Highly Stable Probe for Detailed Long-Term Imaging of Mitochondria Movement

Cy3 has gained prominence as a mitochondrial probe, driven by its low cytotoxicity, favorable biocompatibility, and intrinsic mitochondrial targeting capabilities. Nevertheless, its reliance on electrostatic interactions for mitochondrial targeting frequently results in undesirable off-target effects. Moreover, the inherent instability of the double bonds in Cy3 makes it highly susceptible to reactive oxygen species (ROS) generated during imaging, leading to significant photobleaching. Consequently, these limitations severely impede the application of Cy3 in demanding long-term dynamic mitochondrial imaging. To overcome these challenges, we introduced high-reactivity functional groups into Cy3 for the first time. These functional groups are capable of forming covalent bonds with nucleophilic sites on mitochondrial proteins, thereby mitigating off-target effects. Initially, asymmetric Cy3 derivatives containing carboxylic or hydroxyl groups were synthesized to provide sites for subsequent functionalization. Recognizing that the labeling efficiency of covalent mitochondrial probes is intrinsically linked to the reaction kinetics with target proteins, we systematically evaluated the compatibility of four highly reactive functional groups for Cy3-based mitochondrial labeling: (1) N-hydroxysuccinimide group (NHS), (2) epoxide group, (3) chloroacetamide group, and (4) pyridyl group, which is capable of undergoing protonation to form stable N–S bonds with mitochondrial membrane proteins. To improve photostability, cyclooctatetraene (COT), a widely recognized antiphotobleaching agent, was incorporated into the probe structure. This multifaceted design strategy culminated in the generation of eight distinct covalent mitochondrial probes: Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, and Cy3-COT-EP. Additionally, HZ Mito Deep Red was synthesized by extending the conjugated structure of HZ Mito Red to enable multicolor mitochondrial labeling (Schemeb). The detailed synthetic routes for all probes are shown in Scheme S1–S4↗, and their chemical structures were confirmed by NMR and HRMS analysis, as described in the Supporting Information↗.

The photophysical properties of the novel covalent mitochondrial probes were examined in phosphate-buffered saline buffer (PBS, pH 7.2, Table). Compared to the parent fluorophore Cy3, all probes exhibited a slight red shift in both their maximum absorption and fluorescence peaks (Figure S1a, b↗). These shifts are attributed to the reduced energy difference (ΔE) between HOMO and LUMO in all covalent mitochondrial probes compared to Cy3, which leads to lower emission energy and consequently spectral red shifts (Figure S2↗). Notably, Cy3-EP and Cy3-COT-EP, possessing the strongly electron-donating ether groups, exhibit the smallest energy gaps and the most pronounced red-shifted emissions, with both compounds showing maximum emission wavelengths at 580 nm and corresponding Stokes shifts of 27 and 22 nm, respectively. Moreover, among the probes, HZ Mito Red achieved the highest fluorescence brightness (ε × Φ = 14400 M^–1 cm^–1), with a 1.7-fold increase compared to Cy3 (Table). Subsequently, to evaluate their mitochondrial labeling efficacy, these novel covalent probes were subjected to rigorous cellular assays. Considering the typical working concentration of Cy3-derived mitochondrial probes to be within the 250 nM to 500 nM range, U-2 OS cells were incubated with each probe at a uniform concentration (0.5 μM), and subsequently imaged by spinning disk confocal microscopy. As shown in Figure S3↗, under identical imaging parameters, the fluorescence intensity emanating from cells labeled with all covalent mitochondrial probes was diminished compared to Cy3. This decrease in fluorescence signal is posited to stem from the reduced hydrophobicity of the modified probes, consequently leading to lower transmembrane efficiency. To determine the working concentrations that yield fluorescence intensities comparable to Cy3 for subsequent comparative analysis, concentration gradients were established for each probe, and cell viability was assessed at the corresponding concentrations. As shown in Figure S4↗, intracellular fluorescence intensity exhibited a concentration-dependent increase across all probes. Specifically, when the concentrations of Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, and Cy3-COT-Py were increased to 10 μM, and Cy3-EP and Cy3-COT-EP were increased to 2.5 μM, the labeling brightness was equivalent to that of 0.5 μM Cy3 (Figure S5a, b↗). Furthermore, we performed cell viability assessments using the chemiluminescent CellTiter-Lumi assay at various concentrations of each probe. No obvious cytotoxicity was observed, even at a concentration of 20 μM (Figure S6↗). Subsequently, the effect of extended incubation time with a low concentration of HZ Mito Red (0.5 μM) on intracellular fluorescence intensity was further investigated. The results showed that prolonged incubation significantly increased the average intracellular fluorescence intensity (Figure S7a↗). Meanwhile, no significant cytotoxicity was observed even when the incubation time was extended to 12 h (Figure S8↗). However, a fluorescence intensity comparable to that achieved with 10 μM HZ Mito Red after 30 min of incubation was only observed after 12 h of incubation with 0.5 μM HZ Mito Red (Figure S7b↗). Therefore, considering time efficiency and the absence of cytotoxicity at higher concentrations (2.5 or 10 μM), we chose a 30 min incubation time for each probe at these concentrations in subsequent experiments, rather than extending the incubation time at a lower concentration.

Furthermore, to rigorously evaluate the mitochondrial labeling specificity of Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, and Cy3-COT-EP, colocalization studies were conducted in U-2 OS cells using Mito Tracker Deep Red (MTDR) as a reference mitochondrial marker. As shown in Figurea, b and Figure S9a↗, all probes except Cy3-COT-NHS, which exhibited a PCC of 0.5, showed a high degree of colocalization with MTDR-labeled mitochondria, with PCC values greater than 0.86. Normalized fluorescence intensity profiles further confirmed near-complete overlap between the fluorescence of Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, Cy3-COT-EP, and MTDR (Figurec, d, Figure S9b↗). This indicates that, with the exception of Cy3-COT-NHS, all probes effectively and specifically label mitochondria in living cells.

Stable mitochondrial labeling is essential for long-term dynamic imaging. Therefore, we further evaluated the stability of mitochondrial labeling by various probes. U-2 OS cells, prelabeled with Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, or Cy3-COT-EP, were subjected to glutaraldehyde (GA) fixationa procedure that abolishes MMP and induces off-target effects for probes relying solely on electrostatic adsorption for mitochondrial targeting. In comparison to prefixation conditions, cells labeled with Cy3-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, or Cy3-COT-EP exhibited a fluorescence intensity retention exceeding 60% postfixation, whereas Cy3-labeled control cells retained less than 35% (Figure S9c, d↗). Notably, Cy3-CA and HZ Mito Red outperformed the series, retaining over 85% of their initial fluorescence intensity after fixation (Figuree, f). To further substantiate the mitochondrial origin of fluorescence signals in fixed cells, U-2 OS cells transfected with a Cox4-EGFP plasmid were incubated with Cy3, Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, or Cy3-COT-EP, fixed with GA, and subjected to super-resolution imaging. As shown in Figureg and Figure S9e↗, Cy3-labeled mitochondria displayed a diffuse fluorescence signal distribution upon fixation, with the PCC to Cox4-EGFP significantly dropping to 0.18. Moreover, while all covalent mitochondrial probes exhibited enhanced labeling stability compared to Cy3, the PCC values for Cy3-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, and Cy3-COT-EP decreased to approximately 0.7. In contrast, Cy3-CA and HZ Mito Red maintained high PCC values of 0.86 and 0.9, respectively, after fixation. (Figurei, Figure S10↗). Normalized fluorescence intensity distributions along the line in fixed cells further indicate that Cy3-CA and HZ Mito Red exhibited superior signal-to-noise ratios compared to Cy3 (Figureh, (i). These results indicate that all probes achieved covalent mitochondrial labeling, but Cy3-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, and Cy3-COT-EP were less effective than Cy3-CA and HZ Mito Red. This highlights the higher reaction efficiency of Cy3 with the chloroacetamide group toward mitochondrial proteins, as well as its stronger compatibility compared to NHS group, pyridyl group, and epoxide group. In summary, Cy3-CA and HZ Mito Red achieved highly specific and stable covalent mitochondrial labeling.

Subsequently, we applied Cy3-CA and HZ Mito Red for long-term dynamic super-resolution imaging of mitochondria, comparing their performance with the commercial mitochondrial probe MTR and the widely recognized PK Mito Red. We began by conducting fluorescence spectroscopy on the four probes in solution. Under identical concentrations and detection parameters, all four probes exhibited virtually identical fluorescence intensities (Figure S11a↗). Subsequently, these probes were applied to cells, and SIM super-resolution imaging was performed under consistent parameters. Despite variations in probe concentrations within the cell culture medium during incubation, the super-resolution imaging revealed no significant differences in the average intracellular fluorescence intensity, suggesting that the intracellular concentrations of the four probes were remarkably consistent (Figure S11b↗). The subsequent results of mitochondrial dynamics imaging are presented in Figurea, where after capturing 50 frames of super-resolution images, MTR exhibited significant background signals resembling the endoplasmic reticulum (ER), which intensified with increasing frame count. This phenomenon is attributed to MTR's reliance on electrostatic adsorption for mitochondrial labeling, leading to off-target effects during long-term dynamic super-resolution imaging. This issue is further exacerbated by its inherent phototoxicity, which induces mitochondrial dysfunction. Normalized fluorescence intensity distribution along the line in Sec61β-mEGFP-expressing cells revealed that the off-target MTR accumulated on the ER due to its high lipophilicity. In comparison, PK Mito Red only showed faint background signals after capturing 200 frames, owing to its lower phototoxicity. However, as image acquisition continued, its accumulation in the ER progressively increased, similar to MTR. In striking contrast, Cy3-CA and HZ Mito Red exhibited a complete absence of discernible background signals even after acquiring 500 frames. Normalized fluorescence intensity distribution analyses in merged channels further affirmed the exceptional signal-to-noise ratio afforded by Cy3-CA and HZ Mito Red. These results indicate that both Cy3-CA and HZ Mito Red enable long-term dynamic mitochondrial super-resolution imaging, with labeling stability more than 10 times higher than that of MTR. Furthermore, to quantify photobleaching resistance, we statistically analyzed alterations in average fluorescence intensity during long-term dynamic imaging. As shown in Figureb, Cy3-CA exhibited the most rapid photobleaching kinetics. Conversely, HZ Mito Red and PK Mito Red displayed the lowest photobleaching rates, retaining over 60% of their fluorescence intensity after continuous acquisition over 500 frames. Intriguingly, the pronounced phototoxicity of MTR triggered cellular release of the probe, leading to an initial rapid increase in cellular brightness within the first 20 frames, followed by accelerated photobleaching. However, due to off-target effects, the retained fluorescence signal within the cells was not entirely from the mitochondria. Therefore, we further analyzed the changes in average fluorescence intensity specifically within the mitochondrial region, revealing comparable rapid photobleaching kinetics for Cy3-CA and MTR, with a 20% reduction in mitochondrial fluorescence intensity after approximately 70 and 50 frames, respectively. HZ Mito Red and PK Mito Red maintained 80% of their fluorescence intensity after acquiring nearly 250 frames (Figurec, d). Crucially, beyond 250 frames, HZ Mito Red demonstrated superior photostability compared to PK Mito Red. Additionally, the phototoxicity was further quantified by comparing the full width at half-maximum (fwhm) of the fluorescence intensity distribution across mitochondrial widths (Figure S12a, b↗). On average, HZ Mito Red and PK Mito Red were able to provide ∼400 frames of super-resolution imaging before the mitochondrial width expanded to 125% of its original size, whereas Cy3-CA and MTR could only provide ∼200 frames and 80 frames, respectively, under the same conditions (Figuree). These comprehensive results unequivocally establish that HZ Mito Red can robustly label mitochondria during protracted dynamic imaging, showcasing exceptional photobleaching resistance and minimal phototoxicity. Furthermore, HZ Mito Red demonstrates broad compatibility, enabling mitochondrial labeling across multiple cell lines (Figure S13↗). In summary, HZ Mito Red stands out as a superior probe for stable mitochondrial labeling in long-term dynamic imaging applications.

Ferroptosis, apoptosis, and autophagy represent distinct modes of programmed cell death, wherein mitochondria assume a pivotal regulatory role.− Long-term dynamic monitoring of mitochondrial behavior is essential for gaining deeper insights into the mechanisms of cell death., However, the loss of MMP, coupled with the fact that these processes often span tens of minutes to several hours, makes long-term dynamic imaging of mitochondria highly challenging. Herein, we employed HZ Mito Red to pioneer long-term super-resolution imaging of mitochondrial dynamics during ferroptosis, apoptosis, and autophagy, which were induced using Erastin, Oligomycin A (OA), and CCCP, respectively (Figurea). As a control, PK Mito Red was initially used to track mitochondrial dynamics during autophagy using SIM, acquiring super-resolution images every 1 min. As shown in Figureb, a significant increase in background signal was observed by 20 min, and the imaging signal-to-noise ratio gradually decreased as autophagy progressed, eventually resulting in loss of mitochondrial localization (Figurec). In contrast, cells labeled with HZ Mito Red achieved high signal-to-noise ratio dynamic tracking of mitochondria during autophagy, apoptosis, and ferroptosis under the same imaging conditions. As shown in Figured, no discernible changes in mitochondrial morphology were detected within the initial 20 min of autophagy. Between 20 and 140 min, mitochondria exhibited significant swelling and sustained contraction. Additionally, during apoptosis, mitochondria gradually fragmented and contracted within 30–70 min post-OA induction, culminating in spherical structures (Figuref). Analogously, mitochondria maintained their normal morphology in the first 30 min of ferroptosis (Figureh). Subsequently, from 30 to 180 min, the mitochondria divided into shorter fragments and gradually underwent vacuolization. Long-term dynamic imaging provides a detailed temporal profile of mitochondrial morphological alterations across diverse death models, a capability that surpasses the limitations of traditional single-frame fluorescence imaging. This underscores that long-term dynamic imaging offers more comprehensive and nuanced insights into the intricate processes of mitochondrial structural remodeling. Moreover, analysis of the fluorescence intensity distribution along the line at 140 min (autophagy), 70 min (apoptosis), and 180 min (ferroptosis) confirmed the absence of significant off-target signal for HZ Mito Red, thereby demonstrating exceptional imaging signal-to-noise ratios (Figuree, g, and i). Furthermore, to preclude the possibility that alterations in mitochondrial morphology were attributable to phototoxicity during imaging, control super-resolution imaging of unstimulated cells was performed under identical conditions, revealing sustained mitochondrial morphology over a 3 h period (Figure S14↗). In summary, these results demonstrate that HZ Mito Red is not limited by MMP and enables high signal-to-noise ratio, long-term dynamic imaging of mitochondria during ferroptosis, apoptosis, and autophagy, providing more detailed information on mitochondrial morphological dynamics.

Finally, mitochondrial morphology was quantitatively analyzed across these three cellular demise processes using the established length-to-width ratio (L/W) methodology, classifying mitochondrial shapes into four categories: round (1.0 < L/W < 1.5), intermediate (1.5 < L/W < 2.0), tubular (2.0 < L/W < 5.0), and hyperfused (L/W > 5.0) (Figureg). Automated image analysis using ImageJ software was employed to statistically evaluate mitochondrial morphology. During autophagy, the proportion of elongated mitochondria decreased, while round, intermediate, tubular, and hyperfused mitochondrial morphologies became more uniformly distributed (Figurek). In apoptosis, mitochondria underwent progressive shortening, with over 90% adopting a spherical morphology by the late stage (Figurel). During ferroptosis, mitochondria exhibited a transition toward shorter and rounder forms, with significant shifts in the proportions of round, tubular, and hyperfused morphologies relative to preferroptosis conditions (Figurem).

Finally, to facilitate multicolor mitochondrial imaging, HZ Mito Deep Red was synthesized by extending the π-conjugated system of HZ Mito Red. Photophysical characterization of HZ Mito Deep Red in DMSO revealed maximum excitation and emission wavelengths at 655 and 690 nm, respectively, yielding a Stokes shift of 35 nm (Figurea, Figure S15↗). Additionally, the solubility of HZ Mito Red and HZ Mito Deep Red in PBS buffer (pH 7.2) was further determined to be 3.4 and 2.0 mg/mL, respectively. HZ Mito Deep Red was then applied for mitochondrial labeling in live cells. Surprisingly, at a concentration of 1 μM, the labeling brightness of HZ Mito Deep Red was comparable to that of 10 μM HZ Mito Red (Figure S16a, b↗). This exceptional brightness is attributed to the intrinsic fluorescence properties of HZ Mito Deep Red and its expanded π-conjugated backbone, which mitigates the influence of the chloroacetamide group on the probe's hydrophobicity. Moreover, HZ Mito Deep Red demonstrated negligible cytotoxicity at working concentrations (Figure S17↗). Next, the mitochondrial targeting specificity of HZ Mito Deep Red in live cells was investigated (Figureb). Colocalization assays with the commercial Mito Tracker Green (MTG) affirmed a high degree of overlap between HZ Mito Deep Red and MTG fluorescence, evidenced by a PCC of 0.92. Subsequently, HZ Mito Deep Red was used to label mitochondria in cells transfected with the Cox4-EGFP plasmid. After fixation and disruption of MMP, HZ Mito Deep Red and EGFP fluorescence still exhibited strong overlap, with a PCC of 0.88 (Figurec). These results demonstrate that HZ Mito Deep Red can specifically and covalently label mitochondria while maintaining excellent labeling stability. Further application of HZ Mito Deep Red to long-term dynamic imaging of mitochondria revealed its superior performance. Upon acquiring 500 consecutive frames, HZ Mito Deep Red exhibited a complete absence of discernible background signal (Figured). In contrast, MTDR displayed a substantial increase in background signal after a mere 100 frames. Normalized fluorescence intensity distribution analysis along the line also indicated that HZ Mito Deep Red provided superior imaging signal-to-noise ratio after capturing 500 frames (Figuree). Furthermore, quantitative analysis of fluorescence intensity alterations within mitochondrial regions revealed the enhanced photostability of HZ Mito Deep Red, demonstrating superior retention of mitochondrial fluorescence throughout 500 consecutive frames compared to MTDR (Figuref). To assess the phototoxicity of HZ Mito Deep Red, alterations in mitochondrial width during imaging were evaluated. Mitochondria stained with HZ Mito Deep Red maintained their native morphology for up to 300 frames, whereas MTDR-stained mitochondria exhibited swelling within the initial 20 frames (Figureg). On average, HZ Mito Deep Red allowed continuous imaging of 491 frames before mitochondrial width increased to 120% of the original, whereas MTDR provided only 40 frames under the same conditions, indicating the low phototoxicity of HZ Mito Deep Red (Figureh). Finally, the labeling efficacy of HZ Mito Deep Red across diverse cell lines was evaluated. As shown in Figurei, PCC between HZ Mito Deep Red and MTR fluorescence consistently exceeded 0.89 in COS-7 cells, BHK21 cells, cardiomyocytes cells and A2780 cells, demonstrating the broad applicability of HZ Mito Deep Red for specific mitochondrial labeling across a range of cell lines. In conclusion, HZ Mito Deep Red emerges as a powerful tool for specific, stable, and low-phototoxic mitochondrial labeling, particularly advantageous for multicolor and long-term dynamic imaging applications.

Immunofluorescence staining is a ubiquitous technique for fluorescently tagging target proteins, typically entailing chemical fixation of samples followed by incubation with fluorophore-conjugated antibodies or toxins for specific labeling.− Given the robust mitochondrial labeling proficiency of HZ Mito Red and HZ Mito Deep Red in fixed cells, we proceeded to investigate their compatibility with immunofluorescence staining. Cells prelabeled with HZ Mito Red or HZ Mito Deep Red were fixed with GA, permeabilized with Triton X-100, and subsequently immunostained with antibodies targeting diverse proteins or protein-binding toxins (Figurea). In fixed U-2 OS cells, immunostaining with Anti-Vimentin-Alexa488 visualized both mitochondria and intermediate filaments (IF) (Figureb). Additionally, three-color fluorescence labeling of nucleus, mitochondria, IF, actin and tubulin was achieved across multiple cell lines, including BHK21 cells, cardiomyocytes cells and COS-7 cells (Figurec–h). This demonstrates that the combination of HZ Mito Red or HZ Mito Deep Red with immunofluorescence staining enables comprehensive mapping of spatial relationships between mitochondria and a diverse array of subcellular architectures across various cell types.

Reagents were purchased of the highest commercial quality and used without further purification unless otherwise stated. Unless otherwise stated, all reactions were carried out in a nitrogen atmosphere with dry solvents under anhydrous conditions. Nuclear magnetic resonance (NMR) spectra (¹H NMR and ¹³C NMR) were obtained on a 400 or 600 MHz spectrometer (Bruker, Switzerland). High-resolution mass spectrometric data were obtained using an 1100 LC/MSD Trap 2D liquid chromatography-ion trap mass spectrometer (LC-MS) (Agilent, USA). UV–vis absorption spectra of sample solutions in spectral grade solvents were measured using an Agilent Cary 60 UV–vis spectrophotometer in a 1 cm square quartz cuvette. Emission spectra were measured using an Agilent Cary Eclipse fluorescence spectrophotometer. The CellTiter-Lumi assay was obtained on a VictorX4 (PerkinElmer, USA). The living-cell confocal imaging was performed using a spinning disk confocal microscope (Olympus) equipped with an UltraVIEW VoX 3D live-cell imaging system (PerkinElmer), sCMOS camera, and four-channel excitation lasers (405, 488, 561, and 640 nm). SIM imaging was performed with a high-sensitivity structured illumination microscope (His-SIM, Guangzhou Computational Super-Resolution Biotech Co., Ltd.). All the super-resolution images were reconstructed from 9 raw images. Fiji software was used to analyze the confocal and super-resolution images.

The structures of Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, Cy3-COT-EP and HZ Mito Deep Red are illustrated in Scheme. S1–S4↗. The Supporting Information↗ provide detailed information on the synthetic routes and methods of compounds, as well as the NMR spectroscopy and mass spectrometry.

Stock solutions of Cy3, Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, Cy3-COT-EP and HZ Mito Deep Red (1 mM, 10 mL) were individually prepared in DMSO. A 20 μL of the stock solution was taken and diluted with PBS buffer (pH 7.2) to prepare a 10 μM test solution. The UV absorption spectrum of the probe was measured from 400 to 650 nm using an Agilent Cary 60 UV–vis spectrophotometer, while the fluorescence emission spectrum, excited at 520 nm and recorded from 540 to 680 nm, was obtained using an Agilent Cary Eclipse fluorescence spectrophotometer.

The fluorescence quantum yield (Φ_f) of the sample was calculated using the following formula, with Rhodamine B in PSB (Φ = 0.31) as the reference:ΦsampleΦref=ODref×Isample×d2sampleODsample×Iref×d2ref

Φ: quantum yield of fluorescence.

I: integrated emission intensity.

OD: optical density at the excitation wavelength.

d: refractive index of solvents: d_PBS = 1.336.

The molar extinction coefficients of Cy3, Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, Cy3-COT-EP and HZ Mito Deep Red in DMSO were measured using an Agilent Cary 60 UV–vis spectrophotometer with a 1 cm quartz cuvette.

Human osteosarcoma cell line (U-2 OS) cells were cultured in McCoy's 5A medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and maintained at 37 °C and 5% CO₂ during culturing. The COS-7 cells, BHK21 cells, Cardiomyocyte cells and A2780 cells were kindly provided by AmyJet Scientific (Wuhan, China). We tested the cytotoxicity of Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, Cy3-COT-EP and HZ Mito Deep Red using CellTiter-Lumi assay. CellTiter-Lumi assay: U-2 OS cells were seeded into 96-well plates and incubated for 24 h at 37 °C with 5% CO₂. Following incubation, the cells were first rinsed with PBS, and then stained with various concentrations (1 ∼ 20 μM) of Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, Cy3-COT-EP, or HZ Mito Deep Red, respectively, for 30 min at 37 °C. After staining, the cells were washed twice with FBS-free culture medium. Subsequently, add 100 μL of the commercial reagent CellTiter-Lumi to each well. This reagent assesses cell viability by detecting ATP generated by metabolically active cells. Chemiluminescence was then measured using a multimode microplate reader (Victor X4). Cell viability was calculated using the following formula:

Cell viability (%) = (Luminescence value of test well/Average luminescence value of control well) × 100.

Incubate U-2 OS cells with different concentrations (0.5, 1, 2.5, 5, 10 μM) of Cy3-CA, HZ Mito Red, Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, and Cy3-COT-EP for 30 min, then perform imaging using a confocal microscope. The imaging parameters for the confocal microscope are set to 10× magnification, a laser power of 15 mW, and an exposure time of 100 ms.

Live Cells: U-2 OS cells were first incubated with 10 μM Cy3-CA, 10 μM HZ Mito Red, 10 μM Cy3-NHS, 10 μM Cy3-COT-NHS, 10 μM Cy3-Py, 10 μM Cy3-COT-Py, 2.5 μM Cy3-EP, and 2.5 μM Cy3-COT-EP for 30 min. After incubation, the cells were washed three times with PBS buffer (pH 7.2) and then stained with 0.5 μM MTDR for 10 min, followed by SIM imaging. For the live cell colocalization experiment with 1 μM HZ Mito Deep Red, the same procedure was followed, except MTDR was replaced with MTG.

Fixed Cells: U-2 OS cells transfected with the Cox4-EGFP plasmid were incubated with 10 μM Cy3-CA, 10 μM HZ Mito Red, 10 μM Cy3-NHS, 10 μM Cy3-COT-NHS, 10 μM Cy3-Py, 10 μM Cy3-COT-Py, 2.5 μM Cy3-EP, and 2.5 μM Cy3-COT-EP for 30 min. Subsequently, the cells were fixed with 4% GAfor 10 min, washed three times with PBS, and imaged using SIM. For the fixed cell colocalization experiment with 1 μM HZ Mito Deep Red, the same procedure was used. The PCC was obtained using the colocalization function of ImageJ software.

The cells were first incubated with 0.5 μM MTR, 0.5 μM PK Mito Red, 10 μM Cy3-CA, or 10 μM HZ Mito Red for 30 min. After washing three times with PBS, the cells were imaged using SIM. The imaging conditions for the His-SIM microscope were set as follows: laser power at 5 mW, exposure time at 20 ms, and continuous acquisition without intervals.

The cells were first incubated with 10 μM HZ Mito Red for 30 min, followed by treatment with 10 μM Eastin, 10 μM OA or 10 μM CCCP to induce ferroptosis, apoptosis, and autophagy, respectively. Subsequently, the cells were imaged using SIM. The imaging conditions for the His-SIM microscope were set as follows: laser power at 5 mW, exposure time at 20 ms, and an interval time of 1 min.

The cells were first incubated with 10 μM HZ Mito Red or 1 μM HZ Mito Deep Red for 30 min, then fixed in 4% GA for 10 min. Permeabilization was performed with 0.2% Triton X-100 (10 min), followed by standard immunofluorescence staining and imaging.

The cells were first incubated with 0.5 μM MTDR or 10 μM HZ Mito Deep Red for 30 min. After washing three times with PBS, the cells were imaged using SIM. The imaging conditions for the His-SIM microscope were set as follows: laser power at 5 mW, exposure time at 20 ms, and continuous acquisition without intervals.

Statistical analyses and P-values were computed using GraphPad Prism 8. The statistical differences between experimental and control groups were assessed analyzed by t test analysis. For all data, values of P < 0.05 indicated statistical significance. The number of mitochondrial cristae was quantified using ImageJ.