors review and revise the formatting of all numerical values and units in their supplementary materials to ensure accuracy and consistency. Such attention to detail would enhance the overall quality and readability of the research.\n8. The authors should discuss the potential limitations of their method more thoroughly. For instance, what are the limitations in terms of dye loading efficiency, photostability over extended imaging sessions, and potential artifacts in complex tissue samples? Addressing these points will improve the paper’s balance and credibility.\n9. As a crucial component of the research, it seems appropriate to suggest that the “Materials and Methods” section, currently located in the supplementary files, should be included within the main text of the article.\nIn summary, this is an interesting and well-executed study that presents a promising new tool for mitochondrial imaging. I think the manuscript has the potential to make a high impact in the field. The authors should consider the provided comments and suggestions to further strengthen their work.", "reviewer_2": "In this study, the authors introduced the fluorescent dye PK MitoDeepRed (PKMDR) as a tool for measuring mitochondrial membrane potential (MMP) using fluorescence lifetime imaging microscopy (FLIM). The manuscript is generally well-written, easy to follow, and presents the data clearly. However, the paper lacks significant scientific advancements and does not offer new insights into mitochondrial biology. A key concern is that while PKMDR provides an alternative method for measuring MMP, it does not demonstrate a significant advantages over existing intensity-based fluorescent probes such as TMRM, JC-1, or MitoView 633. For example, TMRM has been employed by several groups, including the O'Rourke lab (10.1074/jbc.M302673200), to monitor the dynamics of mitochondrial oscillations, demonstrating high temporal resolution. It has also shown remarkable spatial resolution, allowing the recording of mitochondrial dynamics at the single-mitochondrion level (mitochondrial flash), as demonstrated by the Wang lab (10.1016/j.cell.2008.06.017). Additionally, intensity-based fluorescent dyes like TMRM and MitoView 633 have been used to assess MMP heterogeneity in isolated cells, such as cardiomyocytes. One of the major challenges in mitochondrial biology remains the measurement of MMP in vivo. If the authors can provide evidence that PKMDR is capable of this, it would represent a significant advancement in the field. Some specific comments are listed below.\n1. One of the advantages of FLIM is that it minimizes the effects of photon scattering in thick sample layers. However, is there a limit to the sample thickness that can be accurately measured using this technique?\n2. The quantitative correlation between dye concentration and   in live cells should be examined.\n3. Figure 2, an inner membrane marker should be used in addition to the outer membrane marker TOM20.\n4. The impact of temperature and pH on the behavior and dynamics of the dye should be examined, including the extent of these effects.", "reviewer_3": "This work introduces PKMDR, a mitochondrial dye previously developed from the corresponding author’s lab, to measure mitochondrial membrane potential (MMP) based on its concentration-dependent change of fluorescence lifetime as a result of quenching. The authors demonstrated the concentration-induced change of fluorescence lifetime of the dye in solutions and in giant unilamellar vesicles (GUV) and in response to metabolic perturbations in tissue culture cells. They further applied the dye to a variety of biological samples and argued that the dye can detect MMP changes across conditions, cell types, single cells and subcellular regions. While this work demonstrated the potential of the dye as a fluorescence-lifetime based sensor for MMP that is in principle superior than intensity based sensors, such as the widely used TMRM dye, due to fluorescence lifetime being a more robust metric against light scattering and variation of sample thickness, some of the data presented in the paper is in contradiction with the literature, calling in questions about the robustness of the probe.\nMajor comments:\n1. The measurement of the spatial patterns of MMP in MII mouse oocytes is in contradiction with the literature (Ref [56]), which reports a lower MMP towards the cell periphery and a higher MMP towards the center measured using TMRM. This contradiction highlights potential confounding factors in the measurement of PKMDR. This paper (PMID: 31579926) has also showed that the enhanced cortical MMP is a measurement artefact. Is the dye pH sensitive? Figure 1c shows a non-monotonic behavior of the fluorescence lifetime, how to ensure the dye is operating in the quenching regime in vivo?\n2. In general, the measurements of PKMDR should be compared with TMRM across all conditions tested in this paper to ensure robustness of the results. The authors did it for some samples but for most samples/conditions the authors compared their measurements with indirect results in the literature. For example, the authors expected MMP to increase under starvation and cited ref 33-35, which do not include any direct measurement of MMP.\n3. A conceptual difficulty for this reviewer is that the authors related MMP directly with OXPHOS activity, which is not always true. For example, oligomycin increases MMP but decreases OXPHOS activity. Proton leak decreases MMP but increases respiration from FCCP treatment. The relation between OXPHOS and MMP is contextual and should not be generalized.\n4. The methods used to do image analysis is not clearly stated. How are FLIM decay curves fitted: how many exponentials are used for each condition? How is mean fluorescence lifetime weighted? Is there any image segmentation before analysis? Without this information it would be difficult to reproduce the results in the paper.\n5. For a new probe, it would be useful to highlight potential limitations and usage of caution to ensure responsible and reproducible usage of the dye.\nOther comments:\n1. Since pH, ionic strength and other factors could vary from cell type to cell type, can the dye be used to directly compare MMP across different cell types, for example between oocyte and granulosa cells as stated in the paper?\n2. Can the dye be calibrated to measure absolute value of MMP. The authors mentioned in line 290 that MMP increases linearly, which needs to be supported with calibration. This paper (PMID: 22495585) provides a calibration protocol for TMRM.\n3. The metabolic heterogeneity in brain organoids and tumor slice as reported in Figure 4 should be quantified.\n4. Methods section need references to the corresponding figures.\n5. Since the dye binds to mitochondrial inner membrane, is the binding reversible? Does the dye measure dynamic changes of MMP, for example, does the dye diffuse out if MMP depolarizes?\n6. It would be useful to show with GUV the full non-monotonic response of fluorescence lifetime to identify the quenching regime.\n7. Figure S6: typo, it does not show fluorescence lifetime.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewers:\nImaging mitochondrial membrane potential via concentration-\ndependent fluorescence lifetime changes\n(Reference number: NCOMMS-24-81168-T)\nDilizhatai·Saimi, Luc Reymond, Tursunjan Aziz, Xuan Shen, Ziying Luo, Shuaibo Pi, Yitong Liu, Song Fu,\nShuangjin Ding, Anming Meng, Liangyi Chen, Hui Jiang, Zhixing Chen*\n* E-mail: zhixingchen@pku.edu.cn\nWe thank the reviewers for their thoughtful and valuable comments on our original manuscript. Their\nfeedback was extremely helpful in revising our manuscript. We have carefully considered all these comments\nand changed the manuscript accordingly. Please find a revised manuscript. Comments have been summarized\nbelow, along with our point-by-point response.\nReviewer #1:\nComments:\nThis article presents a new approach to monitor mitochondrial membrane potential (MMP) using the\nPKMDR probe in combination with fluorescence lifetime imaging microscopy (FLIM). The study\ndemonstrates that PKMDR’s fluorescence lifetime serves as a sensitive readout for MMP, revealing\nmitochondrial metabolic heterogeneity in various cell types, organoids, and tissues. The authors provide a\nthorough literature review on existing mitochondrial imaging tools and their limitations, setting the stage for\ntheir new method. They explain the photophysical properties of PKMDR and how its fluorescence lifetime\nchanges with concentration. The experimental validation, involving both in vitro and in vivo systems, is\nsufficient, demonstrating PKMDR-FLIM’s utility under various metabolic conditions and in different cell\ntypes. The findings regarding metabolic heterogeneity within and between cells, as well as the temporal\ndynamics of mitochondrial membrane potential, are partially novel and have good implications for\nmitochondrial biology and related diseases.\nHowever, some comments and suggestions for improvement could strengthen the manuscript:\nThe fluorescence lifetime of a dye is generally considered to be independent of its concentration, which\nrepresents a significant advantage over fluorescence intensity measurements, as it is not affected by\nvariations in dye concentration within cells. However, the authors present a unique probe, PKMDR, whose\nfluorescence lifetime specifically depends on concentration to characterize mitochondrial membrane\npotential.\nResponse:\nWe are grateful to the reviewer for raising this important point. Actually, PKMDR is not special – we show\nthat the dependence of fluorescence lifetime and concentration is general among cationic dyes (Figure 1).\nMeanwhile, regarding the relationship between fluorescence lifetime and dye concentration, it has been\nreported in prior studies (Berezin & Achilefu, 2010) that fluorescence lifetime remains concentration-\nindependent only within specific concentration ranges.\nComments:\nThis raises an important question: How can the authors ensure that the parameter being characterized (such\nas ion concentration) is independent of the amount of dye taken up by the cells?\nResponse:\nPKMDR is a lipophilic cation, whose cellular uptake is driven by electrochemical potential. Eventually, the\nfluorescence lifetime is dependent of concentration, which is dependent on the dye uptake. Such uptake is\nultimately dependent on the mitochondrial membrane potential.\nComments:\n2. While the authors propose concentration-induced quenching as the mechanism for the decrease in\nPKMDR’s fluorescence lifetime at high concentrations, more experimental evidence supporting this\nmechanism would be valuable. For example, control experiments with structurally similar but non-quenching\ndyes could help confirm that the observed effect is indeed due to quenching and not other factors.\nResponse:\nWe thank the reviewer for raising this question. Our experimental data (Figure 1c-g) demonstrate that the\nconcentration-dependent decrease in fluorescence lifetime is a general property observed across various\nfluorophores, including TMRM, TMRE and Mitorotor-1, rather than a distinctive feature unique to PKMDR.\nComments:\n3. What is the typical imaging speed for FLIM using PKMDR in cells? Generally, TCSPC, integrated in the\nsystem TCS SP8 FALCON or STELLARIS 8 FALCON, often requires relatively long “photon counting”\ndurations. For long-term time-lapse FLIM imaging as seen in Movie S1-3, are the lifetime values in each\nframe accurate, given that sufficient photon counts are essential for high goodness of fit? What are the the\nR-squared values or Chi-squared values used for estimation of goodness of fit throughout the article?\nResponse:\nWe would like to thank the reviewer for raising this question. The typical imaging speed of FLIM using\nPKMDR in cells depends on the parameters set at the time of imaging. When the format is 512 × 512, the\nscan speed is 400 Hz, and the frame repetition is 10, the imaging speed is 1.498 s. For time-lapse FLIM in\nMovie S1-3, there is sufficient photon number in each frame for accurate fluorescence lifetime fitting. The\nχ2 values of all the images in the whole article are less than 1.3.\nWe have made the following changes to the main text and supplementary information:\nThe pinhole was set to 1.0 AU. Data were collected using LAS X (Leica) at 512 × 512 or 1024 × 1024 pixel\nresolution and frame accumulations of 1-10 times, or collecting 500 photons per pixel. (Supplementary Table\nS2). FLIM data were analyzed using the LAS X software (Leica Microsystems) by fitting a mono-, bi-, or\ntriexponential decay model (n-exponential reconvolution) to the decay (χ2<1.3) (Supplementary Table S2).\nIntensity-weighted lifetime was used as mean fluorescence lifetime in each image.\nTable S2. Fluorescence microscopy data acquisition parameters.\n[... table omitted for brevity ...]\nComments:\n4. Some of the data presented are based on a relatively small number of biological replicates (e.g., n=6 cells\nin Fig. 1,2,3. Mostly less than 10). Performing statistical tests to confirm the significance of the observed\ndifferences and including error bars or confidence intervals in the figures would strengthen the conclusions.\nResponse:\nWe have added more data and showed the error bars in the figure. We thank the reviewer for this suggestion.\nWe have made the following changes:\nFigure 2. PKMDR fluorescence lifetime is correlated to mitochondria inner-membrane potential in\nlive cell imaging.\nd, Corresponding bar plots showing the average fluorescence lifetime change of mitochondria treated with\nFCCP (n = 14 cells), antimycin A (n = 14 cells), rotenone (n = 13 cells) or oligomycin (n = 13 cells). Data\nwere presented as the mean ± SEM. P-values were calculated using unpaired Student’s t-test. ****p < 0.0001.\nFigure 3. PKMDR-FLIM signal indicates the activity of mitochondrial oxidative phosphorylation.\na, PKMDR-FLIM images of HeLa cells after FBS starvation and nutrient starvation (cultured in DMEM\nlack of glucose, sodium-pyruvate and glutamine), and HUVECs at senescence conditions. Scale bar, 20 μm.\nb, Plots showing the average mitochondrial fluorescence lifetime under FBS starvation (n = 11 cells),\nnutrient starvation (n = 12 cells), and senescence HUVECs (n = 11 cells).\nc, Schematic illustration of mitochondrial electron transport chain proteins.\nd, PKMDR-FLIM images of wild type, COX4I1 and NDUFS2 knockout 143B cells.\ne, Plots showing the average mitochondrial fluorescence lifetime in wild type (n = 15 cells) and knockout\ncells (n = 15 cells).\nf, Bright field and FLIM images of PKMDR-stained mitochondria in germinal vesicle (GV) stage oocyte\nand granulosa cells.\ng, Plots showing the average mitochondrial fluorescence lifetime in oocyte and granulosa cells (n = 8 cells).\nh, Bright field, intensity and FLIM images of PKMDR-stained mitochondria in GV and metaphase Ⅱ (MⅡ)\noocyte.\ni, Plots showing the average mitochondrial fluorescence lifetime in GV and MⅡ oocyte (n = 19 cells).\nj, FLIM images of PKMDR-stained mitochondria in naïve and activated T cells.\nk, Plots showing the average mitochondrial fluorescence lifetime in naïve and activated T cells (n = 18 cells).\nStatistical analysis was performed with unpaired two-tailed Student’s t-test for b, e, i, k, paired two-tailed\nStudent’s t-test for g. Data were presented as the mean ± SEM. **** indicates p < 0.0001. Scale bars = 20\nμm.\nComments:\n5. In Figure 4b, it is noticed that the TMRM-intensity and PKMDR-FLIM images do not depict 4-cell or 8-\ncell stages, whereas the 3D image does. Is the 3D view a representation of the fluorescence lifetime map?\nAdditionally, the observation of fewer cells in the 2D view raises the question of whether this is due to limited\ndepth of field, and if so, does this represent a limitation of the technique? The apparent discrepancy in the\nnumber of cells observed between the 2D and 3D views may be attributed to the limitations of depth of field\nin the 2D images. 2D images may only capture cells that are within a narrow focal plane, leading to an\nunderestimate of the total cell count.\nResponse:\nWe thank the reviewer for pointing this out. The 3D images represent a reconstructed fluorescence lifetime\nmap generated from Z-stack acquisitions of 2D FLIM images. FLIM is based on confocal microscopy, which\ncaptures only a single focal plane (typically 0.5-1 μm thick). The reduced cell count observed in 2D views\nstems from this limitation—confocal microscopy primarily visualizes cells within a narrow focal plane,\npotentially missing cells above or below that plane. By performing Z-stack confocal imaging, we overcome\nthis limitation, capturing fluorescence lifetime information across multiple axial planes to generate a\ncomprehensive 3D representation of mitochondrial membrane potential dynamics. Simultaneous brightfield\nimaging during confocal acquisition was integrated to provide complementary morphological context,\nensuring accurate cell counting and minimizing potential underestimation of cell numbers due to focal plane\nconstraints.\nComments:\n6. How was the phasor plot in Figure 5 obtained? The authors did not mention the parameter settings in the\nMaterials and Methods section. Given that the phasor plot is a powerful tool for distinguishing between\ndifferent fluorescence lifetime components, it is surprising that the authors did not utilize it to differentiate\nand visualize between cellular components. For the sake of clarity and reproducibility, it would be beneficial\nfor the authors to provide a detailed description of the methodology used to generate the phasor plot,\nincluding all relevant parameter settings, and to explain why it was not employed for cellular component\ndifferentiation in this study.\nResponse:\nWe are grateful to the reviewer for raising this important point. We added how the phasor plot obtained in\nthe Methods and Material.\nIn our study, we employed phasor analysis to demonstrate that mitochondria within individual cells exhibit\nheterogeneous clustering patterns. These distinct clusters likely reflect variations in mitochondrial membrane\npotentials and/or metabolic states. Importantly, our observations suggest a potential spatial correlation\nbetween these functionally distinct mitochondrial subpopulations and their physical distribution within the\ncellular architecture. We have also made the following changes to the main text:\nPhasor plot analysis in Figure 5 is performed in LAS X software (Leica Microsystems). After selecting a\nregion of interest around the cell for analysis, the corresponding phasor plot is displayed, and pixels with\nsimilar lifetimes are grouped into clusters. Distinct clusters emerge when MMPs in an individual cell are\ndifferent. Pixels forming each cluster can be identified in the lifetime image and thus separated. No\nthreshholding is applied.\nComments:\n7. I have noticed that in Supplementary Figures, there is a lack of spaces between some numbers and their\nunits (e.g., “5μM” instead of “5 μM”, 2.25±0.13). This inconsistency in formatting may lead to confusion or\nmisinterpretation of the data, as proper spacing is crucial for clarity and adherence to scientific conventions.\nIt is recommended that the authors review and revise the formatting of all numerical values and units in their\nsupplementary materials to ensure accuracy and consistency. Such attention to detail would enhance the\noverall quality and readability of the research.\nResponse:\nWe thank the reviewer for pointing this out. We have corrected the above error.\nComments:\n8. The authors should discuss the potential limitations of their method more thoroughly. For instance, what\nare the limitations in terms of dye loading efficiency, photostability over extended imaging sessions, and\npotential artifacts in complex tissue samples? Addressing these points will improve the paper’s balance and\ncredibility.\nResponse:\nWe are grateful to the reviewer for raising this important point. We have added the following paragraph to\nthe discussion part:\nPKMDR-FLIM has certain limitations. First, the prolonged data acquisition time required for time-domain\nFLIM imaging, which necessitates sufficient photon accumulation for accurate lifetime calculations,\ninherently restricts temporal resolution. This is a particular issue at low ΔΨm where PKMDR’s fluorescence\nintensity become lower. Another limitation is that PKMDR’s concentration-dependent fluorescence lifetime\nbehavior imposes stringent experimental constraints. The probe’s lifetime-concentration correlation window\ndemand a critical threshold concentration, which can be achieved only under an optimized staining protocol\noutlined in our study. For non-standardized biological models, preliminary dose-response experiments are\nessential. While these limitations are inherent to PKMDR’s design, our methodology incorporates rigorous\ncontrols and validation steps to mitigate their impact on data interpretation.\nComments:\n9. As a crucial component of the research, it seems appropriate to suggest that the “Materials and Methods”\nsection, currently located in the supplementary files, should be included within the main text of the article.\nResponse:\nWe thank the reviewer for this suggestion. We have added the materials and methods section to the main text.\nComments:\nIn summary, this is an interesting and well-executed study that presents a promising new tool for\nmitochondrial imaging. I think the manuscript has the potential to make a high impact in the field. The authors\nshould consider the provided comments and suggestions to further strengthen their work.\nResponse:\nThank you for the positive assessment of this manuscript.\nReviewer #2:\nIn this study, the authors introduced the fluorescent dye PK MitoDeepRed (PKMDR) as a tool for measuring\nmitochondrial membrane potential (MMP) using fluorescence lifetime imaging microscopy (FLIM). The\nmanuscript is generally well-written, easy to follow, and presents the data clearly. However, the paper lacks\nsignificant scientific advancements and does not offer new insights into mitochondrial biology.\nResponse:\nWe are grateful to the reviewer for raising this important point. In this study, one of our key scientific advance\nlies in discovering that dye concentration critically influences fluorescence lifetime, which challenges the\nconventional interpretation that fluorescence lifetime changes reflect mitochondrial viscosity (such as in the\nMitorotor-1 probe paper (Singh et al., 2023). This finding provides a transformative perspective in the field\nof biophotonics. For mitochondrial biology, on the one hand we can map the difference of mitochondrial\nmetabolism in neurons by one-step imaging using PKMDR, which corroborates a biochemistry paper (Wei\net al., 2023), showcasing that simpler imaging protocol can visualize metabolic heterogeneity in neurons. On\nthe other hand, using PKMDR-FLIM technology, we revealed asymmetric MMP among the four blastomeres\nof mouse embryos at the 4-cell stage, a metabolic heterogeneity undetectable by conventional TMRM-\nintensity measurements. We aim to empower biologists with this tool to uncover novel mitochondrial biology\ninsights.\nComments:\nA key concern is that while PKMDR provides an alternative method for measuring MMP, it does not\ndemonstrate a significant advantage over existing intensity-based fluorescent probes such as TMRM,\nJC-1, or MitoView 633. For example, TMRM has been employed by several groups, including the O'Rourke lab\n(10.1074/jbc.M302673200), to monitor the dynamics of mitochondrial oscillations, demonstrating high\ntemporal resolution. It has also shown remarkable spatial resolution, allowing the recording of mitochondrial\ndynamics at the single-mitochondrion level (mitochondrial flash), as demonstrated by the Wang lab\n(10.1016/j.cell.2008.06.017). Additionally, intensity-based fluorescent dyes like TMRM and MitoView 633\nhave been used to assess MMP heterogeneity in isolated cells, such as cardiomyocytes.\nResponse:\nWe appreciate the reviewer’s insightful comments regarding the comparative advantages of PKMDR over\nexisting intensity-based probes like TMRM. PKMDR achieves comparable performance in monitoring\nmitochondrial membrane potential (MMP) dynamics with high spatiotemporal resolution, it uniquely\naddresses critical limitations of traditional intensity-based dyes and provides novel capabilities:\nPhototoxicity artifacts: TMRM's intense phototoxicity causes fluorescence decay during prolonged\nimaging, confounding interpretation of MMP changes (physiological decreased vs. dye-induced damage).\nPKMDR’s low phototoxicity eliminates this ambiguity.\nZ-axis dependence: TMRM intensity shows 16% coefficient of variation across Z-positions due to optical\nsectioning effects.\nPKMDR lifetime exhibits only 1.5% coefficient of variation (Figure R1), enabling reliable MMP\nquantification in 3D samples.\nThick specimen compatibility: Embryonic blastomeres at 4-/8-cell stages reside at different Z-positions.\nTMRM intensity measurements become unreliable due to Z-dependent signal attenuation, whereas PKMDR\nlifetime remains robust. Essentially this brings the measurement of MMP to another dimension.\nConcentration artifacts: TMRM intensity follows a non-monotonic relationship with concentration,\nrequiring strict pre-calibration to avoid misinterpretation(Perry, Norman, Barbieri, Brown, & Gelbard, 2011).\nPKMDR lifetime decreases monotonically with concentration, simplifying experimental design (Figure R2).\nFigure R1. (a), FLIM (upper) and confocal (lower) image of HeLa cells stained with PKMDR and TMRM.\nScale bar, 20 μM. (b), Plots showing the mitochondrial fluorescence intensity and lifetime in HeLa cells at\ndifferent Z position.\nFigure R2. Fluorescence lifetime of PKMDR in DMSO at various concentrations showing a fluorescence\nlifetime decrease.\nComments:\nOne of the major challenges in mitochondrial biology remains the measurement of MMP in vivo. If the\nauthors can provide evidence that PKMDR is capable of this, it would represent a significant advancement\nin the field. Some specific comments are listed below.\nResponse:\nIn principle PKMDR-FLIM should work in vivo. At this stage, however, we are not accessible to an upright\nFLIM microscope to try this experiment. We plan to try this as a future project.\nComments:\n1. One of the advantages of FLIM is that it minimizes the effects of photon scattering in thick sample layers.\nHowever, is there a limit to the sample thickness that can be accurately measured using this technique?\nResponse:\nThank you for raising this important question. Fluorescence Lifetime Imaging Microscopy (FLIM) is\ninherently based on confocal microscopy, where the accurate measurement depth is closely tied to both the\nworking distance (WD) of the objective lens and the scattering parameter of sample. Therefore, it is hard to\nanswer this question with mathematical rigor. The working distance of Leica microscope objective (HCX PL\nAPO 100x/1.40 OIL) that we used in this paper is 0.13 mm. That should also be the practical distance FLIM\ncan reach.\nComments:\n2. The quantitative correlation between dye concentration and τ in live cells should be examined.\nRespons e:\nWe appreciate the reviewer’s insightful comment regarding the relationship between dye concentration and\nτ in live cells. We acknowledge that quantitatively correlating the local dye concentration with τ in this\ncontext rem ains challenging due to the complex microenviron ment of the mitochondrial inner membrane.\nCurrent technical limitations in spatially resolving sub-mitochondrial dye concentrations at high precision\nfurther com plicate this analysis. We agree that this represents an important direction for future investigation\nto fully unravel the biophysical mechanisms in mitochondrial studies.\nComments:\n3. Figure 2, an inner membrane marker should be used in addition to the outer membrane marker TOM20.\nResponse:\nWe are grateful to the reviewer for raising this important point. We tried to co-transfect the inner\nmitochondrial membrane marker MCU-GFP and the outer membrane marker Tomm20-Halo tag to visualize\nPKMDR l ocalization. However, we observed significant cellular stress and altered mitochondrial\nmorphology under these conditions (fragmented and swollen mitochondria), which com promised the\nreliability of the co-localization analysis (Figure R3). While we were unable to achieve s uccessful dual-\nlabeling mitochondria, previous work has proved PKMDR’s mitochondrial localization (Z. Yang et al., 2020).\nFigure R3. SIM imaging of COS-7 cells expressing MCU-GFP and Tomm20-HaloTag stained with\nPKMDR. Scale bar, 5 μm.\nComments:\n4. The impact of temperature and pH on the behavior and dynamics of the dye should be examined, including\nthe extent of these effects.\nResponse:\nWe appreciate the reviewer's insightful suggestion. As shown in Figure R4a, the fluorescence lifetime of\nPKMDR was measured in phosphate buffer solutions across pH 5.8–7.8. The results demonstrated that the\nfluorescence lifetime remained stable and independent of environmental pH within this physiologically\nrelevant range. To evaluate temperature effects, PKMDR was incorporated into DOPC vesicles. The\nfluorescence lifetime exhibited a temperature-dependent change of 0.38 ns over the range of 25°C to 45°C\n(Figure R4b). Importantly, this variation is significantly smaller than the 1.24 ns lifetime shift induced by\nconcentration-dependent aggregation of PKMDR (Figure 1g). While temperature modulates the fluorescence\nlifetime to a measurable extent, its contribution is ~3.3-fold weaker compared to concentration-dependent\neffects. This indicates that PKMDR’s sensing performance is primarily governed by its concentration rather\nthan thermal fluctuations under physiological conditions.\nFigure R4. Fluorescence lifetime plot of PKMDR at various pH (a) and temperature (b). Data were\npresented as the mean ± SEM, n=3.\nReviewer #3 (Remarks to the Author):\nThis work introduces PKMDR, a mitochondrial dye previously developed from the corresponding author’s\nlab, to measure mitochondrial membrane potential (MMP) based on its concentration-dependent change of\nfluorescence lifetime as a result of quenching. The authors demonstrated the concentration-induced change\nof fluorescence lifetime of the dye in solutions and in giant unilamellar vesicles (GUV) and in response to\nmetabolic perturbations in tissue culture cells. They further applied the dye to a variety of biological samples\nand argued that the dye can detect MMP changes across conditions, cell types, single cells and subcellular\nregions. While this work demonstrated the potential of the dye as a fluorescence-lifetime based sensor for\nMMP that is in principle superior than intensity-based sensors, such as the widely used TMRM dye, due to\nfluorescence lifetime being a more robust metric against light scattering and variation of sample thickness,\nsome of the data presented in the paper is in contradiction with the literature, calling in questions about the\nrobustness of the probe.\nMajor comments:\n1. The measurement of the spatial patterns of MMP in MII mouse oocytes is in contradiction with the\nliterature (Ref [56]), which reports a lower MMP towards the cell periphery and a higher MMP towards the\ncenter measured using TMRM. This contradiction highlights potential confounding factors in the\nmeasurement of PKMDR. This paper (PMID: 31579926) has also showed that the enhanced cortical MMP\nis a measurement artefact.\nResponse:\nWe appreciate the reviewer’s insightful question regarding mitochondrial membrane potential in oocytes.\nOur TMRM-based fluorescence measurements align closely with the findings reported in Ref56, confirming\nthe reproducibility of MMP in oocytes (Figure R5). We acknowledge the critical concern raised in\nPMID:31579926 regarding potential artifacts in cortical MMP measurements using JC-1, where\nspatiotemporal redistribution of J-aggregates may confound interpretation. JC-1 measures MMP via\nratiometric spectral shifts (green monomer vs. red J-aggregate), a process inherently susceptible to\naggregation kinetics and spatial heterogeneity. In contrast, PKMDR’s fluorescence lifetime decreases\nmonotonically with increasing concentration (i.e., higher MMP), which independent of probe aggregation.\nThis mechanistic difference ensures that PKMDR’s lifetime signal is stably correlated with MMP.\nOn the other hand, NADH FLIM measurements (Venturas, Yang, Sakkas, & Needleman, 2023; X. Yang,\nHa, & Needleman, 2021) demonstrate that the ETC flux displayed a strong spatial gradient within oocytes,\nwith a higher flux closer to the cell periphery This finding directly corroborates the PKMDR-FLIM results\nshowing higher MMP (shorter lifetimes) at the oocyte periphery, suggesting a coordinated spatial gradient of\nmitochondrial respiration and membrane potential.\nWe acknowledge that further mechanistic studies are needed to dissect how MMP dynamics are\nphysiologically regulated in oocytes and to validate these findings using complementary approaches. And\nwe agree that current methodologies for measuring MMP in oocytes, including widely used dyes such as\nTMRM and JC-1 remain controversial. While our data do not resolve all methodological debates, they offer\nnew experimental evidence and novel insights for studying spatial heterogeneity of MMP.\nWe have made the following changes to the main text:\nNotably, the spatial distribution patterns of MMP in oocytes remain a subject of ongoing debate. Conflicting\nresults have been reported between TMRM measurements (showing MMP gradient decreasing from the\ncentral region to the periphery) and those obtained through JC-1 imaging and NADH FLIM (demonstrating\nan inverse peripheral-to-central gradient) 54-57. Our analysis aligns closely with the spatial patterns observed\nin JC-1 and NADH FLIM studies, which providing support and new insights for the study of spatial\nheterogeneity of MMP.\nFigure R5. FLIM and confocal image of oocyte co-stained with PKMDR and TMRM. Scale bar, 20 μM.\nComments:\nIs the dye pH sensitive?\nResponse:\nWe measured the fluorescence lifetime of PKMDR in phosphate buffer solutions across pH 5.8–7.8. As\nshown in Figure R4a, the fluorescence lifetime of PKMDR is not affected by pH.\nFigure R4. Fluorescence lifetime plot of PKMDR at various pH (a) and temperature (b). Data were\npresented as the mean ± SEM, n=3.\nComments:\nFigure 1c shows a non-monotonic behavior of the fluorescence lifetime, how to ensure the dye is operating\nin the quenching regime in vivo?\nResponse:\nRegarding the concern about the monotonic decrease in PKMDR fluorescence lifetime, we acknowledge that\nFigure 1c might initially appear misleading due to artifacts introduced by the spectrophotometric\nmeasurements. Specifically, radiative energy transport—a phenomenon where re-absorption of emitted\nfluorescence photons by neighboring dye molecules distorts lifetime measurements (Kelley & Kelley, 2022;\nLanghals & Schlücker, 2022)—can artificially inflate fluorescence lifetimes as the concentrations increased,\ncreating an upward trend. However, this artifact does not reflect the true relationship between concentration\nand fluorescence lifetime. As demonstrated in Figure R2, the correlation between PKMDR concentration and\nfluorescence lifetime is actually monotonically decreasing. The vesicle experiments in Figures 1g-j provide\ndirect evidence of this relationship. These results confirm that fluorescence lifetime remains constant below\nthe critical concentration, and only decreases monotonically once this critical concentration is exceeded.\nNotably, PKMDR exhibits a significantly lower critical concentration compared to conventional dyes like\nTMRM or TMRE, owing to its enhanced lipophilicity. This property ensures that at the recommended\nstaining concentrations (optimized in our study), PKMDR’s fluorescence lifetime reliably reflects membrane\npotential changes in live cells, with no artificial artifacts arising from radiative energy transport.\nFigure R2. Fluorescence lifetime of PKMDR in DMSO at various concentrations showing a fluorescence\nlifetime decrease.\nComments:\n2. In general, the measurements of PKMDR should be compared with TMRM across all conditions tested in\nthis paper to ensure robustness of the results. The authors did it for some samples but for most\nsamples/conditions the authors compared their measurements with indirect results in the literature. For\nexample, the authors expected MMP to increase under starvation and cited ref 33-35, which do not include\nany direct measurement of MMP.\nResponse:\nWe thank the reviewer for this suggestion. We have added the experimental results of TMRM to detect the\nchanges of MMP after adding FCCP, antimycin a, rotenone, oligomycin or starvation treatment or induction\nof senescence. We also used TMRM to detect the MMP difference mitochondria of 143 B cells between\nnormal and knockout NDUFS2 and COX4I1. We have made the following changes to the supplementary\ninformation:\nFigure S3. Measuring membrane potential using TMRM.\n(a), Confocal image of HeLa cells treated with FCCP (OXP HOS uncouplers), antimycin A (complex Ⅲ\ninhibitor), rotenone (complex I inhibitor) and oligomycin (A TP synthase inhibitor). Scale bar, 20 μM. (b),\nCorresponding bar plots showing the average fluorescence intensity change of mitochondria treated with\nFCCP (n = 9 cells), antimycin A (n = 11 cells), rotenone (n = 9 cells) or oligomycin (n = 14 cells). Data\nwere presented as the mean ± SEM. P-values were calculated using unpaired Student’s t-test. ****p < 0.0001.\nFigure S6. Mitochondrial membrane potential in starvation and senescence cell.\n(a) TMRM-Intensity images of HeLa cells after FBS starvation and nutrient starvation (cultured in DMEM\nlack of glucose, sodium-pyruvate and glutamine), and HUVECs at senescence conditions. Scale bar, 20 μm.\n(b) Plots showing the average mitochondrial fluorescence intensity under FBS starvation (n = 15 cells),\nnutrient starvation (n = 17 cells), and senescence HUVECs (n = 20 cells).\nFigure S7. Mitochondrial membrane potential in COX4I1 and NDUFS2 knockout 143B cells.\n(a) TMRM-intensity images of wild type, COX4I1 and NDUFS2 knockout 143B cells. Scale bar, 20 μm. (b)\nPlots showing the average mitochondrial fluorescence lifetime in wild type (n = 20 cells) and knockout cells\n(n = 22 cells). P-values were calculated using unpaired Student’s t-test. ****p < 0.0001.\nComments:\n3. A conceptual difficulty for this reviewer is that the authors related MMP directly with OXPHOS activity,\nwhich is not always true. For example, oligomycin increases MMP but decreases OXPHOS activity. Proton\nleak decreases MMP but increases respiration from FCCP treatment. The relation between OXPHOS and\nMMP is contextual and should not be generalized.\nResponse:\nThank you for raising this important conceptual concern. In Figure 2, we explicitly focused on measuring\ndirect changes in MMP induced by mitochondrial drugs (e.g., FCCP, antimycin A, rotenone, oligomycin).\nAll descriptions in this section strictly refer to \"increased/decreased MMP,\" as these compounds directly alter\nproton gradient dynamics (e.g., FCCP dissipates MMP, Oligomycin elevates MMP by inhibiting ATP\nsynthase).\nIn Figure 3, however, the models (e.g., serum/nutrient starvation, senescence, and knockdown of electron\ntransport chain proteins) were selected based on prior literature demonstrating their systemic impact on\noxidative phosphorylation (OXPHOS) activity. For example, knocking down the electron transport chain\nproteins is known to downregulate OXPHOS capacity. Here, we used the term \"OXPHOS activity\" to reflect\nthe broader functional consequences of these interventions.\nWe fully agree that MMP and OXPHOS activity are context-dependent and not universally correlated. For\nexample, as noted, oligomycin elevates MMP while suppressing OXPHOS, and proton leak uncouples MMP\nfrom respiration. Thus, we revised the expression in lines 258, 259, 289. Thank you again for highlighting\nthis critical nuance, which strengthens the interpretation of our findings.\nComments:\n4. The methods used to do image analysis is not clearly stated. How are FLIM decay curves fitted: how many\nexponentials are used for each condition? How is mean fluorescence lifetime weighted? Is there any image\nsegmentation before analysis? Without this information it would be difficult to reproduce the results in the\npaper.\nResponse:\nWe would like to thank the reviewer for raising this question. We analyzed the FLIM image using the LAS\nX software (Leica Microsystems) by fitting a bi-, or triexponential decay model (n-exponential reconvolution)\nto the decay (χ2<1.3) (Supplementary Table S2). No image segmentation processing was performed. Some\nFLIM images selected ROI for analysis. Intensity-weighted lifetime was used as mean fluorescence lifetime\nin each image.\nWe have made the following changes to the main text and supplementary information:\nThe pinhole was set to 1.0 AU. Data were collected using LAS X (Leica) at 512 × 512 or 1024 × 1024 pixel\nresolution and frame accumulations of 1-10 times, or collecting 500 photons per pixel. (Supplementary Table\nS2). FLIM data were analyzed using the LAS X software (Leica Microsystems) by fitting a mono-, bi-, or\ntriexponential decay model (n-exponential reconvolution) to the decay (χ2<1.3) (Supplementary Table S2).\nIntensity-weighted lifetime was used as mean fluorescence lifetime in each image.\nTable S2. Fluorescence microscopy data acquisition parameters.\n[... table omitted for brevity ...]\nComments:\n5. For a new probe, it would be useful to highlight potential limitations and usage of caution to ensure\nresponsible and reproducible usage of the dye.\nResponse:\nWe thank the reviewer for pointing this out. We have added the following paragraph to the discussion part:\nPKMDR-FLIM exhibits certain limitations. First, the prolonged data acquisition time required for time-\ndomain FLIM imaging, which necessitates sufficient photon accumulation for accurate lifetime calculations,\ninherently restricts temporal resolution. At low ΔΨm, PKMDR’s fluorescence intensity decreases\nsignificantly, necessitating repeated scans to achieve adequate signal-to-noise ratios—a trade-off that further\ncompromises temporal resolution. Another limitation is that PKMDR’s concentration-dependent\nfluorescence lifetime behavior imposes stringent experimental constraints. The probe’s lifetime-\nconcentration correlation becomes reliable only above a critical threshold concentration, requiring users to\nstrictly adhere to the optimized staining protocol outlined in our study. For non-standardized biological\nmodels, preliminary dose-response experiments are essential. While these limitations are inherent to\nPKMDR’s design, our methodology incorporates rigorous controls and validation steps to mitigate their\nimpact on data interpretation.\nIn the Material and Methods section, we explicitly emphasized the protocols and usage of caution for\nPKMDR:\nPKMDR labeling for live-cell FLIM imaging\nCells were seeded into a glass-bottom dish (Cellvis) a day before imaging with the cell density at around\n4×104 cells/mL. For labeling of mitochondria, cells were stained with DMEM supplemented with 250 nM\nPKMDR for 20 min at 37 °C. Following the staining procedure, the cells were washed three times with pre-\nwarmed PBS or culture medium to remove the unbound dye. If long-term time-lapse imaging is desired,\nlabeling density should be kept as low as possible for optimal imaging. If the signal is too weak, leading to\nnoisy data or low intramitochondrial contrast, we recommend extending the staining time up to 45 min.\nMoreover, we observed that a washing step of 15 to 30 min following the staining procedure could\nsignificantly increase the intramitochondrial contrast while reducing unspecific ER labeling. For multi-color\nlabeling, we recommend sequential staining if the used dyes require different incubation times.\nOther comments:\n1. Since pH, ionic strength and other factors could vary from cell type to cell type, can the dye be used to\ndirectly compare MMP across different cell types, for example between oocyte and granulosa cells as stated\nin the paper?\nResponse:\nWe are grateful to the reviewer for raising this important point. In our study, we tested PKMDR fluorescence\nlifetime across a range of in vitro pH gradients (pH 5.8–7.8) (Figure R4a), confirming that its lifetime\nresponse remained stable and correlated with MMP changes induced by FCCP, antimycin A or oligomycin\n(see Figure 2). Thus, PKMDR can compare MMP between cell types (e.g., oocytes vs. granulosa cells)\nprovided experimental conditions are strictly standardized.\nComments:\n2. Can the dye be calibrated to measure absolute value of MMP. The authors mentioned in line 290 that MMP\nincreases linearly, which needs to be supported with calibration. This paper (PMID: 22495585) provides a\ncalibration protocol for TMRM.\nResponse:\nWe would like to thank the reviewer for raising this question. Currently, due to technical limitations, we are\nunable to calibrate the dye to report absolute values of MMP at this stage. This aspect represents an important\ndirection for future research and requires further optimization and validation with established reference\nmethods, which are beyond the scope of the current study. Concerning the statement on line 290, we have\nmade the following changes to the main text:\nIn these spreading cells, mitochondrial membrane potential increased from the nuclear region to the cell\nperiphery.\nComments:\n3. The metabolic heterogeneity in brain organoids and tumor slice as reported in Figure 4 should be quantified.\nResponse:\nThe primary objective of this figure is to demonstrate that our PKMDR probe can effectively visualize MMP\nnot only in cultured cells but also in complex biological systems like brain organoids and tumor tissue slices.\nWhile the current data qualitatively reveal the inherent heterogeneity of MMP distribution within these\nmodels, we fully acknowledge the importance of quantitative characterization. Unfortunately, standardized\nmethodologies for spatial quantification of metabolic heterogeneity in organoids and thick tissue specimens\nremain underdeveloped in the field. Addressing this technical challenge will require systematic optimization\nof imaging analysis pipelines and heterogeneity metrics, which we plan to pursue in future studies\nspecifically focused on computational bioenergetics profiling.\nComments:\n4. Methods section need references to the corresponding figures.\nResponse:\nWe thank the reviewer for this suggestion. We have corrected the above error.\nComments:\n5. Since the dye binds to mitochondrial inner membrane, is the binding reversible? Does the dye measure\ndynamic changes of MMP, for example, does the dye diffuse out if MMP depolarizes?\nResponse:\nThank you for raising this important question regarding the reversibility and dynamic response of PKMDR.\nAs a lipophilic cation, PKMDR’s localization to the mitochondrial inner membrane is entirely driven by\nMMP, and its binding is fully reversible. This behavior aligns with the Nernstian distribution mechanism\ntypical of cationic probes, where accumulation and release are governed by changes in MMP. In Figure 6a,\nlong-term live-cell imaging reveals temporal changes in PKMDR fluorescence lifetime, reflecting dynamic\nfluctuations in MMP over time. These observations confirm that PKMDR responds reversibly to\nphysiological variations in MMP. In Figure 2c, PKMDR rapidly dissociates from the mitochondrial inner\nmembrane and diffuses into the cytosol after treated with FCCP. This confirms that PKMDR’s localization\nis directly tied to MMP, and PKMDR will diffuse out if MMP depolarizes.\nComments:\n6. It would be useful to show with GUV the full non-monotonic response of fluorescence lifetime to identify\nthe quenching regime.\nResponse:\nWe are grateful to the reviewer for raising this important point. Regarding the concern about the monotonic\ndecrease in PKMDR fluorescence lifetime, we acknowledge that Figure 1c might initially appear misleading\ndue to artifacts introduced by the spectrophotometric measurements. Specifically, radiative energy\ntransport—a phenomenon where re-absorption of emitted fluorescence photons by neighboring dye\nmolecules distorts lifetime measurements (Kelley & Kelley, 2022; Langhals & Schlücker, 2022)—can\nartificially inflate fluorescence lifetimes as the concentrations increased, creating an upward trend. However,\nthis artifact does not reflect the actual relationship between concentration and fluorescence lifetime.\nAs demonstrated in Figure R2, the actual correlation between PKMDR concentration and fluorescence\nlifetime is indeed monotonically decreasing. The vesicle experiments in Figures 1g-j provide direct evidence\nof this relationship. These results confirm that fluorescence lifetime remains constant below the critical\nconcentration, and only decreases monotonically once this critical concentration is exceeded. Importantly,\nPKMDR exhibits a significantly lower critical concentration compared to conventional dyes like TMRM or\nTMRE, owing to its enhanced lipophilicity. This property ensures that at the recommended\nstaining concentrations (optimized in our study), PKMDR’s fluorescence lifetime reliably reflects membrane\npotential changes in live cells, with no artificial artifacts arising from radiative energy transport.\nFigure R2. Fluorescence lifetime of PKMDR in DMSO at various concentrations showing a fluorescence\nlifetime decrease.\nComments:\n7. Figure S6: typo, it does not show fluorescence lifetime.\nResponse:\nWe thank the reviewer for pointing this out. We have corrected the above error.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\n[... references omitted for brevity ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the concerns raised and provided additional data, clarifications, and methodological details that significantly strengthen the manuscript. In particular, your responses regarding the concentration dependence of PKMDR’s fluorescence lifetime, the validation of quenching mechanisms, and the technical considerations for FLIM imaging (e.g., photon counts, χ² values, and imaging parameters) have satisfactorily resolved the initial concerns. The inclusion of error bars, additional replicates, and a more comprehensive Materials and Methods section further improves reproducibility and transparency.\nThe manuscript now presents a compelling and well-supported case for PKMDR-FLIM as a valuable tool for monitoring mitochondrial membrane potential and metabolic heterogeneity.", "reviewer_3": "The comparison between TMRM and PKMDR measurements across conditions is a significant improvement of the manuscript. The clarification of the methods and discussion of limitations also improve the manuscript. To reviewers #3 and #4, there are still outstanding questions:\n1. The authors mentioned that the non-monotonic dependence of the lifetime on PKMDR concentration in Figure 1c is an artefact and provided a new Figure R2. It is unclear what is the difference of the measurement conditions between these two figures? It is also unclear if this artefact can actually impact the measurement in cells. If Fig. 1C is not representative of the real lifetime-concentration profile of PKMDR due to spectrophotometric artifacts, showing the real profile as in Fig R2, might be more useful. It is also helpful to clarify the inconsistency with Fig. 1g-j and the text lines about the monotonicity of the profile.\n2. The authors addressed the question about the limitations of PKMDR, but few questions remain unanswered. What is the photostability of the dye over time (intensity and lifetime)? Since the PKMDR is washed out before imaging, does it unbind and diffuse out of mitochondria over time? ER labeling is mentioned as an unspecific effect of the dye, are there other artifacts? Given that the article studies the use of PKMDR as a membrane potential dye, it would be relevant to add in the main text the advantages/disadvantages (vs. intensity-based dyes like TMRM) and the possible limitations listed in the reply to the reviewers.\n3. Due to the fact that MMP and OXPHOS activity correlation is context-dependent, the comparison of PKMDR (to measure MMP) with NADH-FLIM measurements in Ref 56 (to estimate OXPHOS activity) might need a reconsideration (specially given that TMRM measurement of MMP in Ref56 also shows the inverse result as the MMP measurements in this article from PKMDR).\n4. Regarding the intercellular MMP heterogeneities in developing embryos, how are cell identities determined? In other words, how are cells numbered?\n5. The authors used different linear combinations of exponential functions for the fitting (mono-, bi-, tri- exponential functions), what is the criteria or reasoning behind the specific choices? Consistency in analysis is always more welcomed. Additionally, in the introduction it should be immediately clarify that “lifetime” refers as the mean lifetime, instead of mentioning it only in the methods. Specially, given that the authors use the intensity-weighted lifetime, which large timescales have higher weight on the final mean, it is unclear if they can be readily comparable (other means, like a weighted harmonic mean, could be mathematically more robust for comparing different n-exponential). If the choice of function is not based on a chemical model of the fluorophore, one must also be aware of overfitting.\n6. It would be also helpful for clarity to provide some basic equations, for example, the definition of “intensity-weighted lifetime”.\n7. Is the χ2 value provided the reduced χ2 or the normal χ2?\n8. “Imaging speed” is an unclear parameter, especially given that the unit is seconds.\n9. Figure R1 and Figure R4 should be added to supplementary figures.\n10. According to Figure S4, PKMDR does seem to respond to changes in membrane tension, how does this impact data interpretation?\n11. In Figure 5, shouldn't the distance be um instead of nm?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewers：\nImaging mitochondrial membrane potential via\nconcentration-dependent fluorescence lifetime changes\n(Reference number: NCOMMS-24-81168A)\nDilizhatai·Saimi, Luc Reymond, Tursunjan Aziz, Xuan Shen, Ziying Luo, Shuaibo Pi,\nYitong Liu, Song Fu, Shuangjin Ding, Anming Meng, Liangyi Chen, Hui Jiang, Zhixing\nChen*\n* E-mail: zhixingchen@pku.edu.cn\nThank you very much for your time, insightful comments, and constructive feedback.\nWe are deeply grateful for the editor’s and reviewers’ thoughtful evaluation of our\nmanuscript (NCOMMS-24-81168A). We have carefully considered all these comments\nand changed the manuscript accordingly. Please find a revised manuscript. Comments\nhave been summarized below, along with our point-by-point response.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nComment：\nThe authors have addressed the concerns raised and provided additional data,\nclarifications, and methodological details that significantly strengthen the manuscript.\nIn particular, your responses regarding the concentration dependence of PKMDR’s\nfluorescence lifetime, the validation of quenching mechanisms, and the technical\nconsiderations for FLIM imaging (e.g., photon counts, χ² values, and imaging\nparameters) have satisfactorily resolved the initial concerns. The inclusion of error bars,\nadditional replicates, and a more comprehensive Materials and Methods section further\nimproves reproducibility and transparency.\nThe manuscript now presents a compelling and well-supported case for PKMDR-FLIM\nas a valuable tool for monitoring mitochondrial membrane potential and metabolic\nheterogeneity.\nResponse:\nThank you for the positive assessment of this manuscript.\nReviewer #3 (Remarks to the Author):\nComments:\nThe comparison between TMRM and PKMDR measurements across conditions is a\nsignificant improvement of the manuscript. The clarification of the methods and\ndiscussion of limitations also improve the manuscript. To reviewers #3 and #4, there\nare still outstanding questions:\n1. The authors mentioned that the non-monotonic dependence of the lifetime on\nPKMDR concentration in Figure 1c is an artefact and provided a new Figure R2. It is\nunclear what is the difference of the measurement conditions between these two figures?\nIt is also unclear if this artefact can actually impact the measurement in cells. If Fig. 1C\nis not representative of the real lifetime-concentration profile of PKMDR due to\nspectrophotometric artifacts, showing the real profile as in Fig R2, might be more useful.\nIt is also helpful to clarify the inconsistency with Fig. 1g-j and the text lines about the\nmonotonicity of the profile.\nResponse:\nWe apologize for the confusion The artifact is clearly explained in Reference 29. This\nartifact only happened when the experiments were performed in the common 90°\ngeometry (Figure RR1a), where the excitation beam enters the cell along one axis and\nthe fluorescence are collected from roughly the middle of the cell, propagating along a\nperpendicular axis. When using this device to detect fluorescence lifetime, the\nabsorption and re-emission of the fluorescence (“radiative energy transport”) lengthens\nthe apparent lifetime at higher concentrations. Therefore, when measuring cells and\nGUVs (Figure 1g-j), due to the shorter and difference of light path, this artifact will not\nexist on the fluorescence lifetime imaging microscope (Figure RR1b). Figure R2 is\nintended to illustrate what the data in Figure 1c should look like in the absence of\nartifacts. In other words, Figure R2 was only illustrative and thus should not be added\nin the manuscript.\nFigure RR1. The light path of fluorescence spectrometer (a) and fluorescence lifetime\nimaging microscope (b).\nComment:\n2. The authors addressed the question about the limitations of PKMDR, but few\nquestions remain unanswered. What is the photostability of the dye over time (intensity\nand lifetime)? Since the PKMDR is washed out before imaging, does it unbind and\ndiffuse out of mitochondria over time? ER labeling is mentioned as an unspecific effect\nof the dye, are there other artifacts? Given that the article studies the use of PKMDR as\na membrane potential dye, it would be relevant to add in the main text the\nadvantages/disadvantages (vs. intensity-based dyes like TMRM) and the possible\nlimitations listed in the reply to the reviewers.\nResponse:\nWe thank the reviewer for raising this question. The photostability of PKMDR has been\ndiscussed in Reference 23. The localization of PKMDR to mitochondria is dependent\non the MMP. Unless the MMP is altered, PKMDR will not diffuse out of mitochondria\nafter washin g. Actually, when using PKMD R for staining, ER labeling hardly occurs,\nand other ar tifacts do not appear either. In the previous review comments, we were\nrequired to highlight potential limitations and usage precautions to ensure the\nresponsible and reproducible application of the dye. Therefore, referring to other\nmitochondrial probes(Liu et al., 2022), we wrote a b o u t th e possible issues and\ncorresponding solutions.\nWe have made the f o l lowing ch a nges to the m ain text a n d supplementary information:\nCompared to the intensity signal of TMRM, PKMDR’s low p hototoxicity eliminates\nfluorescence decay artifacts during prolonged imaging. More over, PKMDR lifetime\nexhibits minimal Z position variation (1.5% coefficient of variation vs. TMRM\nintensity's 16% c o e f f ic i ent of v ar ia ti o n ) , enabling r e li able 3D quantification in\nspecimens like embryonic blastomeres at different depths where TMRM becomes\nunreliable (Figure S11). Furthermore, PKMDR lifetime decreases monotonically with\nconcentration, simplifying experimental design and avoiding the misinterpretation risks\ninherent in TMRM's non-monotonic intensity-concentration relationship.\nFigure S11. Measurement of mitochondrial membrane potential using TMRM-\nintensity and PKMDR-FLIM. (a) FLIM (upper) and confocal (lower) image of HeLa\ncells stained with PKMDR and TMRM. Scale bar, 20 μM. (b) Plots showing the\nmitochondrial fluorescence intensity and lifetime in HeLa cells at different Z position.\nComments:\n3. Due to the fact that MMP and OXPHOS activity correlation is context-dependent,\nthe comparison of PKMDR (to measure MMP) with NADH-FLIM measurements in\nRef 56 (to estimate OXPHOS activity) might need a reconsideration (specially given\nthat TMRM measurement of MMP in Ref56 also shows the inverse result as the MMP\nmeasurements in this article from PKMDR).\nResponse:\nWe are grateful to the reviewer for raising this important point. Our PKMDR-FLIM\ndata revealing mitochondrial heterogeneity in oocytes suggest a potential gradient\ndistribution of mitochondrial membrane potential from the peripheral to central regions\nof oocytes. We agree that this could be controversial, and this phenomenon warrants\nmore in-depth investigation to fully elucidate its underlying mechanisms. We see this\ndata as a new clues and would like to present it to researchers in the oocyte biology\nfield to stimulate further studies.\nComments:\n4. Regarding the intercellular MMP heterogeneities in developing embryos, how are\ncell identities determined? In other words, how are cells numbered?\nResponse:\nWe thank the reviewer for pointing this out. During the 2-cell, 4-cell, and 8-cell stages\nof an embryo, the number of cells can be clearly observed under a microscope, allowing\nfor manual numbering.\nComments:\n5. The authors used different linear combinations of exponential functions for the fitting\n(mono-, bi-, tri- exponential functions), what is the criteria or reasoning behind the\nspecific choices? Consistency in analysis is always more welcomed. Additionally, in\nthe introduction it should be immediately clarify that “lifetime” refers as the mean\nlifetime, instead of mentioning it only in the methods. Specially, given that the authors\nuse the intensity-weighted lifetime, which large timescales have higher weight on the\nfinal mean, it is unclear if they can be readily comparable (other means, like a weighted\nharmonic mean, could be mathematically more robust for comparing different n-\nexponential). If the choice of function is not based on a chemical model of the\nfluorophore, one must also be aware of overfitting.\nResponse:\nWe appreciate the reviewer’s insightful question regarding fluorescence lifetime fitting.\nFor the FLIM analysis, we observed that different samples required distinct exponential\nfunctions to achieve optimal goodness-of-fit. Specifically, some FLIM data were fitted\nwith biexponential decay model to achieve χ² value below our acceptance criterion of\n<1.3, whereas others necessitated tri-exponential functions. This approach aligns with\nestablished practices in FLIM research, as demonstrated in previous studies(Frei, Koch,\nHiblot, & Johnsson, 2022; M. S. Frei et al., 2022; Singh et al., 2023). Furthermore, all\nlifetime values reported in our manuscript, both in images and tables, represent the\nintensity-weighted average fluorescence lifetimes. This is the conventional and widely\naccepted metric for reporting and comparing lifetimes in biological FLIM imaging. To\nensure experimental consistency across comparative analyses, we maintained a uniform\nfitting approach for all relevant figures, thereby preserving the reliability and\ncomparability of our results.\nComments:\n6. It would be also helpful for clarity to provide some basic equations, for example, the\ndefinition of “intensity-weighted lifetime”.\nResponse:\nWe appreciate the reviewer's insightful suggestion. We have made the following\nchanges to the main text:\nFLIM data were analyzed using the LAS X software (Leica Microsystems) by fitting a\nmono-, bi-, or triexponential decay model (n-exponential reconvolution, equation 1) to\nthe decay (χ2<1.3, equation 2) (Supplementary Table S2). Intensity-weighted lifetime\n(equation 3) was used as mean fluorescence lifetime in each image.\n[... equations omitted for brevity ...]\nComments:\n7. Is the χ2 value provided the reduced χ2 or the normal χ2?\nResponse:\nWe are grateful to the reviewer for pointing this out. The χ2 value provided in the\nmanuscript is reduced χ2.\nComments:\n8. “Imaging speed” is an unclear parameter, especially given that the unit is seconds.\nResponse:\nWe thank the reviewer for pointing this out. There was a writing error in the last\nresponse letter, which should be:\nWhen the format is 512 × 512, the scan speed is 400 Hz, and the frame repetition is 10,\nthe imaging speed is 1.498 s/frame.\nComments:\n9. Figure R1 and Figure R4 should be added to supplementary figures.\nResponse:\nWe are grateful to the reviewer for pointing this out. We have made the following\nchanges to the main text and supplementary information:\nMoreover, we also examined the impact of temperature and pH on the lifetime of\nPKMDR. The fluorescence lifetime of PKMDR was measured in phosphate buffer\nsolutions across pH 5.8–7.8. The results demonstrated that the fluorescence lifetime\nremained stable and independent of environmental pH within this physiologically\nrelevant range (Supplementary Fig. S3a). To evaluate temperature effects, PKMDR\nwas incorporated into DOPC vesicles. The fluorescence lifetime exhibited a\ntemperature-dependent change of 0.38 ns over the range of 25°C to 45°C\n(Supplementary Fig. S3b). Importantly, this variation is significantly smaller than the\n1.24 ns lifetime shift induced by concentration-dependent aggregation of PKMDR\n(Figure 1g). While temperature modulates the fluorescence lifetime to a measurable\nextent, its contribution is ~3.3-fold weaker compared to concentration-dependent\neffects. This indicates that PKMDR’s sensing performance is primarily governed by its\nconcentration rather than thermal fluctuations under physiological conditions.\nFigure S3. Fluorescence lifetime plot of PKMDR at various pH (a) and temperature\n(b). Data were presented as the mean ± SEM, n=3\nCom pared to the intensity signal of TMRM, PKMDR’s low phototoxic ity eliminates\nfluor escence decay artifacts during prolonged imaging. Moreover, PKMDR lifetime\nexhibits minimal Z position variation (1.5% coefficient of variation vs. TMRM\nintensity's 16% coefficient of variation), enabling reliable 3D quantification in\nspecimens like embryonic blastomeres at different depths where TMRM becomes\nunreliable (Figure S11). Furthermore, PKMDR lifetime decreases monotonically with\nconcentration, simplifying experimental design and avoiding the misinterpretation risks\ninherent in TMRM's non-monotonic intensity-concentration relationship.\nFigure S11. Measurement of mitochondrial membrane potential using TMRM-\nintensity and PKMDR-FLIM. (a) FLIM (upper) and confocal (lower) image of HeLa\ncells s t a i n ed wi t h P K MDR and TMRM. Scale bar, 20 μM. (b) Plots showing the\nmitochondrial fluorescence intensity and lifetime in HeLa cells at different Z position.\nComments:\n10. According to Figure S4, PKMDR does seem to respond to changes in membrane\ntension, how does this impact data interpretation?\nResponse：\nWe are grateful to the reviewer for raising this important point. In Figure S4, we added\nhypertonic and hypotonic solutions to decrease and increase membrane tension,\nrespectively. If PKMDR responded to membrane tension, it should have induced two\ndistinct changes in fluorescence lifetime. However, our experimental results showed\nthat fluorescence lifetime increased in both hypertonic and hypotonic solutions. We\nattribute this to the deterioration of cellular physiological state and reduction in\nmembrane potential caused by adding solutions with different osmotic pressures, rather\nthan a response to membrane tension.\nComments:\n11. In Figure 5, shouldn't the distance be um instead of nm?\nResponse:\nWe thank the reviewer for pointing this out. We have corrected the above error.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\n[... references omitted for brevity ...]"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have addressed most of my questions and the paper now represents a significant contribution to the bioenergetic field. To improve the presentation of the paper, I have a few remaining questions based on the authors' response to my last review:\n1. The most important point is that since the authors clarified that the increase of PKMDR lifetime with concentration in the solution is an artifact due to the set up of the fluorescence spectrometer, then why not measure the lifetime on a microscope with solution droplets, which is supposedly free of this artifact? Since Figure 1c is an important figure demonstrating the property of the dye, it will improve the paper if one includes a figure free of such artifacts. In addition, a seemingly non-monotonic curve will confuse the downstream interpretation of the data for first time readers.\n2. The authors wrote in the text that TMRM suffers from non-monotonic intensity-concentration relationship, which I think is too general a statement. TMRM can operate in either quenching and non-quenching mode, whose properties have been well-characterized in the literature. I would rephrase this comment in the text in reference to the literature.\n3. FLIM of NADH does not measure MMP, hence I would not use it to support the measurement of PKMDR. I would rephrase it in the text.\n4. The inclusion of equations are very helpful. For eqn (2), why is the chi-squared computed in the frequency domain, shouldn't it be calculated in the time domain?"}, "author_response": "Response to Reviewers：\nImaging mitochondrial membrane potential via\nconcentration-dependent fluorescence lifetime changes\n(Reference number: NCOMMS-24-81168B)\nDilizhatai·Saimi, Luc Reymond, Tursunjan Aziz, Xuan Shen, Ziying Luo, Shuaibo Pi,\nYitong Liu, Song Fu, Shuangjin Ding, Anming Meng, Liangyi Chen, Hui Jiang, Zhixing\nChen*\n* E-mail: zhixingchen@pku.edu.cn\nThank you very much for your time, insightful comments, and constructive feedback.\nWe are deeply grateful for the editor’s and reviewers’ thoughtful evaluation of our\nmanuscript (NCOMMS-24-81168B). We have carefully considered all these comments\nand changed the manuscript accordingly. Please find a revised manuscript. Comments\nhave been summarized below, along with our point-by-point response.\nRemarks to Authors from Referee #3 on the NCOMMS-24-81168B version\nComment：\nThe authors have addressed most of my questions and the paper now represents a\nsignificant contribution to the bioenergetic field. To improve the presentation of the\npaper, I have a few remaining questions based on the authors' response to my last review:\n1. The most important point is that since the authors clarified that the increase of\nPKMDR lifetime with concentration in the solution is an artifact due to the set up of\nthe fluorescence spectrometer, then why not measure the lifetime on a microscope with\nsolution droplets, which is supposedly free of this artifact? Since Figure 1c is an\nimportant figure demonstrating the property of the dye, it will improve the paper if one\nincludes a figure free of such artifacts. In addition, a seemingly non-monotonic curve\nwill confuse the downstream interpretation of the data for first time readers.\nResponse:\nWe appreciate the reviewer's suggestion.\nCuvette-based fluorometer measurements are regarded as more optically rigorous than\nmicroscopy-based measurements due to the higher signal, higher stability, and precise\nconcentrations. That was why we chose it in the very beginning.\nFollowing your suggestions, we tried to measure the lifetime on a microscope with\nsolution droplets. However, we encountered challenges in obtaining reliable lifetime\ndata under these conditions. The primary difficulty arose from the lack of a well-defined\nfocal plane within the droplet for precise lifetime measurements. The dominant signal\noriginated from the reflection at the glass-liquid interface, which precluded accurate\nlifetime determination at the intended sample plane.\nThis limitation underscores why fluorescence spectrometer is the gold standard for\ndetecting various parameters (such as fluorescence lifetime, quantum yield, etc.) of\nfluorescent molecular solutions. Admittedly, artifacts in lifetime measurements arising\nfrom radiative energy transport (RET) in fluorescence spectrometer have been\npreviously reported(Kelley & Kelley, 2022). That said, we still think this spectrometer\ndata are the cornerstone of this work.\nWe have made the following changes to the main text:\nTo test this hypothesis, we measured the fluorescence intensity and lifetime of PKMDR\nat various concentrations of DMSO using a fluorescence spectrophotometer, whose\ncuvette-based setup offers accurate measurements. The relationship between\nfluorescence intensity and dye concentration deviated from linearity starting at 20 μM,\nindicating self-quenching behavior (Supplementary Fig. S1a)28. Accordingly, the\nfluorescence lifetime of PKMDR sharply declined above 20 μM (Figure 1b),\ncollectively supporting the emergence of concentration-induced quenching. Notably,\nwhile PKMDR was mainly at a dispersed state in solution below 20 μM, there is a\nknown artifact of radiative energy transport (RET). Within the long light path of the\ncuvette, the re-absorption of the emitted fluorescence photon at another dye molecule\nwould give rise to an apparent increase of lifetime, accounting for the fluorescence\nlifetime increase between 1 μM to 20 μM29,30.\nIn Figure 1e-h, the data were not fully presented in the original manuscript, so we have\nmade the following changes to the main text:\nComment：\n2. The authors wrote in the text that TMRM suffers from non-monotonic intensity-\nconcentration relationship, which I think is too general a statement. TMRM can operate\nin either quenching and non-quenching mode, whose properties have been well-\ncharacterized in the literature. I would rephrase this comment in the text in reference to\nthe literature.\nResponse:\nWe thank the reviewer for raising this question. We agree that the two modes of TMRM\nare not really its drawback. We therefore deleted the related sentence in the main text:\nFurthermore, PKMDR lifetime decreases monotonically with concentration,\nsimplifying experimental design and avoiding the misinterpretation risks\ninherent in TMRM's non-monotonic intensity-concentration relationship.\nComment：\n3. FLIM of NADH does not measure MMP, hence I would not use it to support the\nmeasurement of PKMDR. I would rephrase it in the text.\nResponse:\nWe thank the reviewer for pointing this out. We have deleted the related words in the\nmain text:\nConflicting results have been reported between TMRM measurements (showing MMP\ngradient decreasing from the central region to the periphery) and those obtained through\nJC-1 imaging and NADH FLIM (demonstrating an inverse peripheral-to-central\ngradient) 54-57. Our analysis aligns closely with the spatial patterns observed in JC-1\nand NADH FLIM studies, which providing support and new insights for the study of\nspatial heterogeneity of MMP.\nComment：\n4. The inclusion of equations are very helpful. For eqn (2), why is the chi-squared\ncomputed in the frequency domain, shouldn't it be calculated in the time domain?\nResponse:\nWe are grateful to the reviewer for raising this important point. χ2 is calculated in the\ntime domain. We have corrected the previously inaccurate equation in the main text:\n𝜒 2 = ∑𝑛 [𝑁(𝑡 𝑘 )−𝑁𝑐(𝑡 𝑘 )]2 (2)\n𝑘=1\n𝑁(𝑡 )\nk\nN(t ): Measured fluorescence decay function\nk\nN (t ): Calculated decay function\nC k\nn: Evaluated across the number of data points\nKelley, A. M., & Kelley, D. F. (2022). Comment on “Dependence of the Fluorescent\nLifetime τ on the Concentration at High Dilution”. The Journal of Physical\nChemistry Letters, 13(51), 11942-11945. doi:10.1021/acs.jpclett.2c02677"}]

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