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. 2025 Aug 18;4(1):105–112. doi: 10.1021/cbmi.5c00085

Silicon Rhodamine-Based Fluorescence Lifetime Probe for Dynamics Mapping Lysosomal Oxidative Stress

Qingshuang Xu ‡, Yiyan Zhang †, Yuxun Tang ‡, Senqiang Lv ‡, Lianfeng Su ‡, Pengfeng Mao ‡, Pengqi Liu †, Yutao Zhang †, Chenxu Yan †, Zhiqian Guo †,§,*
PMCID: PMC12848819  PMID: 41613762

Abstract

Lysosomes are organelles responsible for cellular degradation and recycling. The detection of changes in the lysosomal microenvironment, such as viscosity, oxidative stress, and pH value, as well as their interactions among dynamic organelles, remains an intriguing field that contributes to elucidating intracellular homeostasis. Here, we describe the development of a fluorescent probe tool for uniting fluorescence lifetime imaging microscopy (FLIM) and dual-channel near-infrared (NIR) fluorescence signals, which can simultaneously monitor viscosity and reactive oxygen species (ROS) in lysosomes. SiR-Eda exhibits a viscosity-dependent fluorescence lifetime and ROS-sensitive fluorescence emission, allowing for real-time tracking of lysosomal oxidative stress and viscosity within living cells. We demonstrate the utility of SiR-Eda in detecting changes in lysosomal viscosity and ROS in response to various stimuli including oxidative stress and lysosomal dysfunction. Our probe provides a convenient wash-free multifunctional tool for investigating lysosomal biology and has potential applications in the diagnosis and treatment of lysosome-related diseases.

Keywords: lysosomes, probe, fluorescence lifetime, viscosity, dual-mode imaging

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Introduction

Lysosomes are dynamic organelles that play a central role in cellular digestion and recycling. They are charged for the degradation and recycling of cellular waste materials, encompassing proteins, lipids, and organelles. , Viscosity is a critical parameter that affects lysosomal function, as it influences the diffusion of enzymes and substrates within the lysosomal lumen. When lysosomal dysfunction occurs, macromolecules fail to be degraded and accumulate excessively within the lysosome, ultimately resulting in significant changes in lysosomal viscosity. , Increasing evidence suggests that lysosomal degradation function is closely associated with oxidative stress. Lysosomal dysfunction has been implicated in various diseases, including lysosomal storage diseases, neurodegenerative disorders, and cancer. , Therefore, understanding the dynamic changes in lysosomal viscosity and redox state is essential for elucidating their functions in cellular physiology and pathology. Fluorescent probes are characterized by high sensitivity, strong capabilities for real-time monitoring, and a noninvasive nature, which have led to their widespread application in biomedical research and clinical diagnostics. , Particularly in complex biological environments, such as organelles, fluorescent probes can provide valuable information about the dynamic changes of biomolecules. − Currently, there is a lack of probes that can simultaneously detect viscosity and the redox state in lysosomes.

During biological processes, multiple molecular events often occur simultaneously. Also, changes in cellular viscosity and reactive oxygen species (ROS) levels are synchronized. In principle, the visual tracking of viscosity and ROS can be achieved by using two distinct probes for separate detection. However, the introduction of two probes not only leads to spectral overlap and cross-reactivity but also fails to accurately indicate the potential correlation between ROS and viscosity due to factors such as differing site distributions of the probes within the same cell. To our knowledge, there are relatively few probes capable of simultaneously detecting ROS and viscosity, particularly as no currently available probe can meet the requirements for dual-modal concurrently monitoring ROS and viscosity within specific organelles, such as lysosomes. , Current dual-analyte detection probes primarily rely on changes in fluorescence intensity (FI) at two different wavelengths. This method inevitably involves some spectral overlap, which can be easily interfered with by factors such as the probe concentration, leading to instability in the detection results. Combining the advancements in various current imaging modalities can enhance the sensitivity and spatial resolution of multifunctional probes, thereby obtaining more comprehensive and accurate information. , The fluorescence lifetime provides a quantitative, concentration-independent readout for fluorescence lifetime imaging microscopy (FLIM), enabling the tracking of the dynamics of the labeled species. − Combining fluorescence lifetime mode with fluorescence intensity mode can achieve more precise multianalyte detection, especially in complex biological environments. This prompted us to develop a single fluorescent probe (SiR-Eda) for imaging lysosomal viscosity and •OH via FLIM and FI, aiming to elucidate the biological characteristics of lysosomal dynamics and their functions in complex cellular environments.

Edaravone (Figure ) is a widely used free radical scavenger, which has been introduced into fluorophore for •OH detection, proving its feasibility for ROS detection in cell. − In this work, edaravone is introduced into the median of silicon rhodamine to obtain SiR-Eda, and it is found that it is sensitive to protons and can detect ROS in lysosomes (Figure A). Moreover, due to the spatial molecular conformation, SiR-Eda can also detect the viscosity in lysosomes. Herein, we describe the development of a fluorescent probe tool, SiR-Eda, which enables synchronous detection of viscosity and oxidative stress in lysosomes. Compared to conventional dual-analyte responsive multifunctional probes, our probe possesses wash-free subcellular localization capability and can perform dual-modal imaging, which provides a convenient and rapid tool for monitoring the microenvironment of subcellular organelles. It is important to note that we have demonstrated, using the single probe SiR-Eda, that local viscosity changes in lysosomes are regulated by fluctuations in lysosomal ROS. Furthermore, SiR-Eda enables real-time quantitative assessment of lysosomal viscosity changes in living cells via FLIM.

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Introduction of edaravone makes the molecule capable of ROS detection and viscosity detection. (A) The proposed sensing mechanism of probe SiR-Eda toward •OH.

Results and Discussion

Design and Synthesis of SiR-Eda and SiR-Pyr

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is an efficient antioxidant, and it was approved worldwide for the therapy of cerebral infarction and amyotrophic lateral sclerosis (ALS). , Previous studies indicate that edaravone can be an excellent responsive group for •OH when hybridized with fluorophore (such as coumarin and rhodamine). , However, their emission spectrum falls within the visible light window (<600 nm), which limits their application in the biomedical field. Silicon-substituted rhodamines exhibit outstanding optical properties, including the ability to be excited and emit at near-infrared wavelengths, excellent photostability, and good water solubility. Moreover, they tend to accumulate in lysosomes. − Previous work in our lab suggests that Si-rhodamine dyes can be employed as analyte-sensitive dyes via changing the electron properties of substitution at the meso-position. We propose the hypothesis that the conjugation of edaravone with a silicon-substituted xanthene dye at the meso-position would generate ROS-sensitive indicators with peak excitation above 650 nm and lysosome targeting ability. Furthermore, it has been reported in the literature that changes in molecular spatial conformation can be utilized to design viscosity fluorescent probes. , We speculate that introducing edaravone into rhodamine might result in a viscosity response due to spatial effects such as the rotation of the benzene ring and the V-shaped conformation.

First, through nucleophilic substitution at the meso-position of Cl-rhodamines, we obtained SiR-Eda and SiR-Pyr, respectively. SiR-Eda can be smoothly synthesized in one step by reacting Cl-rhodamines with edaravone (Scheme S1), and their structures were comprehensively confirmed through various analytical methods. Fortunately, we obtained single crystals of SiR-Eda (CCDC:2223367) by slow evaporation from dichloromethane (Figures A and S1, Table S1), which unambiguously indicated the structure of the SiR-Eda. Similarly, we have also obtained single-crystal structural characterization of SiR-Pyr (CCDC:2223377), which confirms the correctness of its structure (Figures B and S1, Table S2). However, due to the presence of an additional benzene ring, the edaravone group is more electron-donating than the pyrazolone group, resulting in a V-shaped conformation of the rhodamine parent structure in SiR-Eda.

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Fluorescence properties of SiR-Eda and SiR-Pyr. Chemical structures and single-crystal structures of (A) SiR-Eda and (B) SiR-Pyr. Note: solvent molecules have been omitted for clarity. Fluorescence intensity at 680 nm as a function of pH for SiR-Eda (C) and SiR-Pyr (G). Fluorescence spectra of SiR-Eda (D) and SiR-Pyr (H) in a mixture of ethanol and glycerol, where the volume fraction of glycerol varies. Fluorescence lifetime spectra at 680 nm of SiR-Eda (E) and SiR-Pyr (I) in a mixture of ethanol and glycerol were obtained, where the volume fraction of glycerol varies. Fluorescence spectra of SiR-Eda (F) and SiR-Pyr (J) for •OH (λex 460 nm) in PBS/MeOH (9:1, v/v, pH = 5) system.

Optical Properties of SiR-Eda and SiR-Pyr In Vitro

We carried out the experiments with excitation and emission spectra of SiR-Eda and SiR-Pyr (Figures and S2–S3, Table S3). SiR-Eda and SiR-Pyr’s fluorescence intensity is highly sensitive to pH. SiR-Eda and SiR-Pyr exhibited an absorption response (λab 670 nm, Figure S4) and an obvious enhanced NIR fluorescence (λem 690 nm, Figure S4) at pH values ranging from 3.0 to 7.0, with pK a values of 4.82 and 6.66 (Figure C,G), respectively, which is in good accordance with the pK a value of the lysosomal microenvironment (4.0–5.5). It is hypothesized that the pK a of SiR-Pyr is higher than that of SiR-Eda, because upon protonation, the five-membered ring of SiR-Pyr attains a more stable state with fully delocalized electrons. This enhanced stability facilitates the protonation process of SiR-Pyr, thereby resulting in a higher pK a value. Furthermore, we found that SiR-Eda exhibits good chemical stability under acidic, neutral, alkaline, or multiple acid–base cycling conditions (Figure S5). Therefore, only the SiR-Eda and SiR-Pyr molecules localized in lysosomes exhibit fluorescence.

Subsequently, NIR fluorescence responses of SiR-Eda and SiR-Pyr to viscosity in the ethanol-glycerol system were investigated (Figures D,H and S6). In low-viscosity ethanol, SiR-Eda exhibits a weak absorption peak at approximately 620 nm. As the viscosity of the solvent medium increases, the absorption peak of SiR-Eda at 620 nm significantly increases and undergoes a red shift to 650 nm. Notably, under excitation at 650 nm, the fluorescence emission of SiR-Eda at 688 nm is enhanced 114-fold as the viscosity of the medium increases (Figure D). The above experimental results show that the SiR-Eda exhibits a good fluorescence intensity response to viscosity.

Inspired by the sharp enhancement of emission at 688 nm induced by viscosity, we speculate that SiR-Eda may exhibit changes in the fluorescence lifetime in the ethanol-glycerol mixture. As illustrated in Figure E, with an increase in the proportion of glycerol in the solvent mixture, the fluorescence lifetime (τ) of SiR-Eda gradually lengthens. While the media was changed from EtOH to glycerol, the fluorescence lifetime of SiR-Eda expanded from 0.572 to 3.058 ns. Specifically, the fluorescence lifetime of SiR-Eda at 680 nm indicated a strong linear correlation with the viscosity of the medium. The linear equation was Vis = 798.57τ – 454.23 with a linear coefficient of 0.991 (Figure S7). Further investigation was conducted on the fluorescence lifetime decay of SiR-Eda under varying pH conditions. As shown in Figure S7, the average fluorescence lifetime remained around 1.36 ns, indicating that the fluorescence lifetime of SiR-Eda did not exhibit significant changes across different pH environments. These results indicate that the fluorescence lifetime of SiR-Eda is solely sensitive to viscosity and is independent of pH changes. Therefore, the viscosity lifetime curve shown in Figure S7 can be used as a calibration curve for FLIM-based viscosity quantification. The speculated fluorescence lifetime mechanism of SiR-Eda is as follows: on the one hand, SiR-Eda has a benzene ring that can act as molecular rotors; on the other hand, due to the introduction of an electron-donating benzene ring, SiR-Eda has a V-shaped molecular configuration. These molecular properties endow SiR-Eda with a viscosity-responsive fluorescence lifetime. However, SiR-Pyr is barely affected toward viscosity changes in either fluorescence intensity mode or FLIM mode (Figure H,I). As such, compared with SiR-Pyr, SiR-Eda exhibits advantages such as high sensitivity, which make it a favorable choice for viscosity assessment in intricate biological environments.

Since edaravone is a widely used free radical scavenger, we studied the response performance of SiR-Eda to •OH. SiR-Eda exhibits high selectivity and high sensitivity for ROS (Figures S8–S9). SiR-Eda in free form displays an absorption band centered at 660 nm with almost no fluorescence at 520 nm in PBS/MeOH (9:1, v/v) solution at pH 5.0. Upon the addition of •OH, the absorption spectrum of SiR-Eda exhibited a noticeable change, with the color shifting from blue to yellow. Specifically, a sharp decrease in the absorption peak at 660 nm was observed, accompanied by an increase in a band centered at 420 nm, with a distinct isosbestic point near 460 nm (Figure S9). Concomitantly, upon excitation at the isosbestic point of 460 nm, a remarkable decrease in the emission spectrum at 680 nm was noted coupled with a sharp rise in the emission spectrum at 520 nm (Figures F and S9). Intracellularly, the pH and viscosity within lysosomes are not constant; thus, the fluorescence intensity at 680 nm is influenced by the lysosomal environment and cannot be used as a quantitative reference in cells. We plotted the fluorescence at 520 nm against •OH concentration and also found a good linear correlation (Figure S10), indicating that SiR-Eda would be a potential tool to monitor endogenous •OH in live cells (Figures S11–S12). To our satisfaction, analogous emission characteristics and noninterference responses were also observed in SiR-Pyr toward •OH (Figures J and S9–S10). All the above results showed that both fluorescence emission and lifetime changes of SiR-Eda in response to •OH and viscosity variations offer a possibility for in situ tracking of ROS and lysosomal viscosity change in living cells.

Dynamic Lysosomal Viscosity through Lifetime Imaging at the Subcellular Level

Inspired by the photophysical properties of SiR-Eda, we subsequently studied the applicability of fluorescence lifetime imaging to viscosity changes within lysosomes. Before imaging experiments were conducted, the biocompatibility of SiR-Eda was assessed. According to the MTT assay performed on HeLa cell lines, SiR-Eda demonstrated no significant cytotoxicity, maintaining cell viability above 88%, even at concentrations as high as 128 μM after 24 h of incubation (Figure S13). As anticipated, SiR-Eda exhibited the capability to image HeLa cells within the red emission spectrum of 660–720 nm under excitation at 640 nm. To elucidate the subcellular localization of SiR-Eda within fluorescence imaging, we performed costaining of HeLa cells with SiR-Eda and a panel of commercially available organelle-specific dyes, including Lysosome Tracker Green, Mitochondria Tracker Green, and Endoplasmic Reticulum Tracker Green. These costained cells were then subjected to continuous colocalization imaging. As shown in Figure , SiR-Eda accumulated in HeLa cell lysosomes, exhibiting red dot fluorescence that showed excellent colocalization with lysosomal green fluorescence, as evidenced by a Pearson’s correlation coefficient of 0.90. However, Pearson’s colocalization coefficients with mitochondria and endoplasmic reticulum were determined to be 0.48 and 0.62, respectively. The specific targeting of SiR-Eda to lysosomes can be attributed to its acidic pH response characteristic. Similarly, due to its acidic response, SiR-Pyr can also be specifically localized in lysosomes for imaging (Figure S14). The above colocalization investigation confirmed that SiR-Eda and SiR-Pyr can stain lysosomes specifically.

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Subcellular organelle targeting in HeLa cells by SiR-Eda. The colocalization images of HeLa cell co-incubated with SiR-Eda for the red channel (λex 640 nm, λem 660–720 nm), while the associated organelle-targeting dyes for the green channel (ER-Tracker Green (λex 504 nm, λem 510–570 nm) (A); MitoTracker Green (λex 490 nm, λem 500–570 nm) (B) and LysoTracker Green (λex 504 nm, λem 510–570 nm) (C)).

SiR-Eda demonstrates utility in the real-time monitoring of dynamic changes in the lysosomal viscosity. It has been documented that the local viscosity within lysosomes increases subsequent to treatment with dexamethasone. The feasibility of employing SiR-Eda for detecting dynamic viscosity changes within lysosomes under dexamethasone treatment was systematically evaluated. RAW 264.7 cells were stained with SiR-Eda, followed by incubation with dexamethasone. In order to observe the dynamic changes of cells in situ from a fixed perspective, a catheter was inserted into the cell dish as an indwelling needle to inject SiR-Eda and dexamethasone into a cell culture dish (Figure A,B). Three minutes after injection of the probe, fluorescence at 680 nm was observed (Figure C), which proved that the probe had good biocompatibility, fast pH response, and wash-free characteristic. Six minutes after probe injection, the fluorescence intensity decreased significantly, accompanied by a notable extension in fluorescence lifetime (from 1.82 to 2.46 ns, Figure C), which is speculated to be due to a large number of probes entering the lysosomes, leading to an increase in pH and viscosity. At 12 min, the probe reached equilibrium within the lysosomes, with the fluorescence lifetime decreasing from 2.46 ns back to 1.59 ns (Figure C). After 15 min, dexamethasone was applied to the cell culture dish, and a gradual increase in the fluorescence lifetime of the cells was observed, rising from 1.59 to 1.92 ns (Figure C,D). The above experiments demonstrate that SiR-Eda enables real-time imaging of dynamic lysosomal viscosity changes under dexamethasone stimulation. The observations indicate that the application of SiR-Eda in FLIM for mapping lysosomal viscosity changes in live cells represents a valuable approach in lysosome-related pharmacological studies.

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FLIM investigation of viscosity changes in the RAW 264.7 cell by SiR-Eda. In situ real-time equipment photo (A) and schematic drawing (B). (C) Detection of lysosomal viscosity dynamics via FLIM imaging using SiR-Eda (10 μM) in RAW 264.7 cell treated with 5 μM dexamethasone. (D) FLIM output of (C).

NIR Fluorescence Imaging of Oxidative Stress Variations

To explore whether SiR-Eda could work well on ROS detection in living systems, we utilized SiR-Eda to visualize ROS in living RAW 264.7 cells. Previous studies suggest that high concentrations of LPS can cause oxidative stress. Therefore, RAW 264.7 cells were pretreated with LPS to initiate oxidative stress. Confocal images of those cells (Figure A) showed much brighter fluorescence in 520 nm than the cells without treatment. At the same time, the fluorescence lifetime in 680 nm LPS-treated cells also increased significantly. To further confirm the increase in fluorescence in 520 nm upon the capture of ROS by SiR-Eda, we used the H2O2 generating ROS. Strong fluorescence in 520 nm was observed in the cells treated with H2O2 (Figure A), with the fluorescence lifetime increased to 2.13 ns. The analysis of fluorescence intensity I 520nm and fluorescence lifetime in 680 nm of these three groups shows that with the gradual enhancement of oxidation pressure, fluorescence intensity I 520nm increases and fluorescence lifetime increases (Figure B). These results demonstrate that SiR-Eda can be used to sensitively identify intracellular ROS fluctuations.

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Fluorescence imaging and FLIM investigation in RAW 264.7 cells by SiR-Eda. (A) Dual-channel fluorescence imaging and fluorescent lifetime imaging of SiR-Eda in cells (λex 460 nm) under different oxidative stress conditions. (B) Normalized fluorescence intensity I 520 nm and FLIM output of (A).

Real-Time Dual-Modal FLIM and NIR Imaging of Lysosomal Viscosity Increase Induced by ROS Accumulation within Lysosomes

Motivated by the intracellular FLIM and fluorescence responses, we further investigated the integration of dual-modal FLIM and fluorescence imaging for the in situ evaluation of lysosomal state changes in living cells. Since the fluorescence lifetime is independent of probe concentration, the fluorescence lifetime at 680 nm of the remaining probe after ROS consumption can still indicate the viscosity level at that location. To investigate whether lysosomal viscosity dynamics are regulated by fluctuations in lysosomal ROS, we simultaneously imaged ROS and lysosomal viscosity by applying SiR-Eda under ROS oxidation via FI and FLIM. In this experiment, RAW 264.7 cells were pretreated with probe SiR-Eda and then incubated with H2O2. As shown in Figure , upon the addition of H2O2, both the green fluorescence and the lifetime of SiR-Eda were increased in the RAW 264.7 cell, which illustrated that the addition of H2O2 prompted a rise in the intracellular lysosomal viscosity. Therefore, the aforementioned results elucidate that excessive ROS within lysosomes can modulate lysosomal viscosity changes, a critical factor contributing to lysosomal viscosity fluctuations.

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In situ fluorescence imaging and fluorescence lifetime imaging during cellular oxidation process in RAW 264.7 cells by SiR-Eda. (A) Detection of lysosomal oxidation levels (520 nm) and viscosity dynamics (FLIM) by SiR-Eda (10 μM) in 5 μM H2O2-treated RAW 264.7 cells. (B) Normalized fluorescence intensity I 520 nm and FLIM output of (A).

After the addition of H2O2, the fluorescence lifetime gradually increased over time, rising from an initial 1.35 to 1.94 ns. Applying the equation in Figure S7C, our results indicate that the average lysosomal viscosity increased from 624 to 1095 cP in RAW 264.7 cells under ROS oxidation. Our measurements appear to fall within a reasonable range consistent with previously reported values. The aforementioned experimental results indicate that our probe not only facilitates the elucidation of the relationship between ROS and viscosity changes within lysosomes through dual-modal imaging but also enables the real-time quantitative measurement of lysosomal viscosity. Therefore, SiR-Eda can be a convenient and efficient tool for monitoring the microenvironment of lysosomes.

Conclusions

In summary, we reported a novel lysosomal probe SiR-Eda that can simultaneously monitor ROS and lysosomal viscosity via NIR fluorescence and FLIM. SiR-Eda possesses acid-activated fluorescence at 680 nm, ROS-activated fluorescence at 520 nm, and a viscosity-dependent fluorescence lifetime at 680 nm. Importantly, we demonstrated that the local viscosity in the lysosome is regulated by its ROS fluctuations, and SiR-Eda achieves dynamic quantification of lysosomal viscosity variations through FLIM in live cells. For the first time, dual-modal imaging of lysosomal viscosity and oxidation levels was accomplished by using a single probe. Our study opens up exciting avenues for lysosomal probes to unravel complex lysosome-related processes using intracellular NIR fluorescence and FLIM dual-modal imaging.

Supplementary Material

im5c00085_si_001.pdf (1MB, pdf)

Acknowledgments

This work was supported by National Key Research and Development Program (2023YFA1802000), NSFC/China (22225805, 32121005, 32394001, and 22378122), Shanghai Science and Technology Innovation Action Plan (No. 23J21901600), and Shanghai Frontier Science Research Base of Optogenetic Techniques for Cell Metabolism (Shanghai Municipal Education Commission, grant 2021 Sci & Tech 03-28).

Glossary

Abbreviations

FLIM

fluorescence lifetime imaging microscopy

NIR

near-infrared

ROS

reactive oxygen species

FI

fluorescence intensity

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00085.

  • Experimental section; figures of photophysical properties; 1H NMR, 13C NMR, and HRMS spectra of the compounds; and tables of crystal data and photophysical data (PDF)

The manuscript was collaboratively written with contributions from all authors, and all authors have approved the final version of the manuscript.

The authors declare no competing financial interest.

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