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. 2025 Oct 6;4(3):394–401. doi: 10.1021/cbmi.5c00123

All-Dielectric Metasurface-Enhanced Fluorescence for Probing Mitochondrial Membrane Potential Dynamics

Xiaoqing Luo †, Qian Yuan ‡,§, Rong Shi ∥, Shangjun Lin †,⊥, Yunhui Liu ‡,#,*, Guangyuan Li ¶,*
PMCID: PMC13014337  PMID: 41889467

Abstract

Fluorescence probes are the primary tools for monitoring mitochondrial membrane potential (MMP), a key indicator of mitochondrial function and cellular health. Although metasurfaces offer significant potential for enhancing fluorescence biosensing, all-dielectric metasurfaces with their inherent advantages remain largely unexplored for this application. Here, we demonstrate a fluorescence probe for MMP dynamics, significantly enhanced by an all-dielectric metasurface. Designed to support bound states in the continuum (BICs) via folded Brillouin zones, this metasurface achieves both a high quality factor and strong near-field enhancement. Experiments reveal an order-of-magnitude fluorescence intensity enhancement for cells on the metasurface compared to those off it, irrespective of forskolin stimulation. This approach enables dynamic monitoring of cellular health through MMP fluctuations at subthreshold concentrations.

Keywords: all-dielectric metasurface, biosensor, fluorescence probe, bound state in the continuum, brillouin zone folding, mitochondrial

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1. Introduction

Mitochondria are indispensable eukaryotic organelles, widely recognized as the “powerhouses” of the cell due to their pivotal role in cellular energy metabolism. − Their principal functions encompass adenosine triphosphate (ATP) synthesis, metabolic regulation, ion transport, apoptosis modulation, inflammatory response control, and the inheritance of mitochondrial deoxyribonucleic acid (DNA). , Central to their function is oxidative phosphorylation, a process critically dependent on the electrochemical proton gradient across the inner mitochondrial membrane. The mitochondrial membrane potential (MMP), which is defined as the voltage difference across the inner membrane, is a fundamental physiological metric to evaluate mitochondrial function and cellular health. , MMP directly drives essential processes such as ATP synthesis, import of mitochondrial proteins, and metabolite transport. , A decline in MMP is emblematic of cellular senescence and mitochondrial disorders; therefore, faithful monitoring of MMP offers a robust indicator of mitochondrial health and cellular viability. It should be noted that MMP is subject to modulation by environmental stimuli and intracellular signaling cascades; even within mitochondria exhibiting functional impairment, MMP may be partially restored or enhanced through targeted therapeutic intervention. −

Fluorescence-based probes represent the most widely utilized techniques for MMP measurement. These include voltage-sensitive dyes such as 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), tetramethylrhodamine methyl ester (TMRM), and rhodamine-2 acetoxymethyl ester (Rhod-2 AM), typically used in conjunction with fluorescence microscopy or flow cytometry. − For example, JC-1 forms red fluorescent J aggregates in mitochondria under high MMP, whereas it exists as green fluorescent monomers when MMP is low. Therefore, the red to green fluorescence intensity ratio serves as a surrogate marker for MMP levels. Despite their widespread application, these fluorescence methods are limited by intrinsic dye properties, such as concentration dependence and photostability, and by the sensitivity thresholds of detection instruments. Crucially, current technologies struggle to detect low-intensity “sub-threshold” fluorescence signals that arise under low MMP conditions-signals that are vital for a comprehensive evaluation of mitochondrial functional integrity and cellular viability. Accordingly, the development of technology capable of sensitive, real-time detection of low-intensity fluorescence would enable more complete and dynamic monitoring of MMP, thereby allowing more accurate assessment of cellular health and viability.

Recently, metasurfaces have emerged as an exciting platform for enhanced biomedical imaging and sensing, especially fluorescence-based biosensors. − Compared to their plasmonic counterparts, , all-dielectric metasurfaces based on Mie resonance can provide comparable sensitivity with superior resonance bandwidth, Q factor, figure-of-merit, and lower resonance-induced heating. , Quite recently, bound states in the continuum (BICs) provided a convenient approach to achieve high Q factors, especially for all-dielectric metasurfaces. − Brillouin zone folded (BZF) BICs have high Q factors over a large region around the Γ point in the momentum space and are robust against structure disorders. , Taking advantage of the high-Q BIC-based metasurfaces, ultralow-weight molecules and biological molecules can be detected with high sensitivity. − Iwanaga demonstrated highly sensitive detection of antibody/antigen and nucleic acid targets based on all-metasurface-enhanced fluorescence. Further applications in examination for improving the accuracy of early diagnosis based on cell morphology and tissue imaging has also been widely investigated. − However, high-Q all-dielectric metasurface-enhanced fluorescence biosensors remain largely unexplored to date.

In this work, we report an all-metasurface enhanced fluorescence biosensor to evaluate MMP dynamics, which serves as a critical indicator of cellular status and viability. We design a high-Q all-dielectric metasurface based on Brillouin zone folded (BZF) BICs, which can achieve extreme robustness against disorder while maintaining ultrahigh Q factors. − The resonance wavelengths of the quasi-BICs are engineered to overlap with the fluorescence wavelength of a dye from the rhodamine family that is widely used as MMP indicators. We fabricate a series of monocrystalline silicon metasurfaces to compensate the fabrication imperfections, and pick out a sample of the best performance to maximize the fluorescence enhancement, to perform subthreshold MMP detection, and to investigate the effects of forskolin (FSK). Therefore, we expect that the high-Q BIC-based all-dielectric metasurface’s ability to sensitively and dynamically monitor MMP fluctuations will be of great significance for assessing cellular health.

2. Results and Discussion

Figure a illustrates the unit cell of the designed metasurface with a square lattice of periodicity a. It is composed of four silicon nanodisks with the diagonal two (the left-top and the right-bottom) slightly shifted by S along the y axis from the centers of the quadrisected square. Specially, when S = 0 nm, the minimum unit cell reduces to a square square of half the periodicity in both the x and y directions with only one nanodisk located at the center. Therefore, by slightly shifting the diagonal nanodisks, the lattice period of the minimum unit cell doubles, resulting in the folding of the first Brillouin zone (FBZ), as shown by the transverse electric (TE) and magnetic (TM) band structures in Figure c. The TE1, TM1 and TM2 bands of the unperturbed and perturbed metasurfaces are shown by solid gray curves and dots, respectively. The eigen-field distributions of TE11 and TM11 without any perturbation are shown in Figures b and S1. It is clear that due to the shift perturbation, the FBZ size is reduced by half along both directions: the X (or Y) point is folded into Γ point, and the midpoint between Γ and X (or Y) points of the unperturbed design becomes the X (or Y) point of the perturbed metasurface, which is labeled X′ (or Y’′). As a result, the TE1, TM1, and TM2 bands on the right side of X′ are folded into the left side, as seen by the colored dots.

1.

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(a) Schematics of the metasurface supporting Brillouin zone folding BICs after perturbation of shift. The unit cell with period a = 610 nm composes of four Si nanodisks with height h = 100 nm and radius r = 110 nm, of which the diagonal are shifted by S along y direction. (b) The eigenfield distributions H z and E z of TE11 and TM11 respectively. (c) Calculated band diagram of the TE and TM modes supported by unperturbed (in curves) and perturbed (in dots, S = 10 nm) designs. X is the edge of the Brillouin zone of the unperturbed design, and X′ is that of the perturbed design. Inset shows the FBZ of the unperturbed metasurface presented by the black square, shrinking to its half size by folding along the dashed yellow lines into the FBZ (white area) of the perturbed metasurface. (d,e) Simulated Q factors of two folded bands, TE11 and TM11, versus in-plane k x for different shifts S = 5, 10, 20, 30 nm.

Figure d,e shows the Q factors of the folded TE11 and TM11 modes as functions of the in-plane wavevector for different shift perturbations S. For the TE11 mode, the Q factors are almost flat in the momentum space, whereas for the TM11 mode, the Q factors exhibit a flat feature only when S is relatively large (S ≥ 30 nm), but decrease slowly for smaller shifts. All these Q factors approach infinity at Γ point, and decrease with the shift perturbation S in an inverse quadratic function

Q∝1/S2 1

Figure a–c show the evolution of the simulated transmittance, reflectance, and absorptance spectra of the designed metasurface with varying shift perturbation S, which is under normal incidence of plane wave with unitary electric field polarized along the x direction. Within the spectral range of interest, Figure a shows that there exist two pronounced transmittance dips when S = 25 nm, corresponding to the folded modes TE11 and TM11. As S approaches 0 nm, i.e., the structure reduces to the unperturbed design, both dips narrow down but become shallower and finally disappear at S = 0 nm. These spectral features further validate the BIC characteristics of the folded modes TE11 and TM11. As S increases, the first dip representing the folded mode TE11 is red-shifted and quickly becomes wider, suggesting a rapid drop of the Q factor, consistent with Figure c; in contrast, the folded mode TM11 (the second dip) remains resonant almost at λ = 720 nm and slowly becomes wider, which suggests a slow drop of the Q factor, consistent with Figure d. Interestingly, when S = 75 nm these two dips merge at λ = 720 nm.

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(a–c) Evolution of simulated transmittance, reflectance, and absorptance spectra of the designed metasurface as shift S varies. (d) Calculated maximum local electric field intensity |E|2 of TE11 and TM11 at the resonant wavelengths for different S. (e) Near-field electric field distributions |E| of TE11 and TM11 modes for the unit cell at the half height plane of nanodisks with S = 10, 30, 50, and 70 nm from top to bottom.

Figure b shows that the reflectance spectra have two peaks with evolution similar to the transmittance dips. The absorptance spectra in Figure c, which are calculated with A(λ) = 1 – T(λ) – R(λ), show that the TE11 mode exhibits large absorptance around S = 10 nm, whereas the TM11 mode has narrow absorptance between S = 20 and 70 nm.

Figure e shows the evolution of the near-field electric field distributions |E| for the TE11 and TM11 modes as the shift perturbation S varies. For small perturbations such as S = 10 nm, the TE11 mode features the electric quadrupole confined outside the nanodisk, whereas the electric fields of the TM11 mode are confined to the nanodisks. Because the TE11 and TM11 modes gradually merge as S increases to 75 nm, their electric field distributions gradually evolve into the same profile, of which the greatly enhanced electric field is mainly confined to the small vertical gaps between two closest nanodisks. Interestingly, the electric field strength for the TE11 mode first decreases to |E| = 17 when S ≤ 50 nm, and then slightly increases to |E| = 19 when S increases to 70 nm. In contrast, for the TM11 mode, the electric field strength keeps increasing and reaches the maximum value of |E| = 17 when S = 70 nm. These behaviors are better visualized in Figure d, where the maximum electric field intensities |E|2 are plotted, and that the TE11 mode always has larger field enhancement than the TM11 mode. Note that here the electric field intensity |E|2 indicates the near-field enhancement factor because the incident plane wave has unitary amplitude. The great near-field enhancement suggests significantly enhanced light–matter interactions, making the designed metasurface attractive for enhancing the fluorescence.

Figure d also shows that the TE11 mode has the largest |E|max for tiny shift perturbations, consistent with the Q factor behaviors shown in Figure c,d. However, in Figure b we find that the reflectance peaks are not pronounced for tiny shifts, indicating weak coupling efficiency between the TE11 mode and free-space light. For large shifts, the gap between the two neighboring nanodisks will be too small. For example, for S = 70 nm, this gap is only 10 nm, imposing challenges in nanofabrication. As a result, after comprehensive consideration of the efficiency of free-space coupling, the near-field electric field enhancement, and the convenience of nanofabrication, we adopted S = 20, 30, and 40 nm for fabricating the metasurfaces that are used to enhance fluorescence sensing.

For each shift S, we need to slightly vary the nanodisk radius around 110 nm so that the TE11 resonance can locate closest to λ = 645 nm, which is the peak FL wavelength of the Mito-Tracker Deep Red 633 (MTDR633). In order to compensate the fabrication deviations, we fabricated a series of silicon metasurfaces based on a silicon-on-insulator (SOI) substrate (see Methods). This is because the top silicon of the SOI substrate is monocrystalline and has relatively low absorption loss in the visible regime.

Figure a,b compare the simulated and measured reflectance spectra for different nanodisk radii and different shifts. Here we measured the reflectance because the bottom silicon film of the SOI-based metasurfaces was not removed, which acts as a mirror for the metasurface. For S = 20 nm and r = 104 nm, Figure a shows that there exist two peaks in the simulated reflectance spectrum: a pronounced narrow peak around λ = 640 nm, and a weak narrow peak around 720 nm, which correspond to the TE11 and TM11 modes, respectively. As r increases, these two peaks are red-shifted. In general, the measured reflectance spectra agree with the simulations except that the resonances are red-shifted by ∼20 nm and the line widths are slightly broadened. The good agreement between the simulations and the experiments is better visualized by the resonance wavelengths λpeak and the Q factors for both TE11 (in red) and TM11 (in blue) modes in Figure c,d, which are extracted from the reflectance spectra. Results show that the measured λpeak’s are about 20 nm smaller than the simulated values, and that the measured Q factors are about half of the simulation data. These deviations may originate from the nanofabrication imperfections, including variations in shapes and sizes, and side-wall tilting.

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(a) Simulated and (b) measured reflectance spectra for different radii r = 104, 110, and 116 nm. (c,d) Comparison of simulated and measured (c) resonance wavelengths and (d) Q factors of TE11 and TM11. From top to bottom, the shifts are S = 20, 30, 40 nm, respectively.

Figure a,b show that for both S = 30 and 40 nm, together with which the nanodisk radius is slightly varied, the simulated resonance wavelengths of the TE11 mode are around 660 nm, whereas the measured resonance wavelengths are blue-shifted to ∼640 nm. Except for the resonance wavelength deviations and the drop of the measured Q factors due to nanofabrication imperfections, the experimental results show good agreement with the simulation data.

In Figure b we find that the measured resonance wavelength of the TE11 mode supported by the as-fabricated metasurface of S = 30 nm and r = 114 nm is λ = 653 nm with Q factor of 48, and is the closet to the peak FL wavelength of MTDR633 (λ = 645 nm), which is indicated by the vertical dashed lines. Therefore, we adopted this metasurface, of which the scanning electron microscope (SEM) image is shown in Figure a, to maximize the enhancement of the fluorescence from the cells. For this purpose, we first incubated cells on the metasurface with MTDR633 of different concentrations, and then covered the metasurface with a coverslip on top of the metasurface for fluorescence microscopy imaging (see Methods), as illustrated by Figure b.

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(a) SEM image of the SOI-based metasurface, the scale bar indicates 200 nm. (b) Schematics of FL sensing for evaluating mitochondrial membrane potential dynamics, the cell culture, fluorescence probe with or without FSK treatment and FL image capture. Created with BioRender.com. (c) FL images taken at 5 s exposure time for different Mito-Tracker concentrations increasing from 0 pM to 1 μM. The metasurface region, labeled as “Meta”, is brighter than outside background region, labeled as “BG”. Insets show zoomed-in views taken with extended exposure time of 15 s so that the boundaries of cells can be better visualized. (d) FL images for different FSK concentrations of 10 nM, 100 nM, 1 μM, and 10 μM. (e,f) Average FL intensities of (e) the Meta and BG regions, and (f) a cell on top of the Meta and BG regions, and their ratios. (g,h) Average FL intensities as functions of the FSK concentration. Fitting was performed with linear functions in a logarithmic scale.

In this work, we selected MTDR633 as the fluorescent probe due to its spectral compatibility with the designed metasurface structure, which exhibits fluorescence enhancement at specific wavelengths. MTDR633 is a far-red fluorescent probe that selectively accumulates in the mitochondria of live cells in a membrane potential-dependent manner. It passively diffuses across the cell membrane and is retained in the mitochondrial matrix, driven by the electrochemical gradient maintained by metabolically active mitochondria, emitting a bright far-red fluorescence. The probe exhibits maximum excitation and emission wavelengths of 622 and 648 nm, respectively. MTDR633 exhibits fixation-compatible staining, allowing enduring observations of mitochondrial morphology, and the emitted deep red fluorescence possesses superior capability of penetration in the cells and tissues.

To evaluate the metasurface’s capability to enhance weak fluorescence signals, we performed subthreshold MMP detection using gradient concentrations of MTDR633. Specifically, HEK293FT cells were incubated with MTDR633 at concentrations ranging from 0 to 1 μM for 30 min, followed by fluorescence imaging. Remarkably, at an ultralow concentration of 100 pM, well-defined fluorescence signals delineating cellular morphology were observed exclusively within the metasurface region, which is labeled “Meta”, whereas no discernible signals were detected in the control region outside the metasurface, which is labeled “BG”. As the dye concentration increased, the fluorescence intensity enhances correspondingly. Throughout the entire concentration range under test, the background regions consistently showed negligible fluorescence signals, as shown by Figure c. These results confirm the strong fluorescence enhancement capability empowered by the metasurface.

In order to quantify the FL enhancement due to the metasurface, we averaged the intensities over the Meta region and over the BG region, respectively. We also averaged the intensities of a cell on top of the Meta and BG regions, respectively, as illustrated by the inset of Figure c, showing the zoomed-in images with extended exposure time to better visualize the boundaries of cells. Figure e shows that the average intensities of the Meta and BG regions increase with the MTDR633 concentration, and that the average intensity of the Meta region is always higher than those of the BG region. Their ratios are about 3 for different concentrations. For the average intensities of the cells in the Meta and BG regions, Figure f shows similar behaviors, except that the intensity of the cell in the Meta region increases significantly. As a result, the ratio increases from ∼3 to ∼10. In other words, the FL intensity of the cell can be enhanced by an order of magnitude if it is located within the metasurface region.

Since the dynamic monitoring of subthreshold MMP levels is crucial for a comprehensive assessment of mitochondrial functionality and cellular vitality, we further investigated the effects of FSK, which is known as a cellular stimulant, on MMP levels in MTDR633-labeled cells. Cells were treated with FSK at concentrations of 10 nM, 100 nM, 1 μM, and 10 μM, and corresponding fluorescence images were acquired. Notably, even at a low concentration of 10 nM FSK, distinct fluorescence signals were observed only in the metasurface region. The signal intensity increases progressively with the FSK concentration. In stark contrast, negligible fluorescence was detected in the control regions under all treatment conditions, as shown by Figure d.

Figure g,h shows that the average intensities of the Meta and BG regions, and those of the cells in these two regions all increase with the FSK concentration. Interestingly, the results can be fitted with linear functions when the FSK concentration is on a logarithmic scale. The slopes for the Meta and for the cell in the Meta region are larger than those for the BG and for the cell in the BG region, respectively. For a linear calibration curve, the LOD can be estimated according to the following expression

LOD=3Sa/b 2

where S a can be estimated by the standard deviation of the intercepts in the fittings, and b is the slope of the fitting lines. The LODs are estimated to be 1.7 pM and 20 pM based on the intensities of the Meta and BG regions, respectively, and 4 pM and 16 pM based on the intensities of the cells in the Meta and BG regions, respectively. It is clear that the LODs in the Meta region are much lower than those in the BG region. These findings provide compelling evidence that the metasurface significantly enhances fluorescence signals, thereby enabling the sensitive detection of dynamic changes in subthreshold MMP levels.

3. Conclusions

In conclusion, we have designed and fabricated high-Q all-dielectric metasurfaces based on BZF BICs. We have engineered the structural parameters so that the folded mode TE11, which has sustainable high Q factors, large near-field enhancement, and relatively high coupling efficiency with free-space light, is resonant close to the peak FL wavelength of the MTDR633. Based on the high-Q BZF-BIC metasurface, we have demonstrated that the FL intensity of the cell can be increased by an order of magnitude when the cell locates within the metasurface region. Under the stimulation of FSK, we have also observed higher FL intensities for cells in the metasurface region. We therefore expect that the enhanced FL signals by the metasurface will be useful for real-time monitoring of cellular health via the sensitive detection of subthreshold MMP levels.

4. Methods

4.1. Simulations

In order to simulate the transmittance and reflectance spectra, T(λ) and R(λ), as well as the near-field distributions of the silicon metasurfaces in Figures and we adopted a home-built package of rigorous coupled wave analysis (RCWA), which was developed following. − In all simulations, the same wavelength-dependent permittivities of crystalline silicon are tabulated in Palik.

4.2. Fabrication of Silicon Metasurfaces

The designed metasurfaces were fabricated with state-of-the-art nanofabrication techniques. We started with an SOI substrate, spin-coated a thin film of electron-beam resist (ZEP520A) on the top, and patterned it with electron-beam lithography. We then deposited a 40 nm film of chromium using electron-beam evaporation, followed by liftoff, resulting in a chromium metasurface of the same design. With the chromium nanodisks serving as the hard mask, we etched the top silicon layer of the SOI using inductively coupled plasma-reactive ion etching (ICP-RIE). Finally, we removed the chromium mask with wet-etching, and obtained the designed metasurface composed of silicon nanodisks that covers a large area of 1 mm × 1 mm.

4.3. Cell Culture and Treatment

HEK293FT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were kept in a humidified incubator at 37 °C with 5% CO2. For mitochondrial membrane potential (MMP) detection, cell suspensions at a density of 2 × 105 cells per well were seeded into 6-well plates preloaded with metasurface substrates. When cells reached approximately 75% confluence, various concentrations of MTDR633 were added and incubated for 30 min under standard culture conditions. After incubation, the metasurface substrates bearing the cell samples were transferred to a fluorescence microscope for imaging and subsequent analysis.

To investigate the effects of varying degrees of cellular activation on dynamic changes in MMP and to further evaluate the fluorescence enhancement performance of the metasurfacecells were treated with a mixture containing 100 nM MTDR633 and FSK at varying concentrations (gradient determined experimentally). After coincubation (duration consistent with or based on prior incubation conditions), fluorescence imaging was performed to assess MMP-related signal variations.

4.4. Optical Measurements

For the measurement of the reflectance spectra, the fabricated SOI sample containing a series of metasurfaces with different structural parameters was mounted on a computer-controlled three-dimensional translation stage. A linearly polarized collimated beam from a continuous laser impinges on the sample under normal incidence. The reflected light was spatially filtered and then directed to an optical spectrometer (AQ6370D, Yokogawa).

The FL images were acquired with a Zeiss fluorescence microscope (Axio scope.A1) at an exposure time of 5 s with the camera gain set to be 3. The samples were illuminated by 532 nm blue light with a spot diameter of 2 mm, then FL light is collected by a 10× objective lens with a numerical aperture of 0.25.

Supplementary Material

im5c00123_si_001.pdf (223KB, pdf)

Acknowledgments

This research was sponsored by Shenzhen Medical Research Fund (A2303003), the National Natural Science Foundation of China (32271073, 62275261), the Natural Science Foundation of Guangdong Province (2024A1515011835, 2514050002345), the Guangdong Provincial Key Laboratory of Brain Connectome and Behavior (2023B1212060055), and Shenzhen Science and Technology Program (JCYJ20220818101600001, JCYJ20240813154717023, JCYJ20220531100206014).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00123.

  • Figure S1. The eigenfield distributions of TE11, TM11 and TM21 (PDF)

∇.

X.L. and Q.Y. contributed equally.

The authors declare no competing financial interest.

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