Near-Infrared Probes Designed with Hemicyanine Fluorophores Featuring Rhodamine and 1,8-Naphthalic Derivatives for Viscosity and HSA Detection in Live Cells

Abstract

This paper presents the development of near-infrared (NIR) fluorescent probes, A and B, engineered from hemicyanine dyes with 1,8-naphthalic and rhodamine derivatives for optimized photophysical properties and precise mitochondrial targeting. Probes A and B exhibit absorption peaks at 737 nm and low fluorescence in phosphate-buffered saline (PBS) buffer. Notably, their fluorescence intensities, peaking at 684 (A) and 702 nm (B), increase significantly with viscosity, as demonstrated through glycerol-to-PBS ratio experiments. This increase is attributed to restricted rotational freedom in the fluorophore and its linkages to rhodamine or 1,8-naphthalic groups. Theoretical modeling suggests nonplanar configurations for both probes, with primary absorptions in the rhodamine and hemicyanine cores (A: 543; B: 536 nm), and additional transitions to 1,8-naphthalic (A: 478 nm) and rhodamine (B: 626 nm) groups. Probe A is also responsive to human serum albumin (HSA), a key biomarker, with fluorescence increasing in HeLa cells as HSA concentrations rise. In contrast, probe B shows no response to HSA, likely due to steric hindrance from its bulky rhodamine group, illustrating a selectivity difference between the probes. Probe B, however, excels in mitochondrial imaging, confirmed through cellular and in vivo studies. In HeLa cells, it tracked viscosity changes following treatment with monensin, nystatin, and lipopolysaccharide (LPS), with fluorescence increasing in a dose-dependent manner. In fruit flies, probe B effectively detected monensin-induced viscosity changes, demonstrating its stability and in vivo applicability. These findings highlight the versatility and sensitivity of probes A and B as tools in biological research, with potential applications in monitoring mitochondrial health, detecting biomarkers like HSA, and investigating mitochondrial dynamics in disease.

Graphical Abstract

INTRODUCTION

Mitochondria, the organelles responsible for cellular energy production, are indispensable for cellular metabolism, energy production, and regulation of apoptosis. These organelles convert nutrients into adenosine triphosphate (ATP) through oxidative phosphorylation and play vital roles in generating reactive oxygen species (ROS), calcium signaling, and regulating cell death pathways.^1–9 Given their central role, mitochondrial dysfunction is implicated in various illnesses, encompassing neurodegenerative diseases, cancer, and metabolic syndromes.^1–9 Consequently, understanding mitochondrial dynamics and properties in live cells is essential for both basic research and clinical applications.^1–9 Fluorescence microscopy has gained recognition as an effective tool for real-time observation of intracellular processes. Near-infrared (NIR) fluorescent probes offer significant advantages, such as reduced phototoxicity, enhanced tissue penetration, and minimal autofluorescence, making them ideal for live-cell imaging. Developing NIR probes that specifically target subcellular compartments like mitochondria enables detailed visualization and quantification of mitochondrial dynamics and functions.^10–16 Hemicyanine dyes have recently gained attention as promising candidates for NIR fluorescent probes due to their large quantum yields and wide Stokes shifts, tunable absorption and emission wavelengths, and excellent photostability and biocompatibility.^17–35 Such characteristics render them ideal for live-cell imaging and for creating probes optimized for a range of biological uses.^17–19 An essential aspect of mitochondrial health that can be assessed with these probes is mitochondrial viscosity. Viscosity within mitochondria reflects their physical state and functionality, impacting processes such as oxidative phosphorylation and permeability transitions. It is also closely linked to the respiratory and metabolic states within the organelle, with deviations in viscosity often observed in pathologies such as neurodegenerative diseases, diabetes, and atherosclerosis.

Beyond mitochondrial function, monitoring human serum albumin (HSA) levels in cells provides critical insights into systemic health. HSA, one of the most abundant proteins in the body, is vital for maintaining osmotic pressure, balancing nutrition, and transporting a wide range of drugs and metabolites. Normal HSA concentrations in blood serum range from 35 to 50 g/L, while levels in urine remain below 30 mg/L in healthy individuals due to kidney filtration. Abnormal HSA levels serve as reliable indicators of liver and kidney function, with elevated levels in urine (albuminuria) often signaling early kidney impairment, and reduced blood levels (hypoproteinemia) commonly linked to liver diseases such as cirrhosis and chronic hepatitis.³⁶ Consequently, there is a growing need for rapid, specific, and cost-effective tools to monitor HSA levels in body fluids, as fluctuations in HSA can reflect early stages of disease and guide preventive care. Therefore, developing multifunctional NIR probes that can simultaneously monitor mitochondrial viscosity and HSA levels in live cells would offer a significant advancement in the real-time assessment of cellular and mitochondrial health. Such probes could serve as invaluable tools for disease diagnosis, treatment evaluation, and clinical applications, paving the way for more comprehensive insights into mitochondrial and systemic functions.^{27–34,37–51}

In this manuscript, we describe the creation of innovative near-infrared (NIR) fluorescent probes, named probes A and B, which are derived from hemicyanine dyes with 1,8-naphthalic and rhodamine derivatives (Scheme 1). These probes were designed to enhance photophysical properties, including sensitivity to environmental factors such as viscosity, and to facilitate targeted cellular imaging. Probes A and B feature distinct absorption peaks at 684 and 741 nm, and 685 and 737 nm, respectively, and show minimal fluorescence in phosphate-buffered saline (PBS) buffer. Notably, their fluorescence intensity increases significantly with rising viscosity, demonstrated by modulating glycerol-to-PBS ratios in solution. This behavior is attributed to the restricted rotational freedom within the hemicyanine fluorophore and the constrained linkages between hemicyanine and 1,8-naphthalic or rhodamine groups, underscoring the probes’ sensitivity to microenvironmental viscosity. To expand their potential applications, we investigated probes A and B for their ability to detect human serum albumin (HSA), a protein vital for numerous physiological functions and an important marker for assessing liver and kidney health. Probe A displayed a progressive increase in fluorescence intensity at 731 nm with increasing HSA concentrations in PBS buffer (pH 7.4) under 680 nm excitation, highlighting its high sensitivity to HSA and suggesting its potential for real-time HSA monitoring in biological systems. The binding affinity between HSA and Probe A, as determined through molecular docking, is −11.3 kcal/mol. In contrast, probe B exhibited no fluorescence response within this concentration range, likely due to steric hindrance from its bulky rhodamine residue, which may impede effective hydrophobic interactions with HSA. Beyond HSA detection, probe B was applied in cellular and in vivo imaging to evaluate its effectiveness in sensing viscosity changes. In HeLa cells, probe B successfully detected viscosity changes induced by treatments with monensin, nystatin, and bacterial lipopolysaccharide (LPS). Monensin, known to disrupt ion gradients, induced a dose-dependent increase in fluorescence, signifying increased cellular viscosity. Similarly, nystatin and LPS treatments resulted in enhanced fluorescence, consistent with elevated viscosity. Probe B also demonstrated strong performance in live fruit fly models, where monensin treatment correlated with a measurable increase in fluorescence intensity, further validating probe B’s utility for in vivo applications. Additionally, probe A was tested for HSA detection in live cell contexts. Overall, probe B represents a significant advancement in studying mitochondrial viscosity and cellular dynamics. Its high selectivity, excellent photostability, and low cytotoxicity make it a valuable tool for investigating mitochondrial function and stress responses across cell culture and live organisms. This work highlights the potential of probes A and B for expanding our understanding of mitochondrial biology and their promising biomedical applications.

Scheme 1.

NIR Fluorescent probes Derived from Hemicyanine Dyes Featuring Rhodamine and 1,8-Naphthalic Derivatives for Viscosity and HSA Detection

EXPERIMENTAL SECTIONS

Synthesis of probes A and B.

Synthesis of probe A.

¹H NMR (500 MHz, CDCl₃), δ (ppm): 1.02 (3H, t, J = 7.5 Hz), 1.07 (3H, t, J = 7.5 Hz), 1.74 (6H, s), 1.78 (2H, m), 1.87 (2H, m), 1.95 (2H, t, J = 5.0 Hz), 2.66 (2H, t, J = 5.0 Hz), 2.77 (2H, t, J = 7.5 Hz), 3.85 (2H, t, J = 7.5 Hz), 4.17 (2H, t, J = 7.5 Hz), 5.76 (1H, d, J = 10.0 Hz), 6.92 (1H, d, J = 10.0 Hz), 7.12 (3H, m), 7.39 (1H, s), 7.63 (1H, t, J = 7.5 Hz), 7.73 (1H, d, J = 10.0 Hz), 8.16 (1H, d, J = 10.0 Hz), 8.24 (1H, d, J = 15.0 Hz), 8.59 (1H, d, J = 5.0 Hz), 8.63 (1H, d, J = 5.0 Hz). ¹³C NMR (125 MHz, CDCl₃) δ (ppm): 11.12, 11.53, 11.55, 11.74, 20.31, 20.33, 21.23, 21.41, 21.43, 24.47, 24.49, 28.21, 28.67, 41.84, 45.07, 48.06, 48.10, 99.99, 104.24, 104.28, 108.66, 108.74, 115.53, 115.66, 121.81, 122.24, 122.60, 126.38, 128.22, 128.27,128.43, 128.50, 130.50, 130.51, 130.69, 130.90, 131.02, 133.52, 133.55, 139.69, 144.27, 164.31, 164.47, 164.49. LCMS m/z = 649.50, calculated m/z = 649.31.

Synthesis of probe B.

¹H NMR (500 MHz, CDCl₃), δ (ppm): 1.0 (3H, t, J = 7.5 Hz), 1.15 (6H, t, J = 7.5 Hz), 1.70 (6H, s), 1.76 (2H, m), 1.86 (2H, t, J = 7.5 Hz), 2.56 (2H, t, J = 5.0 Hz), 2.70 (2H, t, J = 5.0 Hz), 3.29 (4H, t, J = 7.5 Hz), 3.73 (2H, t, J = 7.5 Hz), 5.79 (1H, d, J = 10.0 Hz), 6.36 (2H, d, J = 10.0 Hz), 6.59 (1H, d, J = 10.0 Hz), 6.74 (1H, d, J = 10.0 Hz), 6.90 (1H, d, J = 5.0 Hz), 7.13 (1H, t, J = 7.5 Hz), 7.19 (1H, d, J = 10.0 Hz), 7.29 (4H, m), 7.38 (1H, s), 7.59 (4H, m), 8.01 (1H, d, J = 10.0 Hz), 8.18 (1H, d, J = 10.0 Hz). ¹³C NMR (125 MHz, CDCl₃) δ (ppm): 11.62, 12.52, 12.55, 12.57, 20.36, 24.14, 24.49, 28.29, 28.52, 44.42, 44.45, 63.41, 84.24, 97.55, 104.26, 105.06, 108.20, 117.55, 117.57, 117.68, 122.34, 122.37, 124.30, 124.31, 124.74, 126.98, 127.47, 127.49, 128.23, 128.24, 128.67, 128.89, 128.90, 129.49, 134.90, 149.56, 151.30, 152.93, 153.25, 169.70. LCMS m/z = 781.50, calculated m/z = 781.36.

RESULTS AND DISCUSSION

Probe Synthetic Strategy.

Hemicyanine dyes are renowned for their exceptional photostability and biocompatibility, attributes that make them particularly well-suited for live-cell imaging applications. Their photophysical properties, including tunable absorption and emission wavelengths, can be finely adjusted to optimize performance for various biological applications.^17–34 To further enhance their sensitivity to viscosity changes, we designed and synthesized hemicyanine dyes incorporating 1,8-naphthalic and rhodamine moieties through a Suzuki-palladium-catalyzed reaction, aiming to create probes with improved viscosity sensitivity (probes A and B) (Scheme 2). The construction of probe A involved the introduction of an N-propyl-1,8-naphthalimide moiety into a hemicyanine dye. This was achieved through a two-step Suzuki-palladium-catalyzed process. Initially, we prepared 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-propyl-1,8-naphthalimide (5) by coupling 4-bromo-N-propyl-1,8-naphthalimide (3) with bis(pinacolato)diboron. This intermediate served as a key component for the subsequent reaction. In the second step, intermediate 5 was coupled with a bromo-functionalized hemicyanine dye (6) using the Suzuki-palladium-catalyzed reaction, resulting in probe A. This probe features a hemicyanine dye with an N-propyl-1,8-naphthalimide residue, designed to respond to changes in viscosity by altering its photophysical properties. Similarly, probe B was synthesized by incorporating a rhodamine moiety into the hemicyanine dye structure. We first synthesized 3′-(diethylamino)-6′-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one (10) through a Suzuki-palladium-catalyzed coupling reaction of 3′-(diethylamino)-6′-iodospiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one (9) with bis(pinacolato)diboron (Scheme 2). This intermediate was then reacted with a bromo-functionalized hemicyanine dye (6) via another Suzuki-palladium-catalyzed reaction, yielding probe B (Scheme 2). This probe incorporates a rhodamine moiety, designed to provide sensitivity to changes in viscosity by modulating its fluorescence properties. All intermediate and final probes were rigorously studied using nuclear magnetic resonance (NMR) and mass spectral analysis (Figures S1–S11). NMR spectra confirmed the molecular structures and purity of the intermediates and probes (Figures S1–S5 and S7–S10). Mass spectrometry provided additional verification of molecular weights and the integrity of the synthesized compounds (Figures S6 and S11). These probes were designed to enhance the sensitivity of hemicyanine dyes to viscosity changes, making them suitable for detailed studies of mitochondrial viscosity in live cells (Figures S12). The introduction of specific moieties through well-established synthetic methods ensures that the probes have optimized photophysical properties for precise and reliable measurement of viscosity changes, contributing valuable insights into mitochondrial dynamics and function.

Scheme 2.

Strategic Synthetic Route to Prepare Hemicyanine Dyes Featuring Rhodamine and 1,8-Naphthalic Derivatives for Mitochondrial Viscosity Detection (probes A and B)

Probe Optical Sensing of Viscosity.

To evaluate the fluorescence responsiveness of probes A and B to viscosity alterations, a series of experiments were performed using PBS buffer mixed with varying glycerol concentrations. Probes A and B were prepared at a 10 μM concentration in PBS buffer (pH 7.4). The viscosity of the solutions was systematically adjusted by incorporating glycerol in proportions ranging from 0 to 100%. In only PBS buffer, the probes displayed broad absorption peaks featuring two maxima, probes A (684, 741 nm) and B (685, 737 nm) (Figures 1 and 2). Under these solvent conditions, probes A and B exhibited minimal fluorescence, with weak emissions observed at 682 and 692 nm, respectively (Figures 1 and 2), As the glycerol concentration increased, notable shifts in absorbance and enhancements in fluorescence intensities were observed for both probes (Figures 1 and 2). A strong linear relationship is observed between log (I_max) and log (η) for probes A and B, with correlation coefficients of 0.9870 and 0.9857, respectively (Figure S12). This relationship supports their use for the quantitative determination of viscosity. The absorbance spectrum in 100% glycerol, probe A featured two peaks at 619 and 671 nm and probe B contained two defined peaks at 629 and 680 nm (Figure 1). Under these conditions, probe A exhibited a marked increase in fluorescence emission at 690 nm (740 nm, shoulder), while probe B demonstrated similar enhancement at 702 nm (747 nm, shoulder) (Figure 2). This shift in absorbance in the probes is attributed to aggregation effects in water with the broad peak and to isolated molecules in 100% glycerol, resulting in sharper better-defined absorbances. It is known that aggregation quenches fluorescence,⁵² thus in the buffer solution, the probes do not emit much. However, the enhancement in fluorescence could be attributed to the increased viscosity, which restricts the rotational freedom of the carbon–carbon bond connections between the hemicyanine fluorophore and the rhodamine or 1,8-naphthalic moieties. This restriction reduces nonradiative decay pathways and leads to increased fluorescence emissions. The data confirms that both probes A and B are highly sensitive to viscosity changes (Figures 1 and 2). Their fluorescence response to varying glycerol concentrations provides a reliable and measurable indicator of viscosity (Figure S12). This high sensitivity underscores the potential of these probes for applications in monitoring viscosity-related phenomena in biological systems. The capability to detect real-time viscosity changes makes probes A and B crucial aids in studying mitochondrial viscosity and other cellular processes where viscosity plays a critical role.

Figure 1.

Absorbance (left) and emission (right) profiles of 10 μM probe A in mixture containing pH 7.4 PBS buffer and glycerol in different proportions, captured with 600 nm excitation.

Figure 2.

Absorbance (left) and emission (right) profiles of 10 μM probe B in solutions containing pH 7.4 PBS buffer and glycerol in different proportions, captured with 600 nm excitation.

We further evaluated the ability of probes A and B to determine human serum albumin (HSA). Probe A presents a broad near-infrared absorption peak at 715 nm, a visible absorption peak at 352 nm, and a weak near-infrared fluorescence peak at 731 nm in the absence of HSA (Figure 3). Upon gradually increasing HSA concentrations from 0 μM to 80 μM in PBS buffer (pH 7.4), the emission intensity of probe A at 731 nm increases progressively with linear response up to 80 μM under 680 nm excitation (Figures 3 and S13). In contrast, probe B does not exhibit any fluorescence response to HSA within the same concentration range. This lack of response is likely due to the bulky rhodamine residue in probe B, which may hinder effective hydrophobic interactions with HSA.

Figure 3.

Absorbance (left) and emission (right) profiles of 10 μM probe A with and without different HSA concentrations in pH 7.4 phosphate buffers using 680 nm excitation.

Theoretical Calculations.

The procedures for carrying out these calculations are outlined in the Supporting Information and adhere to the methods described earlier.^53,54 Probe A is constituted of three sections, the central rhodamine section linked on the end to a 1,8-naphthalic derivative (i.e., 2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione) with an interplanar twist of 64.1° and at the other end coplanar with a dimethyl hemicyanine (Figure S20). In contrast, probe B consists of coplanar central rhodamine and hemicyanine sections but a more complex rhodamine (3′-(diethylamino)-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one) is attached at the other end at an interplanar angle of 44.0° (Figure S23).

Current density illustrations for the lowest energy calculated absorptions for the probes are detailed in Figure 4. Interestingly, the calculated absorption spectrum for probe A, Figure S21 consisted in part of a broad absorption at 543 nm with a very weak peak at 478 nm. The representation of the current density in Figure 4 together with the LCAOs for the transition (Figure S22) suggests that the main absorption is due to the movement of electron density essentially localized on the central rhodamine and the dimethyl hemicyanine moieties. The much weaker transition was due to a transfer of electron density from the rhodamine and dimethyl hemicyanine section to the 1,8-naphthalic derivative which, because these groups were not planar, resulted in the very weak oscillation factor of 0.0079, Table S2 and Figure S22.

Figure 4.

Schematic representations of disparities in current density for electronic transitions are illustrated as isosurfaces for probe A (left) and probe B (ES1, middle, ES2, right). The quantity of energized states (ES), including both the theoretically predicted and empirically determined wavelengths, as well as the transitions, are outlined along with their relative contribution values. The values related to the color grading at the top of the figure are also specified. To provide additional clarity, diagrams of the LCMOs marked with numerical identifiers are included in the Supporting Information.

By contrast, probe B consisted of a broad peak at 536 nm with a shoulder peak at 626 nm. The shoulder peak is due to the transfer of electron density from the coplanar regions consisting of the central rhodamine and the dimethyl hemicyanine moieties to the more complex rhodamine on the left of the illustration, Figures 4 and S24. The nature of the electronic transition of the broad peak at 536 is similar in flow to that for probe A described above suggesting that increasing viscosity may result in less rotation around the C–C atom single bond linkage with the central group and the entities to the left of both probes resulting in a more efficient transfer of electron density and increased fluorescence as displayed in Figures 1 and 2. The molecular docking analysis reveals a binding affinity of −11.3 kcal.mol between HSA and Probe A (Table S5 and Figures S26–S34).

Probe Selectivity, Photostability, pH Effect, and Cytotoxicity.

The high selectivity of the probes toward viscosity was rigorously tested against a wide array of common interfering substances, including cations, anions, amino acids, and biothiols. In a pH 7.4 PBS buffer, the emission intensity of probe A (10 μM) was measured upon excitation at 600 nm, while probe B (10 μM) was measured upon excitation at 650 nm. Probes A and B exhibit extremely weak emission at 786 and 788 nm, accordingly, and maintain stability without sensitivity to pH changes between 5.7 and 8.5 in PBS buffers (Figure S18). The selectivity assessment involved introducing various potentially interfering species at a concentration of 100 μM. These species included anions (SO₃²⁻, HCO₃²⁻, NO₃⁻, CN⁻, Cl⁻), cations (Mg²⁺, Fe²⁺, Co²⁺, Na⁺, K⁺), biothiols (glutathione (GSH)) and amino acids (DL-valine, methionine (Met), L-cysteine (Cys), DL-lysine). The results demonstrated that both probes A and B maintained a significant fluorescence response to viscosity changes with minimal interference from these substances (Figure S14). Additionally, glycerol (100% concentration) was used as a viscosity control to confirm the probes’ sensitivity to viscosity. These findings highlight the exceptional selectivity of probes A and B, making them well-suited for monitoring viscosity-related phenomena in biological systems. Similarly, probe A was tested to assess its selectivity for HSA in the presence of the corresponding ions (Figure S15).

The photostability of the probes was evaluated to evaluate their resilience under continuous light exposure, a critical attribute for reliable long-term imaging applications. Both probes were dissolved in glycerol solution at a concentration of 10 μM. The emission intensity of probe A was measured upon excitation at 600 nm, and probe B at 650 nm. The results, depicted in Figure S16, illustrate that both probes maintained 95% of their initial fluorescence intensity even after 2 h of continuous excitation. This high level of photostability ensures that probes A and B can deliver stable and accurate fluorescence signals during extended imaging sessions, making them exceptionally suitable for monitoring dynamic biological processes where prolonged light exposure is necessary. The robust photostability further underscores the utility of these probes in real-time, live-cell imaging applications, facilitating the investigation of viscosity-related changes in biological systems with minimal photobleaching concerns.

The cytotoxicity of probe B was quantified with the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay to ensure their biocompatibility for live-cell imaging applications.^8,22,55–63 HeLa cells were subjected to varying probe concentrations, with a particular focus on the 50 μM concentration (Figure S17). The cell viability was determined after 24 h of incubation. The analysis confirmed that more than 87% of HeLa cells remained viable at a 50 μM concentration of probe B, indicating low cytotoxicity. This high level of cell viability confirms that probe B is well-tolerated by live cells, making them suitable for long-term studies without compromising cell health. These findings support the potential of these probes for use in dynamic biological environments, where maintaining cell viability is crucial for accurate and reliable imaging. The MTT assay analysis underscores biological compatibility of probe B, further validating their application in live-cell imaging to monitor viscosity-related changes and other cellular processes.

Cellular Fluorescence of HeLa Cells.

Monensin acts as an ionophore, disrupting ion gradients across cellular membranes by facilitating the transport of sodium (Na⁺) and potassium (K⁺) ions.⁶⁴ This disruption leads to an imbalance in ion homeostasis, causing cellular stress and changes in the cytoplasmic environment.⁶⁴ One cellular response to this stress is an increase in intracellular viscosity, attributed to macromolecular aggregation, cytoskeletal reorganization, and altered organelle dynamics.^30,65 In this study, HeLa cells were exposed to monensin at concentrations of 0, 5, 10, and 20 μM (with 0 μM as control) in cell culture media for 30 min to induce changes in cellular viscosity. Following monensin treatment, the cells were cultured with probe B (5 μM) in growth medium for an additional 20 min to ensure sufficient probe uptake and fluorescence response. Images were recorded through fluorescence microscopy within the 680 to 780 nm wavelength range using a 630 nm excitation source to monitor fluorescence changes in response to different levels of monensin-induced viscosity variations (Figure 5). The fluorescence microscopy images revealed a substantial elevation of cellular fluorescence intensity when the monensin concentration increased (Figure 5). In the control sample (0 μM monensin), minimal fluorescence was observed, indicating a baseline viscosity with probe B. At 5 μM monensin, a noticeable increase in fluorescence was detected, suggesting a moderate rise in cellular viscosity. With 10 μM monensin, there was a further increase in fluorescence intensity, correlating with a more significant viscosity enhancement. The highest fluorescence intensity was observed at 20 μM monensin, indicating a substantial increase in cellular viscosity. The increased fluorescence intensity of probe B with rising monensin concentrations demonstrates the probe’s effectiveness in sensing these viscosity changes (Figure 5). Probe B’s fluorescence mechanism relies on its unique molecular structure, which includes a hemicyanine fluorophore and a rhodamine fluorophore linked by a single bond. As viscosity increases, the restricted rotational freedom of these single bonds within the hemicyanine and at the hemicyanine-rhodamine junction reduces nonfluorescent relaxation pathways, leading to enhanced fluorescence emission. This restriction reduces nonradiative decay pathways, resulting in enhanced fluorescence emissions. This property makes probe B an excellent tool for detecting and monitoring viscosity changes within live cells. The ability to monitor such changes provides valuable insights into cellular processes and conditions where viscosity plays a crucial role, such as drug interactions, disease states, and metabolic activities.

Figure 5.

Fluorescence images of HeLa cells exposed to increasing concentrations of Monensin (from 0 to 20 μM) for a duration of 30 min, Afterward, the cells were under exposure to probe B (5 μM) in Dulbecco’s modified eagle medium (DMEM) for 20 min. Fluorescence was detected in the 680–780 nm range, using a 630 nm excitation. The scale bar is equivalent to 50 μm.

To evaluate our hypothesis that probes A and B selectively target mitochondria because of their positive charge, which promotes electrostatic attraction to the negative charge across the mitochondrial membrane, we performed colocalization imaging studies. In these experiments, HeLa cells were pretreated with 5 μM Nystatin for 30 min to influence cellular conditions, subsequently incubated with either probe A (Figure S19) or probe B, along with the mitochondrial-selective dye MitoView 405. The findings revealed a considerable overlap between probe B and MitoView 405, yielding a high Pearson correlation coefficient of 0.966 (Figure 6). This high degree of colocalization confirms that probe B is efficiently targeted to mitochondria, supporting the proposed targeting mechanism based on electrostatic interactions. This strong correlation underscores the effectiveness of probe B’s mitochondrial targeting, suggesting it can reliably accumulate within the mitochondria of live cells without additional targeting groups. Such targeting is critical for studying mitochondrial dynamics and assessing changes related to mitochondrial function in real time. Given probe B’s high correlation with MitoView 405, it represents a promising tool for mitochondrial imaging applications, allowing researchers to track mitochondrial behavior and alterations under various conditions. This selective localization capability provides a robust foundation for exploring mitochondrial biology, including the investigation of dysfunctions and cellular stress responses in both physiological and pathological contexts.

Figure 6.

Emission images of HeLa cells with a 30 min treatment with 5 μM Nystatin, followed by a 20 min stimulation with 5 μM probe B and 5 μM MitoView 405. The blue fluorescence, gotten ranging from 425 to 475 nm upon 405 nm excitation, corresponds to MitoView 405, marking mitochondrial site. The red fluorescence, recorded from 680 to 780 nm with 630 nm excitation, represents probe B, indicating the intracellular distribution of the probe. Scale bars are 50 μm.

Nystatin, an antifungal agent, disrupts cellular membrane integrity by binding to sterols, particularly ergosterol, within the cell membrane.^66,67 This interaction disturbs the normal fluidity of the lipid bilayer, leading to significant alterations in membrane dynamics and organization.^66,67 As nystatin integrates into the membrane, it increases the lipid bilayer’s viscosity, resulting in a more viscous cellular environment. Furthermore, Nystatin-induced stress can trigger cellular responses that contribute to macromolecular aggregation and changes in the cytoskeleton, further elevating intracellular viscosity.⁶⁸ To investigate the effect of nystatin on cellular viscosity, we conducted experiments analogous to those performed with monensin. HeLa cells were treated with varying concentrations of nystatin for 30 min to induce changes in cellular viscosity. Following this treatment, cells were treated with probe B (5 μM) for 20 more minutes to allow for adequate probe uptake and fluorescence response (Figure 7). The fluorescence intensity of probe B was directly correlated with viscosity alterations. Our results demonstrated a progressive increase in fluorescence intensity corresponding to higher nystatin concentrations. In the control sample (0 μM nystatin), fluorescence was minimal, reflecting baseline cellular viscosity (Figure 7). Treatment with 5 μM nystatin led to a noticeable emission increase, indicating a moderate rise in viscosity. The fluorescence intensity further increased at 10 μM and peaked at 20 μM nystatin, showing a significant elevation in cellular viscosity (Figure 7). The enhanced fluorescence of probe B in response to Nystatin treatment reflects these viscosity changes. In summary, nystatin induces increases in cellular viscosity similar to monensin. These findings deliver essential insights into how agents like Nystatin influence cellular viscosity and highlight the potential of viscosity-sensitive probes for studying cellular responses to diverse compounds.

Figure 7.

Fluorescence images of HeLa cells were exposed to increasing concentrations of Nystatin for 30 min, then, treated with probe B (5 μM) in DMEM for a further 20 min. Emission was detected within the 680–780 nm range using a 630 nm excitation wavelength. Scale bars indicate 50 μm.

Bacterial-derived lipopolysaccharide (LPS) was employed to investigate its role in cellular viscosity changes in HeLa cells, serving as an alternative to nystatin. LPS, a vital element of the Gram-negative bacterial outer membrane,^69,70 is known to trigger potent immune responses and induce cellular stress through the activation of Toll-like receptor 4 (TLR4).^71,72 This activation leads to inflammatory pathways that affect various cellular processes, including membrane fluidity and intracellular viscosity. HeLa cells were exposed to different concentrations of LPS for 30 min to induce changes in cellular viscosity. Following this treatment, the cells were incubated with probe B (5 μM) for an extra 20 min to allow sufficient probe uptake and fluorescence response. The microscopy images revealed a marked increase in emission intensity correlating with higher concentrations of LPS (Figure 8). The control sample (0 μg/mL LPS) exhibited minimal fluorescence, indicating baseline cellular viscosity. A noticeable increase in fluorescence was observed at 5 μg/mL LPS, suggesting a moderate increase in viscosity. Fluorescence intensity continued to rise with increasing LPS concentrations, peaking at 20 μg/mL and indicating a substantial increase in cellular viscosity. The findings with LPS treatment were consistent with those obtained using nystatin, confirming that LPS effectively increases cellular viscosity. LPS causes stress that affects membrane dynamics and the intracellular environment, leading to macromolecular aggregation and alterations in cytoskeletal structure, which in turn increase viscosity (Figure 8). Utilizing LPS in this experiment provided valuable insights into how inflammatory stimuli influences cellular viscosity. The consistent fluorescence responses across different viscosity-inducing agents, such as LPS and nystatin, validate the efficacy of probe B in detecting viscosity changes and underscore its utility for studying cellular responses to stress conditions. This approach enhances our understanding of viscosity’s role in cellular processes under inflammatory and stress conditions.

Figure 8.

Fluorescence images showing HeLa cells exposed to different concentrations of LPS for 30 min, proceeding with a 20 min incubation with probe B (5 μM) in DMEM. Fluorescence was caught within the 680–780 nm range using a 630 nm excitation wavelength. Scale bars denote 50 μm.

To investigate how cellular starvation impacts viscosity,^25,73 HeLa cells were treated with probe B (5 μM) in fetal bovine serum-free medium. Time-course fluorescence microscopy was employed to observe changes in viscosity over different intervals. Images were captured at 0, 20, 40, and 60 min after the addition of probe B. Figure 9 presents fluorescence microscopy images showing an increase in fluorescence intensity of probe B over time. At 0 min, fluorescence intensity was relatively low, but it progressively increased at 20, 40, and 60 min. This gradual rise in fluorescence intensity indicates that cellular viscosity increases as starvation progresses. Cellular starvation triggers several stress responses, including mitophagy, which is the selective degradation of damaged or dysfunctional mitochondria.⁷⁴ As cells undergo mitophagy, there is an accumulation of cellular debris and alterations in the cellular environment, which can contribute to increased viscosity. Mitophagy often involves significant biochemical and structural changes within cells. As damaged mitochondria are processed and removed, the cellular environment can become more viscous due to the accumulation of waste products, reduced water content, and increased macromolecular aggregation. These changes can restrict molecular motion and enhance interactions with probe B, which is sensitive to viscosity variations. By tracking fluorescence intensity over time, we can infer the impact of starvation-induced cellular processes on viscosity (Figure 9). The results highlight probe B’s utility in studying cellular stress responses and provide insights into the relationship between mitophagy and viscosity changes, advancing our understanding of how cellular stress conditions affect cellular dynamics.

Figure 9.

Time-course emission images of HeLa cells exposed to probe B (5 μM) in fetal bovine serum-free medium. Images were captured at different times post-treatment. Fluorescence emission was detected in the 680–780 nm range with excitation at 630 nm. Scale bars denote 50 μm.

To investigate the capability of probe A to detect human serum albumin (HSA) within a cellular environment, we assessed its fluorescence response in HeLa cells exposed to differing concentrations of HSA (20, 40, and 50 μg/mL). Following a 30 min treatment of HeLa cells with different HSA concentrations, the cells were treated with 10 μM probe A for a further 30 min (Figure 10). The results reveal a clear enhancement in cellular emission intensity with rising HSA concentrations (20, 40, and 50 μg/mL), demonstrating that probe A’s fluorescence is responsive to intracellular HSA levels. At 0 μg/mL HSA, used as the control, minimal fluorescence was observed, whereas incremental increases in HSA concentrations to 20, 40, and 50 μg/mL led to a corresponding enhancement in fluorescence intensity (Figure 10). This trend suggests a concentration-dependent interaction between probe A and HSA within the cellular environment. The observed fluorescence increase with higher HSA concentrations may be attributed to enhanced binding of probe A to HSA molecules within the cells. This interaction likely induces a conformational change in probe A that leads to fluorescence enhancement, making it sensitive to variations in HSA levels. The responsiveness of probe A to HSA within the physiological range indicates its potential as a valuable tool for monitoring HSA levels in live cells.

Figure 10.

Emission images of HeLa cells exposed to varying HSA concentrations on HeLa cells for 30 min, proceeding with a further 30 min incubation with 10 μM probe A. Emission were gained within 700–800 nm range using a 633 nm excitation. Scale bars denote 50 μm.

To explore the effects of monensin on viscosity within living organisms, fruit fly larvae were exposed to monensin at 20 μM and 40 μM levels for 4 h. After the monensin treatment, the larvae were then treated with 10 μM probe B for a further 2 h. For comparison, a control group was treated solely with 10 μM probe B for 2 h without monensin treatment. Figure 11 illustrates the confocal fluorescence microscopy images under these various conditions. The fluorescence intensity in the images increased with higher concentrations of monensin, showing progressively stronger fluorescence in flies treated with 20 and 40 μM monensin compared to the control group. The observed increase in fluorescence intensity in monensin-treated flies indicates a rise in viscosity within these specimens. The mechanism by which monensin affects cellular viscosity may involve several factors. Disruption of cellular ion balance and membrane integrity can induce cellular stress responses that alter the viscosity of the cellular environment. Additionally, monensin may cause the accumulation of cellular components or modifications in intracellular structures that contribute to increased viscosity. The results verify that probe B is greatly effective in detecting viscosity changes in vivo. The correlation between monensin level and fluorescence intensity demonstrates the probe’s sensitivity to variations in viscosity, highlighting its utility as a tool for studying changes in cellular and tissue environments induced by chemical treatments (Figure 11). In summary, the increased fluorescence observed with higher monensin concentrations validates probe B’s capability to monitor viscosity changes in biological systems, offering valuable insights into how chemical treatments impact cellular and tissue properties.

Figure 11.

Fluorescence images of probe B in fruit flies under various treatment conditions. Fruit flies were treated with Monensin at concentrations of 20 and 40 μM for 4 h, incubated thereafter with 10 μM probe B for a further 2 h. As a control, fruit flies were given exposure to 10 μM probe B alone for 2 h without Monensin treatment. Fluorescence images were got in the 680–780 nm emission range using 630 nm excitation. Scale bar = 200 μm.

CONCLUSIONS

This research introduces an innovative approach to mitochondrial imaging with the development of two near-infrared (NIR) fluorescent probes, probes A and B, based on hemicyanine dyes integrated with rhodamine and 1,8-naphthalic derivatives. These probes exhibit optimized photophysical properties that allow for precise detection of mitochondrial viscosity, an essential marker for cellular health and function. The unique absorption and emission responses of the probes to varying viscosity levels underscore their sensitivity and versatility. Notably, probe B has shown exceptional performance in both cellular and in vivo contexts, accurately detecting and quantifying mitochondrial viscosity changes. This ability was validated through experiments with monensin, nystatin, and bacterial lipopolysaccharide (LPS), demonstrating the probe’s robustness across diverse biological conditions. Additionally, probe A exhibited sensitivity to human serum albumin (HSA) levels, as demonstrated by its fluorescence enhancement in HeLa cells exposed to increasing concentrations of HSA. This result indicates the promise of probe A in real-time monitoring of HSA, a crucial biomarker for liver and kidney health, in live cellular systems. Overall, the high selectivity, photostability, and low cytotoxicity of probe B make it a valuable tool for exploring mitochondrial function and stress responses, while probe A’s sensitivity to HSA expands its utility in monitoring essential biomolecules in live cells. These findings not only advance our understanding of mitochondrial and cellular dynamics but also open new avenues for research into mitochondrial-related and albumin-associated diseases, underscoring the potential of NIR fluorescent probes in both basic and applied biomedical research.