Polarity-sensitive Probe Research Articles

Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress.

Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission maximum, as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy experiments with reference organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quantitative description of the lipid order in each organelle of interest. Cholesterol extraction/enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mechanical stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to physical and chemical stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.

An ultrasensitive polarity-specific two-photon probe for revealing autophagy in live cells during scrap leather-induced neuroinflammation process.

A polarity-sensitive fluorescence probe AMN was developed to demonstrate the role of autophagy inhibitory drugs in the process of leather residue-induced neuroinflammation, promoting the knowledge of the relationship between autophagy and neuroinflammation. AMN showed a turn-on fluorescent signal in the process of autophagy inhibition via two-photon confocal imaging, which is different from the current popular autophagy probes. Therefore, AMN can offer high-sensitive imaging analysis of the autophagy inhibition process to better understand the role of autophagy in the process of neuroinflammation. The model of scrap leather-induced neuroinflammation using PC12 cells demonstrated that neuroinflammation can induce autophagy by releasing reactive oxygen species (ROS), and autophagy can alleviate neuroinflammation significantly via ROS scavenging.

Phase-selective staining of model and cell membranes, lipid droplets and lipoproteins with fluorescent solvatochromic pyrene probes

The push-pull solvatochromic pyrene derivatives PA and PK have been applied to the study of model membrane vesicles, cells and purified human serum lipoproteins, using both confocal fluorescence microscopy and fluorescence spectroscopy. These polarity-sensitive probes provide information similar to that obtained by Laurdan or Prodan, i.e. mainly lipid order in biomembranes, but they have the essential advantage of being excitable by a standard 405 nm laser light, bypassing the use of multiphoton excitation. In addition, they are brighter and much more photostable than those dimethylamino naphthalene derivatives. Our results with model membrane spectroscopy (multilamellar vesicles) and with microscopy (giant unilamellar vesicles) showed the capacity of PA and PK to report differently on liquid-disordered, liquid-ordered and gel phase bilayers. Moreover, a ratiometric parameter, the Red/Blue Intensity Ratio (RBIR) could be used for inter-domain, inter-vesicle and even inter-technique comparison, and the appropriate microscopy-spectroscopy conversion coefficients could be estimated. In studies at the cellular level, PA probe stained almost exclusively the plasma membrane of red blood cells, revealing its high degree of lipid order. Using Chinese Hamster Ovary cells PA was shown to be an excellent probe for the detection of cytoplasmic lipid droplets, superior to Nile Red in that PA provides simultaneously a detailed information of membrane order in the whole cell, in which the lipid droplets appear with a very good contrast. Moreover, spectrofluorometric data of PA-stained serum lipoproteins indicated an essentially identical value of RBIR for lipid droplets and for high-density lipoproteins.

Exploring NIR Aza-BODIPY-Based Polarity Sensitive Probes with ON-and-OFF Fluorescence Switching in Pluronic Nanoparticles.

Because of their deep penetration capability in tissue, red or near infrared (NIR) fluorophores attract much attention in bio-optical imaging. Among these fluorophores, the ones that respond to the immediate microenvironment (i.e., temperature, polarity, pH, viscosity, hypoxia, etc.) are highly desirable. We studied the response of six NIR aza-BODIPY-based and structurally similar fluorophores to polarity and viscosity for incorporation inside Pluronic nanoparticles as switchable fluorescent probes (SFPs). Based on our results, all of these fluorophores were moderately to strongly sensitive to the polarity of the microenvironment. We concluded that attaching amine groups to the fluorophore is not necessary for having strong polarity sensitive probes. We further studied the response of the fluorophores when embedded inside Pluronic nanoparticles and found that four of them qualified as SFPs. We also found that the switching ratio of the fluorophore-encapsulated Pluronic nanoparticles (ION-to-IOFF) is related to the length of the hydrophobic chain of the Pluronic tri-block copolymers. As such, the highest switching ratio pertained to F-68 with the lowest hydrophobic block poly (propylene oxide) (PPO chain of only 30 units).

Predicting Spectral Properties of Polarity Sensitive Dyes with QM/MM Simulation

Polarity sensitive, lipophilic dyes such as Laurdan report lipid packing in biomembranes, as the emission spectrum is red shifted in more polar environments. In simple membranes, the dye is more accessible to solvent in more disordered membranes, and the spectral shift is well-explained by dipolar relaxation of the solvent. However, in more complex systems other factors may contribute, especially hydrogen bonding between the environment and the chromophore. Disentangling the factors which control the spectral shift is complicated by the fact that polar environments likely stabilize a charge transfer-like state, and so predicting the emission requires accurate modeling of the relaxation of the environment around the excited state. An approach has been developed in which (i) the local environment is sampled by classical molecular dynamics (MD) simulation of the dye; (ii) prediction of the absorption and excited state by numerical quantum mechanics (QM); (iii) parameterization of an excited state MD model (iv) relaxation of the environment around the excited state by MD; (v) prediction of the emission by QM. The QM steps are computed using GW-BSE (as implemented in Versatile Object-oriented Toolkit for Coarse-graining Applications: Exciton Transport Simulations (VOTCA-XTP)) with the environment modeled as fixed point charges, sampled in the MD simulation steps. Comparison to time dependent DFT shows that GW-BSE yields much more quantitative predictions for both absorption and emission, as expected for charge transfer states, because GW more accurately captures the effective electron-hole interactions following an excitation. The results reveal the mechanisms responsible for the spectral shift of Laurdan, and map an approach for similar applications to other polarity sensitive probes.

Optical Dyes to Monitor Tension and Growth in Model Membranes

Cells have been shown to dynamically regulate physical properties of the membrane in response to mechanical cues. In particular, membranes have been shown to undergo surface area changes in response to membrane tension, a process which plays a role in cellular growth, migration and signaling. The ability to simultaneously monitor membrane strain and membrane surface area changes would be beneficial in understanding this process, yet we lack robust methods to accomplish this. Here, we introduce an optical technique to monitor membrane tension and surface area changes in real time. Through the combinatorial use of two types of optical dyes, which localize to distinct regions of the bilayer membrane, we can monitor changes in lipid separation through the processes of membrane stretch and lipid uptake. Laurdan, a polarity-sensitive probe, is localized to the hydrophobic region of the membrane, while phospholipid-conjugated FRET pairs localize to the hydrophilic head region. These dyes act in concert to indicate changes in water permeation and phospholipid head separation, respectively, as membrane properties change. We observed linear shifts in fluorescence in response to applied tension, indicating the potential of this system as a membrane tension sensor. We then used these probes to observe the process of tension-induced lipid uptake, and discovered that membranes under strain take up excess lipid faster and in greater amounts. Ultimately, the combined use of these probes offers a more complete picture of membrane dynamics, and allows us to remotely track changes in tension and surface area in model membranes.

A flavonoid-based fluorescent probe enables the accurate quantification of human serum albumin by minimizing the interference from blood lipids.

We herein provide a simple design strategy to improve the sensing specificity towards human serum albumin by incorporating a nitrobenzene quencher into a traditional polarity-sensitive probe in responding to the interference from blood lipids.

The polarity sensitivity factor of some fluorescent probe molecules used for studying supramolecular systems and other heterogeneous environments

Fluorescence spectroscopy provides an excellent technique for investigating heterogeneous systems, due to its high sensitivity and the large effect of the local environment on molecular emission. In addition, the use of polarity-sensitive fluorescent probes as guests in supramolecular host–guest inclusion complexes can be exploited in fluorescent sensors. This paper identifies, tabulates, and quantifies a series of useful polarity-sensitive fluorescent probes, with a wide range of polarity-dependent fluorescence responses. The degree of polarity sensitivity is quantified using the polarity sensitivity factor (PSF), developed in our laboratory. In most cases, such polarity-sensitive probes show increased emission as the local polarity is decreased (PSF > 1); 10 such probes are described. However, less commonly, “reverse polarity dependence” can occur in which probe emission decreases with decreasing polarity (PSF < 1); four such probes are described. The mechanism for the observed polarity-induced fluorescence changes will also be discussed in selected representative cases. The purpose of this paper is to present details on a broad arsenal of polarity-sensitive fluorescence probes with varying properties, with potentially useful applications in the study of heterogeneous systems, including inclusion phenomena, and in practical applications such as fluorescent sensors, which will be useful to researchers studying supramolecular and other heterogeneous systems using fluorescence spectroscopy.

Glucose-sensitive silicone hydrogel contact lens toward tear glucose monitoring.

Accurate and reliable monitoring of blood glucose is needed for the treatment of diabetes, which has many challenges, including lack of patient compliance. Measuring tear glucose is an alternative to traditional finger-stick tests used to track blood sugar levels, but glucose sensing using tears has yet to be achieved. We report a methodology for possible tear glucose monitoring using glucose-sensitive silicone hydrogel (SiHG) contact lenses, the primary type of lenses available in today's market. Initially, we assessed the interpenetrating polymer network, with nearly pure silicone and water regions, existing in the SiHGs using a polarity-sensitive probe Prodan. We then synthesized a glucose-sensitive fluorophore Quin-C18 with a hydrophobic side chain for localization of probe at the interfacial region. Using our glucose-sensing contact lens, we were able to measure varying concentrations of glucose in an in-vitro system. The Quin-C18 strongly bound to the lenses with insignificant leaching even after multiple rinses. The lenses displayed a similar response to glucose after three months of storage in water. This study demonstrates that it may be possible to develop a contact lens for continuous glucose monitoring in the near term, using our concept of fluorophore binding at the silicone-water interface.

Simultaneous Fluorescence Visualization of Endoplasmic Reticulum Superoxide Anion and Polarity in Myocardial Cells and Tissue.

Diabetic cardiomyopathy (DCM) is a critical complication of diabetes, the accurate pathogenesis of which remains elusive. It is widely accepted that endoplasmic reticulum (ER) stress and abnormal fluctuations of reactive oxygen species (ROS) are considered to be closely associated with progress of DCM. In addition, DCM-induced changes of myocardial tissue and ROS-derived oxidation of proteins will cause changes of the hydrophilic and hydrophobic domains and may further seriously alter the myocardial cell polarity. Thus, real-time detection of ROS and polarity in ER of live cells and in tissue will contribute to revealing the exact molecular mechanisms of DCM. In this article, we first present an ER-targetable fluorogenic probe termed ER-NAPC for sensitive and selective detection of superoxide anion (O2•-). ER-NAPC can precisely target ER and visualize the increase of O2•- level in a live H9c2 cardiomyocyte cell during ER stress. Meanwhile, by combining ER-NAPC with a polarity-sensitive probe, ER-P, we accomplish the simultaneous fluorescence visualization of O2•- and polarity in ER of live cells and diabetic myocardial tissue. The dual-color fluorescence imaging results indicate that the O2•- level and polarity will synergistically rise during ER stress in live cells and diabetic myocardial tissue. The proposed dual-color imaging strategy may offer a proven methodology for studying coordinated variation of different parameters during ER stress oriented disease.

Fluorescence Microspectroscopy with Nanometer Peak Position Resolution: Novel Applications of Environment-Sensitive Probes

Fluorescence microspectroscopy (FMS) with environment-sensitive probes provides information about local molecular surroundings at microscopic spatial resolution. Until recently, only probes exhibiting large spectral shifts due to local changes have been used. Herein, we show that appropriate measuring procedure and data analysis enable nanometer spectral peak position resolution, even for photosensitive fluorophores [1]. The reach of our approach is demonstrated in several examples. The first application shows how we can distinguish lipid vesicles in different lipid phases with two commonly used polarity-sensitive probes. A synthesized NBD-based fatty acid red-shifted its emission maximum by 1.5-2 nm going from gel to liquid-disordered phase in DPPC. Between these two phases Laurdan exhibits a large 50 nm red-shift. We therefore chose a more challenging combination - gel and liquid ordered phase, realized by DPPC and DPPC/Chol (40 mol%), respectively, where we were able to detect a 3 nm blue-shift with Laurdan [1]. The second example shows application of a synthesized rhodamine-based pH-activatable probe that is sensitive to aggregation. We studied a receptor-mediated internalization in dendritic cells and measured a 3 nm aggregation-induced emission spectral shift due to probe accumulation in endosomes and lysosomes [2,3]. The results show that peak position resolution, characteristic for spectrofluorimetric measurements on bulk samples, could readily be achieved at micrometer spatial scale. [1] I. Urbančič, Z. Arsov, A. Ljubetič, D. Biglino, J. Štrancar, Opt. Express 21:25291–25306 (2013). [2] Z. Arsov, U. Švajger, J. Mravljak, S. Pajk, A. Kotar, I. Urbančič, J. Štrancar, M. Anderluh, ChemBioChem 16:2660–2667 (2015). [3] Z. Arsov, I. Urbančič, J. Štrancar, Spectrochim. Acta A Mol. Biomol. Spectrosc., https://doi.org/10.1016/j.saa.2017.09.067 (2017).

The dynamics of intracellular water constrains glycolytic oscillations in Saccharomyces cerevisiae

We explored the dynamic coupling of intracellular water with metabolism in yeast cells. Using the polarity-sensitive probe 6-acetyl-2-dimethylaminonaphthalene (ACDAN), we show that glycolytic oscillations in the yeast S. cerevisiae BY4743 wild-type strain are coupled to the generalized polarization (GP) function of ACDAN, which measures the physical state of intracellular water. We analysed the oscillatory dynamics in wild type and 24 mutant strains with mutations in many different enzymes and proteins. Using fluorescence spectroscopy, we measured the amplitude and frequency of the metabolic oscillations and ACDAN GP in the resting state of all 25 strains. The results showed that there is a lower and an upper threshold of ACDAN GP, beyond which oscillations do not occur. This critical GP range is also phenomenologically linked to the occurrence of oscillations when cells are grown at different temperatures. Furthermore, the link between glycolytic oscillations and the ACDAN GP value also holds when ATP synthesis or the integrity of the cell cytoskeleton is perturbed. Our results represent the first demonstration that the dynamic behaviour of a metabolic process can be regulated by a cell-wide physical property: the dynamic state of intracellular water, which represents an emergent property.

A Stimuli-Responsive Metallohydrogel Exhibiting Cyclohexane-Like Hydrophobicity.

Silver(I) forms a hydrogel in the presence of cholate with unusual properties, which are not observed with other cations. Polarity-sensitive probes have shown that the spherical aggregates observed in the gel have 'pockets' with hydrophobicity comparable to that of degassed cyclohexane. The gel exhibited thermo- and mechanoresponsive properties. Color tunability from blue to cyan and green was observed with prodan. The two sol phases of the gel formed by applying stress and temperature showed very different properties.

Laurdan and Di-4-ANEPPDHQ probe different properties of the membrane

Lipid packing is a crucial feature of cellular membranes. Quantitative analysis of membrane lipid packing can be achieved using polarity sensitive probes whose emission spectrum depends on the lipid packing. However, detailed insights into the exact mechanisms that cause the changes in the spectra are necessary to interpret experimental fluorescence emission data correctly. Here, we analysed frequently used polarity sensitive probes, Laurdan and di-4-ANEPPDHQ, to test whether the underlying physical mechanisms of their spectral changes are the same and, thus, whether they report on the same physico-chemical properties of the cell membrane. Steady-state spectra as well as time-resolved emission spectra of the probes in solvents and model membranes revealed that they probe different properties of the lipid membrane. Our findings are important for the application of these dyes in cell biology.

A single fluorescent probe enables clearly discriminating and simultaneously imaging liquid-ordered and liquid-disordered microdomains in plasma membrane of living cells.

Liquid-ordered (Lo) and liquid-disordered (Ld) microdomains in plasma membrane play different yet essential roles in various bioactivities. However, discrimination of the two microdomains in living cells is difficult, due to the similarity in their constituents and structures. Up to now, polarity sensitive probes are the only tool for imaging the two microdomains, but their small difference between emission spectra in the two microdomains (less than 50nm) limited their application in living cells. In this work, we first presented an aggregation/monomer type of fluorescent probe (2,7-9E-BHVC12) with much larger separation in emission wavelength (up to 100nm), for dual-color visualizing the two membrane microdomains in living cells. The probe can form red-emissive aggregates and yellow-emissive monomers when induced by Lo and Ld microdomains, respectively, and thus enables clear visualization of the two membrane microdomains in living cells with dual colors, and thus high-fidelity images of substructures of plasma membrane have been obtained. According to the images of three kinds of normal cells and three kinds of cancer cells stained with 2,7-9E-BHVC12, significant difference in plasma membrane microstructure of cancer cells was found. In terms of 2,7-9E-BHVC12, normal cells were mainly consisted of either Lo or Ld microdomains all over their membranes, while cancer cells all clearly display coexistence of Lo and Ld membrane microdomains. Therefore, 2,7-9E-BHVC12 can serve as a powerful tool for studies of membrane microdomains, and the different results of normal and cancer cells would also deepen our understanding in cancer science.

Complexation in Weakly Attractive Copolymers with Varying Composition and Topology: Linking Fluorescence Experiments and Molecular Monte Carlo Simulations

The intramolecular complex formation occurring in miktoarm star polymers and block copolymers is investigated experimentally and by means of Monte Carlo simulations. The driving force of the complex formation is an attractive interaction between the different blocks. In the simulations, a simple bead–spring model with an attractive Lennard-Jones potential is used to mimic the complexation. The effect of topology (arm number) and composition (arm length) is simulated. Experimentally, star-shaped polymeric systems and diblock copolymers of poly(propylene oxide) (PPO) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) of varying degrees of polymerization are investigated. By quantifying the complex formation with help of fluorescence spectroscopy of a polarity-sensitive probe, we show that a complex is formed preferably at high degrees of polymerization of PDMAEMA and a high number of PDMAEMA arms. These results are in accordance with the simulations: we show that the actual amount of PPO in the complex cor...

D-PET-controlled "off-on" Polarity-sensitive Probes for Reporting Local Hydrophilicity within Lysosomes.

Polarity-sensitive fluorescent probes are powerful chemical tools for studying biomolecular structures and activities both in vitro and in vivo. However, the lack of “off-on” polarity-sensing probes has limited the accurate monitoring of biological processes that involve an increase in local hydrophilicity. Here, we design and synthesize a series of “off-on” polarity-sensitive fluorescent probes BP series consisting of the difluoroboron dippyomethene (BODIPY) fluorophore connected to a quaternary ammonium moiety via different carbon linkers. All these probes showed low fluorescence quantum yields in nonpolar solution but became highly fluorescent in polar media. BP-2, which contains a two-carbon linker and a trimethyl quaternary ammonium, displayed a fluorescence intensity and quantum yield that were both linearly correlated with solvent polarity. In addition, BP-2 exhibited high sensitivity and selectivity for polarity over other environmental factors and a variety of biologically relevant species. BP-2 can be synthesized readily via an unusual Mannich reaction followed by methylation. Using electrochemistry combined with theoretical calculations, we demonstrated that the “off-on” sensing behavior of BP-2 is primarily due to the polarity-dependent donor-excited photoinduced electron transfer (d-PET) effect. Live-cell imaging established that BP-2 enables the detection of local hydrophilicity within lysosomes under conditions of lysosomal dysfunction.

Micellar dipolar rearrangement is sensitive to hydrophobic chain length: Implication for structural switchover of piroxicam

The interfacial properties of the membrane are exceptionally vital in drug-membrane interaction. They not only select out a particular prototropic form of the drug molecule for incorporation, but are also potent enough to induce structural switchover of these drugs in several cases. In this work, we quantitatively monitored the change in dipolar rearrangement of the micellar interface (as a simplified membrane mimic) by measuring the dielectric constant and dipole potential with the micellization of SDS at pH 3.6. The dielectric constant and dipole potential were measured utilizing the fluorescence of polarity sensitive probe, pyrene and potential-sensitive probe, di-8-ANEPPS, respectively. Our study demonstrates that the change in dipolar rearrangement directly influences the switchover equilibrium between the anionic and neutral from of piroxicam. We have further extended our work to evaluate the effect of hydrophobic chain length of the surfactants on the dipolar rearrangement and its effect on the structural switchover of piroxicam. It is interesting that the extent of switchover of piroxicam is directly correlated with the dipolar rearrangement induced bythe varying hydrophobic chain length of the surfactants. To the best of our knowledge, our results constitute the first report to show the dependence of dipole potential on the hydrophobic chain length of the surfactant and demonstrate that the dipolar rearrangement directly tunes the extent of structural switchover of piroxicam, which was so far only intuitive. We consider that this new finding would have promising implication in drug distribution and drug efficacy.

Temperature effect on 4-aminophtalimide fluorescence in n-alcohols

The compound 4-aminophthalimide (4-AP) is a well-known dye used as an environment polarity sensitive probe e.g. in solvation studies. This paper presents the effect of temperature on 4-aminophthalimides steady-state and time-resolved fluorescence in five n-alcohols. It is shown that the hydrogen bonding ability of n-alcohols affects the shifts of steady-state absorption and fluorescence spectra of 4-aminophthalimide at room temperature, and the shifts of fluorescence also at temperatures from the range 180 to 323K. Temperature is shown to affect the change in hydrogen bond energy that follows 4-AP excitation, in a way dependent on the n-alcohol alkyl chain length. On the other hand, time-resolved results indicate that the temperature dependence of 4-AP deactivation follows mainly from the energy-gap dependent non-radiative deactivation rate. Fluorescence transition dipole moments at room temperature have been found to be slightly dependent on the solvent, but nothing proves that these changes are connected to different hydrogen bonding character of each n-alcohol. Therefore, while the steady-state results provide clear evidence of hydrogen bonding between 4-AP and n-alcohols, the time-resolved results do not show any evident sign of hydrogen bonding, besides the influence of the position of fluorescence emission on the radiative and non-radiative rates.

Organisation and complexation of mono- and bis-β-cyclodextrins without chromophores with a fluorescence-sensitive probe in aqueous solutions

The behaviour of mono- and bis-β-cyclodextrin (CD) derivatives, namely 6I-deoxy-6I-[4-(hept-6-ynyl)-1H-1,2,3-triazolyl]-β-CD and 1,5-bis((1-(6I-deoxy-β-CD-6I-yl)-1H-1,2,3-triazol-4-yl)methoxy) pentyl, whose appended groups and inter-CD linkers, respectively, do not contain any chromophore, and their complexation with dimethyl-2,6-naphthalenedicarboxylate (DMN), a fluorescent polarity-sensitive probe in aqueous solutions, were investigated. Steady-state, time-resolved fluorescence, circular dichroism techniques, molecular mechanics (MM) and molecular dynamics (MD) simulations were employed. DMN appeared to slightly interact with the mono-β-CD and only unstable non-covalent dimers were formed. On the contrary, stable 1:1 and 2:1 stoichiometry complexes were obtained with bis-β-CD and DMN. A certain cooperativity due to the presence of both cavities and the linker favoured the formation of the complex 2:1 DMN:CD. DMN appeared to be axially oriented inside both CD cavities. MM and MD calculations also demonstrated the stability of the 1:1 and 1:2 stoichiometry complexes.