Visualizing mitochondrial membrane potential with FRET probes: Integrating fluorescence intensity ratio and lifetime imaging

doi:10.21203/rs.3.rs-4721479/v1

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Visualizing mitochondrial membrane potential with FRET probes: Integrating fluorescence intensity ratio and lifetime imaging

https://doi.org/10.21203/rs.3.rs-4721479/v1

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Mitochondrial membrane potential (MMP) is crucial for mitochondrial function and serves as a key indicator of cellular health and metabolic activity. Traditional lipophilic cationic fluorescence intensity probes are unavoidably influenced by probe concentration, laser intensity, and photobleaching, limiting their accuracy. To address these issues, we designed and synthesized a pair of fluorescence molecules, OR-C8 and SiR-BA, based on the Förster Resonance Energy Transfer (FRET) mechanism, for dual-modality visualization of MMP. OR-C8 anchors to the inner mitochondrial membrane through strong hydrophobic interactions, while SiR-BA is expelled from mitochondria when MMP decreases, thereby regulating the FRET process. During MMP reduction, the fluorescence intensity and lifetime of OR-C8 increase, while the fluorescence intensity of SiR-BA decreases. By combining changes in fluorescence intensity ratio and fluorescence lifetime, dual-modality visualization of MMP was achieved. This method not only accurately reflects MMP changes but also provides a novel tool for in-depth studies of mitochondrial function and related disease mechanisms, offering significant potential for advancing mitochondrial research and therapeutic development.

Mitochondrial membrane potential

FRET

Fluorescence intensity

Fluorescence lifetime

Dual-modality

Mitochondria, often referred to as the powerhouses of the cell [1, 2], play a pivotal role in various cellular processes, including energy metabolism, signaling cascades, and programmed cell death [3, 4]. Central to the functionality of mitochondria is the maintenance of MMP, a critical parameter indicative of cellular health and metabolic activity [5, 6]. MMP governs several essential cellular processes, including ATP synthesis, calcium signaling, and reactive oxygen species (ROS) generation, highlighting its significance in cellular physiology and pathology [7, 8]. Given its multifaceted roles, accurate visualization and quantification of MMP are imperative for understanding mitochondrial function and its implications in health and disease.

Traditional lipophilic cationic fluorescence intensity probes, such as TMRE, TMRM, and RHO123, achieve transmembrane equilibrium according to the Nernst equation [9]. Consequently, their accumulation within mitochondria is inversely proportional to the mitochondrial membrane potential (MMP). These probes have been extensively utilized for monitoring MMP. Manley et al. elucidated the role of MMP in mitochondrial fission by observing variations in TMRM fluorescence intensity, revealing that MMP at mitochondrial fission sites was lower compared to other regions [10]. However, fluorescence intensity-based probes are susceptible to factors such as probe concentration, laser intensity, and photobleaching, complicating the interpretation of experimental results. To achieve optimal results, it is essential to rigorously control technical parameters such as probe concentration, staining time, and excitation light intensity to ensure accurate interpretation of staining outcomes.

In contrast, ratiometric probes can effectively mitigate external interferences, enabling high-contrast imaging and quantitative monitoring of mitochondrial microenvironments [11, 12]. Wei et al. developed a pair of probes based on FRET principles, utilizing changes in the fluorescence intensity ratio to visualize and detect MMP alterations effectively [13]. While current ratiometric probes predominantly rely on fluorescence intensity ratio changes to visualize MMP, they still face challenges related to photobleaching. Fluorescence lifetime, an intrinsic property of fluorophores, is not affected by probe concentration, laser intensity, or photobleaching. With the advancement of Fluorescence Lifetime Imaging Microscopy (FLIM) technology [14, 15], future research combining fluorescence intensity ratio changes with fluorescence lifetime for MMP visualization will more accurately reflect MMP. This approach holds significant promise for advancing our understanding of mitochondrial physiological processes.

In this work, a pair of FRET molecules, OR-C8 and SiR-BA, have been designed and synthesized for the visualization of MMP. The donor, OR-C8, is formed by conjugating octanoic acid with rhodamine through an amide condensation reaction. It achieves reaction-free anchoring to the inner mitochondrial membrane via strong hydrophobic interactions with the C8 alkyl chain and the phospholipid bilayer. The acceptor, SiR-BA, is synthesized by linking butyric acid to silicon rhodamine through an amide condensation reaction. Its shorter alkyl chain is employed to adjust the dye’s transmembrane time. Both molecules are cationic rhodamine dyes, enabling efficient mitochondrial targeting imaging. OR-C8 and SiR-BA can undergo a FRET process within the inner mitochondrial membrane. As the MMP decreases, the efflux of SiR-BA hinders the FRET process, resulting in an increase in the fluorescence intensity and lifetime of OR-C8, while the fluorescence intensity of SiR-BA decreases. Thus, dual-modality visualization of MMP changes can be achieved through variations in fluorescence intensity ratio and lifetime.

Synthesis and characterization

The specific synthetic routes for OR-C8 and SiR-BA are detailed in the supporting information. The chemical structures of these molecules have been confirmed by Nuclear Magnetic Resonance (NMR) and High-Resolution Mass Spectrometry (HRMS), with detailed results provided in the supporting information.

Cell viability assay

The cytotoxicity of OR-C8 and SiR-BA was evaluated using a CCK8 assay. HeLa cells were seeded at a density of 1×10^4 cells per well in a 96-well plate and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco), 5% CO₂ at 37°C, penicillin (100 U/mL), and streptomycin (100 µg/mL) for 24 hours. Subsequently, the cells were treated with varying concentrations of OR-C8 or SiR-BA, while cells without dye treatment served as controls. After an additional 24-hour incubation period, the cells were exposed to CCK8 solution for 1 hour, and the absorbance at 450 nm was measured using a Biotek/800TS microplate reader (Berten, USA). Cell viability (%) was calculated using the formula: Viability = (mean absorbance of treated wells / mean absorbance of control wells) × 100%.

Co-localization experiment

HeLa cells were co-stained with MTG (0.2 µM) and OR-C8 (0.4 µM) or SiR-BA (0.4 µM) for 30 min, followed by imaging using confocal microscopy. Subsequently, the cells were treated with 10 µM CCCP or a fixative solution (4% PFA and 1% GA) for 30 min and observed again using confocal microscopy.

Visualizing MMP decline via fluorescence intensity ratio

HeLa cells were co-stained with OR-C8 (0.4 µM) and SiR-BA (0.4 µM) for 30 min, followed by treatment with CCCP (5 µM). Confocal microscopy was performed using a 559 nm laser, with dual channels (570–600 nm and 650–750 nm) to capture the fluorescence signals of OR-C8 and SiR-BA, respectively. Time-lapse imaging sequences were acquired at 30 s intervals over a total duration of 10 min.

Visualizing MMP decline via fluorescence lifetime imaging

Variations in fluorescence lifetime of OR-C8 and SiR-BA at different ratios in model lipid membrane solutions were assessed. Stock solutions of OR-C8 (10 mM) and SiR-BA (10 mM) in DMSO were prepared, with OR-C8 diluted to 10 µM in the model membrane solution and incremental additions of SiR-BA. Fluorescence lifetime changes were measured using an Edinburgh FLS980 series fluorescence spectrometer. For cellular imaging, HeLa cells were stained with OR-C8 (0.4 µM) or co-stained with OR-C8 (0.4 µM) and SiR-BA (0.4 µM) for 30 min, and imaging was performed using a Leica STELLARIS 8 confocal microscope. Time-lapse fluorescence lifetime imaging was done using a Leica TCS SP8 confocal microscope on cells co-stained with OR-C8 (0.4 µM) and SiR-BA (0.4 µM) for 30 min and treated with 5 µM CCCP over 8 min.

Statistical Analysis

Experiments were independently performed with at least three replicates. The data are expressed as mean ± standard deviation (SD). Data analyses and group comparisons were conducted using GraphPad Prism 8 software. A two-sided t-test was employed to assess statistical significance between two groups. Significance levels were indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001, ns p > 0.05.

Photophysical characteristics of FRET pairs

Initially, the absorption and emission spectra of probes OR-C8 (10 µM) and SiR-BA (10 µM) in PBS solution were studied, as shown in Fig. 1A and B. Probe OR-C8 exhibited a distinct absorption peak at 555 nm and an emission peak at 578 nm, whereas probe SiR-BA showed a prominent absorption peak at 660 nm and an emission peak at 679 nm. The overlap between the emission peak of OR-C8 and the absorption peak of SiR-BA suggests a potential FRET process between these two dyes. To verify this process, a mixed solution of OR-C8 and SiR-BA in PBS was prepared. As a comparison, a mixed solution of OR-C8 and SiR-BA in lipid membrane solution (DMPC/DMPG) was also prepared to simulate the interaction of the dyes in a mitochondrial lipid environment. As illustrated in Fig. 1C, the mixed solution in PBS displayed two distinct absorption peaks at approximately 555 nm and 660 nm, attributed to OR-C8 and SiR-BA, respectively. When excited with a 520 nm laser, the fluorescence spectrum showed only the emission peak of OR-C8 at 578 nm, indicating low FRET efficiency due to the difficulty in achieving a distance of less than 10 nm between the dye molecules in PBS, driven by Brownian motion and electrostatic repulsion.

In contrast, in the lipid membrane solution, not only were there significant absorption peaks at around 560 nm and 660 nm (Fig. 1D), but the fluorescence spectrum under 520 nm laser excitation also exhibited notable emission peaks at approximately 580 nm and 680 nm. This indicates a clear FRET process between OR-C8 and SiR-BA, suggesting stronger interactions between dye molecules and lipids in the lipid environment, allowing the distance between dye molecules to fall below 10 nm, facilitating effective FRET. Furthermore, the FRET process at different ratios of the two dyes in the lipid membrane solution was explored. As shown in Fig. 1E, under 520 nm laser excitation, the fluorescence spectrum of OR-C8 alone exhibited a peak at 580 nm. As the proportion of SiR-BA increased, the fluorescence intensity of OR-C8 at 580 nm gradually decreased, while the fluorescence intensity of SiR-BA at 680 nm increased. This trend demonstrates that with the addition of SiR-BA, more OR-C8 molecules transferred their energy to SiR-BA through non-radiative transitions, leading to a decrease in OR-C8 fluorescence and an increase in SiR-BA fluorescence.

As fluorescent probes for monitoring MMP, it is crucial to investigate the influence of bioactive substances on their performance. As depicted in Fig. 2, after incubation with 50 µM bioactive substances, there were no significant changes in the UV absorption and fluorescence emission of OR-C8 and SiR-BA. The results indicate that the optical properties of OR-C8 and SiR-BA are not affected by bioactive substances.

Intracellular localization of OR-LA and SiR-BA

To confirm the mitochondrial targeting and localization ability of the probes OR-C8 and SiR-BA, fluorescence co-localization experiments were conducted using the commercial dye MTG with both OR-C8 and SiR-BA. As shown in Fig. 3A and D (left), the fluorescence signals of MTG in HeLa cells overlapped well with those of OR-C8 and SiR-BA, with Pearson co-localization coefficients of 0.90 and 0.89, respectively, indicating that OR-C8 and SiR-BA indeed target mitochondria in live cells.

Considering that during the decrease in MMP, OR-C8 remains anchored to the mitochondrial inner membrane independently of MMP while SiR-BA is expelled from the mitochondria, the localization of the probes during MMP decline should be tested. The commercial mitochondrial dye MTG, which contains a benzyl chloride group that covalently binds to mitochondrial inner membrane proteins, allows for MMP-independent imaging. However, MTG does not support mitochondrial imaging in fixed cells. Therefore, the localization of OR-C8 and SiR-BA was examined under conditions of CCCP-induced MMP reduction and cell fixation. HeLa cells were co-stained with MTG and OR-C8 or SiR-BA, followed by CCCP treatment. As shown in Fig. 3B and D (middle), after 30 minutes of CCCP treatment, the fluorescence signals of MTG and OR-C8 still overlapped well, with co-localization coefficients of 0.85. In contrast, the fluorescence signal of SiR-BA significantly decreased, and its co-localization coefficient with MTG dropped below 0.2, indicating that OR-C8 remains localized in mitochondria under conditions of CCCP-induced MMP reduction, while SiR-BA is expelled from the mitochondria. Subsequently, the intracellular localization of MTG, OR-C8, and SiR-BA in fixed cells was further investigated. As shown in Fig. 3C and D (right), after fixation of HeLa cells with 4% PFA and 1% GA, OR-C8 still exhibited bright mitochondrial fluorescence signals, while the fluorescence signals of MTG and SiR-BA nearly disappeared, indicating that OR-C8 remains localized in mitochondria under cell fixation conditions. These results demonstrate the MMP-independent mitochondrial targeting imaging capability of OR-C8 and the MMP-dependent imaging capability of SiR-BA.

FRET process of OR-C8 and SiR-BA in vivo

To further confirm that FRET occurs between OR-C8 and SiR-BA in mitochondria and that their emission spectra are well-separated, preventing fluorescence crosstalk, we conducted specific staining experiments. HeLa cells stained solely with OR-C8, as shown in Fig. 4, were excited with a 559 nm laser. Clear OR-C8 fluorescence signals were observed in the 570–600 nm channel, while no fluorescence was detected in the 650–750 nm channel. Subsequently, HeLa cells stained only with SiR-BA were excited with a 559 nm laser, resulting in no fluorescence signal in the 650–750 nm channel; however, upon excitation with a 633 nm laser, significant SiR-BA fluorescence signals were observed in the 650–750 nm channel. When HeLa cells were co-stained with OR-C8 and SiR-BA and excited with a 559 nm laser, bright fluorescence signals of OR-C8 and SiR-BA were detected in the 570–600 nm and 650–750 nm channels, respectively. These results indicate that there is no fluorescence crosstalk between OR-C8 and SiR-BA and that FRET from OR-C8 to SiR-BA occurs in mitochondria.

Monitoring MMP Decline via Fluorescence Intensity Ratio

HeLa cells were co-stained with OR-C8 and SiR-BA, followed by CCCP treatment to induce a decrease in MMP. As shown in Fig. 5A, during the decline of MMP, the fluorescence intensity of OR-C8 gradually increased while that of SiR-BA gradually decreased, indicating a reduction in FRET efficiency. This allows the visualization of MMP changes through fluorescence intensity. Additionally, the changes in fluorescence intensity ratio of OR-C8 and SiR-BA was quantified and plotted (Fig. 5B), facilitating the quantitative analysis of the MMP decrease. It was clearly observed that within the first minute of CCCP induction, there was a significant drop in MMP, followed by a slower decline.

Tracking MMP Decline Using Fluorescence Lifetime Imaging

When FRET occurs between the two dye molecules, a portion of the energy from the donor's radiative transition is transferred to the acceptor through non-radiative transition, resulting in decreased fluorescence intensity and lifetime of the donor. Fluorescence lifetime is typically absolute and unaffected by factors such as excitation light intensity or dye concentration, depending solely on the microenvironment of the dye. Therefore, utilizing fluorescence lifetime microscopy for imaging the decrease in MMP provides more accurate image information and is more advantageous for obtaining molecular state and spatial distribution information. The change in fluorescence lifetime of the donor was investigated in vitro using lipid membrane solutions (DMPC/DMPG) when energy transfer occurred in the FRET pair. As shown in Fig. 6A, the fluorescence lifetime of OR-C8 decreased continuously with the increasing proportion of SiR-BA, indicating that this FRET pair is highly suitable for visualizing changes in MMP. Subsequently, HeLa cells stained solely with OR-C8 exhibited an average fluorescence lifetime of 1.822 ns under fluorescence lifetime microscopy (Fig. 6B). When cells were co-stained with OR-C8 and SiR-BA, the fluorescence lifetime of OR-C8 decreased to 1.315 ns, demonstrating effective FRET between the two dyes within the inner mitochondrial membrane. Following this, HeLa cells co-stained with OR-C8 and SiR-BA were treated with CCCP to induce a decrease in MMP. It was observed that as the MMP decreased, the fluorescence lifetime of OR-C8 increased from approximately 1.036 ns to about 1.775 ns (Fig. 6C). Additionally, the changes in fluorescence lifetime were quantified and plotted (Fig. 6D), facilitating the quantitative analysis of the MMP decrease. This indicates that during the decrease in MMP, SiR-BA is continuously expelled from the mitochondria, leading to a reduction in FRET efficiency with OR-C8, allowing the fluorescence lifetime of OR-C8 to recover. This method of visualizing and monitoring changes in MMP through fluorescence lifetime variation provides a novel approach for mitochondrial research.

In this study, we have developed a pair of mitochondria-targeted fluorescent molecules based on the FRET mechanism. The donor, OR-C8, is anchored to the mitochondrial inner membrane through strong hydrophobic interactions between C8 alkyl chain and the phospholipid bilayer of the inner mitochondrial membrane. The FRET process between the donor OR-C8 and the acceptor SiR-BA is regulated by MMP. By monitoring changes in the fluorescence intensity ratio and the fluorescence lifetime of the donor during the decrease in MMP, we achieve dual-modality visualization of MMP. This approach lays the foundation for in-depth studies on intracellular energy regulation mechanisms and the development of therapeutic strategies for mitochondrial-related diseases.

Author Contributions Fei Peng: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Xiangnan Ai: Formal analysis, Data curation, Validation. Xiaoyu Bu: Investigation, Formal analysis. Zixuan Zhao: Formal analysis. Baoxiang Gao: Writing – review & editing, Visualization, Resources, Methodology, Investigation, Conceptualization.

Funding This work was supported by the National Natural Science Foundation of China (22177024), Natural Science Foundation of Hebei Province (B2019201231), and Postgraduate’s Innovation Fund Project of Hebei University (HBU2024BS026).

Data Availability The article contains the data that were utilized to
support the results of this investigation.

Ethical Approval Not applicable.

Competing Interests The authors declare no competing interests.

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No competing interests reported.

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You are reading this latest preprint version

Visualizing mitochondrial membrane potential with FRET probes: Integrating fluorescence intensity ratio and lifetime imaging

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Version 1

Abstract

Figures

Introduction

Experimental Section

Synthesis and characterization

Cell viability assay

Co-localization experiment

Visualizing MMP decline via fluorescence intensity ratio

Visualizing MMP decline via fluorescence lifetime imaging

Statistical Analysis

Results

Photophysical characteristics of FRET pairs

Intracellular localization of OR-LA and SiR-BA

FRET process of OR-C8 and SiR-BA in vivo

Monitoring MMP Decline via Fluorescence Intensity Ratio

Tracking MMP Decline Using Fluorescence Lifetime Imaging

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1