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The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non‐canonical nucleic acid structures in live cells and other relevant biological settings. [ABSTRACT FROM AUTHOR]

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non‐canonical nucleic acid structures in live cells and other relevant biological settings. [ABSTRACT FROM AUTHOR]

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non‐canonical nucleic acid structures in live cells and other relevant biological settings. [ABSTRACT FROM AUTHOR]

Transition and lanthanide metal complexes have rich photophysical properties that can be used for cellular imaging, biosensing and phototherapy. One of the applications of such luminescent compounds is the detection and visualisation of nucleic acids. In this brief review, we survey the recent literature on the use of luminescent metal complexes (including ReI, RuII, OsII, IrIII, PtII, EuIII and TbIII) as DNA optical probes, including examples of compounds that bind selectively to non-duplex DNA topologies such as quadruplex, i -motif and DNA mismatches. We discuss the applications of metal-based luminescent complexes in cellular imaging, including time-resolved microscopy and super-resolution techniques. Their applications in biosensing and phototherapy are briefly mentioned in the relevant sections. [ABSTRACT FROM AUTHOR]

Measuring viscosity and temperature on the microscale is a challening yet very important task, in materials sciences and in biology alike. In this perpsective we review and discuss fluorescent microviscosity sensors, termed ‘molecular rotors’, that offer a convenient way of measuring microscopic viscosity and sometimes may even be used to measure microscopic temperature in addition to viscosity. We discuss how temperature in combination with various solvent properties can affect microviscosity measurements and we review possible action mechanisms that make molecular rotors sensitive to multiple parameters of their environment. Overall, we reveal a complicated, yet exciting, behaviour of molecular rotors at different viscosity, temperature and solvent properties on the microscale and how this behaviour can be explained and exploited. [ABSTRACT FROM AUTHOR]

Diffusion-mediated cellular processes, such as metabolism, signalling and transport, depend on the hydrodynamic properties of the intracellular matrix. Photodynamic therapy, used in the treatment of cancer, relies on the generation of short-lived cytotoxic agents within a cell on irradiation of a drug. The efficacy of this treatment depends on the viscosity of the medium through which the cytotoxic agent must diffuse. Here, spectrally resolved fluorescence measurements of a porphyrin-dimer-based molecular rotor are used to quantify intracellular viscosity changes in single cells. We show that there is a dramatic increase in the viscosity of the immediate environment of the rotor on photoinduced cell death. The effect of this viscosity increase is observed directly in the diffusion-dependent kinetics of the photosensitized formation and decay of a key cytotoxic agent, singlet molecular oxygen. Using these tools, we provide insight into the dynamics of diffusion in cells, which is pertinent to drug delivery, cell signalling and intracellular mass transport. [ABSTRACT FROM AUTHOR]

Singlet molecular oxygen, O2(a1 Δg), can be created in a single cell from ground-state oxygen, O2(X3Σg-), upon focused laser irradiation of an intracellular sensitizer. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a1Δg →X3Σg-) with subcellular spatial resolution. The singlet oxygen lifetime determines its diffusion distance and hence the intracellular volume element in which singlet-oxygen-initiated perturbation ofthe cell occurs. In this study, the time-resolved phosphores- cence of singlet oxygen produced by the sensitizers chlorin (ChI) and 5,10,1 5,20-tetrakis(N-methyl-4-pyridyl)- 21 H,23H-porphine (TMPyP) was monitored. These molecules localize in different domains of a living cell. The data indicate that (I) the singlet oxygen lifetime and (ii) the rate constant for singlet oxygen quenching by added NaN3 depend on whether ChI or TMPyP was the photosensitizer. These observations likely reflect differences in the chemical and~physical constituency of a given subcellular domain (e.g., spatially dependent oxygen and NaN3 diffusion coefficients), thereby providing evidence that singlet oxygen responds to the inherent heterogeneity of a cell. Thus, despite a relatively long intracellular lifetime, singlet oxygen does not diffuse a great distance from its site of production. This is a consequence of an apparent intracellular viscosity that is comparatively large. [ABSTRACT FROM AUTHOR]

The results of electrochemical measurements, density-functional theory calculations, emission and time-resolved IR (TRIR) spectroscopic studies for fac-[ReCl(CO)[sub3](dppz-X[sub2])), (dppz = dipyrido[3,2-a:2',3'-cjphenazine; X = CH[sub3], H, F, Cl, CF[sub3]) are reported. For all complexes the calculations show that the lowest unoccupied molecular orbital (LUMO) is a phenazine based orbital localized on the dppz ligand. We observe that three different excited states, IL ππ*, metal-to-Ilgand charge-transfer (MLCT) (phen), and MLCT (phz), are formed depending upon the substituent on the dppz ligand and on the nature of the solvent. This means that both the energy and the nature of the photophysically active state(s) can be tuned by both chemical modification of dppz ligand and solvent properties. The excited-state dynamics in these systems is directly related to the mechanism of the "light switch effect" and ps-TRIR has allowed a deeper insight into this mechanism by being able to directly monitor the change in the population of the higher lying emissive phen-type [sup3]MLCT and IL ππ* states and the dark [sup3]MLCT (phz) state depending on the different environmental factors. [ABSTRACT FROM AUTHOR]

Two photon ionisation of 2-aminopurine (2AP) has been monitored following 267 nm irradiation in neutral and acidic aqueous solutions using picosecond time-resolved infrared spectroscopy (ps-TRIR). The transient infrared spectra obtained in neutral and acidic conditions show significant differences that are consistent with the formation of different species, namely the 2AP radical cation, 2AP+˙, in acidic conditions and the uncharged radical, 2AP˙(-H+), in neutral conditions. The ps-TRIR data indicate that deprotonation of 2AP+˙ in neutral solution takes place within <2 ps following photoionisation. DFT calculations (EDF1/6–31+G*) were used to support the assignment of the intermediates observed in these spectroscopic experiments. [ABSTRACT FROM AUTHOR]

We report the synthesis, spectroscopic properties and intracellular imaging of recombinant antibody single chain fragment (scFv) conjugates with photosensitizers used for photodynamic therapy of cancer (PDT). Two widely-studied photosensitizers have been selected: preclinical pyropheophorbide-a (PPa) and verteporfin (VP), which has been clinically approved for the treatment of acute macular degeneration (Visudyne®). Pyropheophorbide-a and verteporfin have been conjugated to an anti-HER2 scFv containing on average ten photosensitizer molecules per scFv with a small contribution (≤20%) from non-covalently bound molecules. Confocal fluorescence microscopy demonstrates good cellular uptake of PPa conjugate with the HER2-positive cell line, SKOV-3, while negligible cell uptake is demonstrated for the HER2-negative cell line, KB. For the VP conjugate, increased rate of cellular uptake and prolonged retention in SKOV-3 cells is observed compared to free photosensitizer. In clinical applications this could provide increased potency and desired selectivity towards malignant tissue, leaving surrounding healthy tissue unharmed and reducing skin photosensitivity. The present study highlights the usefulness of photosensitizer immunoconjugates with scFvs for targeted PDT. [ABSTRACT FROM AUTHOR]

We report a systematic study of the photophysical parameters relevant to photodynamic therapy (PDT) by a new type of sensitizers, conjugated porphyrin oligomers. Due to the strong nonlinear properties of oligomers containing 2, 4 and 8 porphyrin units, these molecules are attractive candidates for PDT via multiphoton excitation. The triplet state energy levels for all molecules have been determined by the triplet quenching method, phosphorescence measurements and DFT calculations. We find that the triplet energies of all the oligomers are sufficient to generate singlet oxygen, >94 kJ mol−1. However, low singlet oxygen quantum yields are observed for the tetramer and the octamer, as compared to the conjugated dimer and monomeric porphyrin, reflecting the decrease in triplet yield. Thus the conjugated porphyrin dimer is the most promising core structure for PDT applications via multiphoton excitation. [ABSTRACT FROM AUTHOR]

The nucleotide 5'-dGMP and polynucleotide poly(dGdC)·poly(dGdC) have been irradiated by using a 200-fs, 200-nm laser pulses and spectrally characterized by using time-resolved infrared spectroscopy. Under the experimental conditions, 200-nm excitation generates both electronic excited states and radical cations through photoionization; the former decay rapidly to vibrationally hot ground state. By using infrared signatures we have been able to follow these processes, and at time scales of > 1 ns we observe an infrared marker band at 1,702 cm-1 within both 5'-dGMP and the polynucleotide assigned to a photoionized product of guanine. This transient has also been reproduced through indirect chemistry through the reaction with photogenerated carbonate radical with 5'-dGMP. The ability to use time-resolved infrared spectroscopy in this way paves the way for developing solution-phase studies to investigate both direct and indirect radiation chemistry of DNA. [ABSTRACT FROM AUTHOR]

The photophysics of the fac-[Re(Cl)(CO)3(CO2Et-dppz)] (CO2Et-dppz = dipyrido[3,2a:2′,3′c]phenazine-11-carboxylic ethyl ester) was studied with picosecond time-resolved infrared (ps-TRIR) spectroscopy in metal carbonyl (2100–1800 cm-1) and organic ester (1800–1600 cm-1) spectral regions. The ps-TRIR spectra in both regions give evidence for the formation of a metal-to-ligand charge transfer (MLCT) excited state. The magnitude of ν(C&z.tbnd6;O) shift in the metal carbonyl region of the excited state relative to those of the ground state allows the excited state to be described more precisely as a dπ(Re) → π* (phenazine) 3MLCT state. [Copyright &y& Elsevier]

The efficacy of many drugs can be limited by undesirable properties, such as poor aqueous solubility, low bioavailability, and "off‐target" interactions. To combat this, various drug carriers have been investigated to enhance the pharmacological profile of therapeutic agents. In this work, we demonstrate the use of mechanical protection to "cage" a DNA‐targeting metallodrug within a photodegradable rotaxane. More specifically, we report the synthesis of rotaxanes incorporating as a stoppering unit a known G‐quadruplex DNA binder, namely a PtII‐salphen complex. This compound cannot interact with DNA when it is part of the mechanically interlocked assembly. The second rotaxane stopper can be cleaved by either light or an esterase, releasing the PtII‐salphen complex. This system shows enhanced cell permeability and limited cytotoxicity within osteosarcoma cells compared to the free drug. Light activation leads to a dramatic increase in cytotoxicity, arising from the translocation of PtII‐salphen to the nucleus and its binding to DNA. [ABSTRACT FROM AUTHOR]

The efficacy of many drugs can be limited by undesirable properties, such as poor aqueous solubility, low bioavailability, and "off‐target" interactions. To combat this, various drug carriers have been investigated to enhance the pharmacological profile of therapeutic agents. In this work, we demonstrate the use of mechanical protection to "cage" a DNA‐targeting metallodrug within a photodegradable rotaxane. More specifically, we report the synthesis of rotaxanes incorporating as a stoppering unit a known G‐quadruplex DNA binder, namely a PtII‐salphen complex. This compound cannot interact with DNA when it is part of the mechanically interlocked assembly. The second rotaxane stopper can be cleaved by either light or an esterase, releasing the PtII‐salphen complex. This system shows enhanced cell permeability and limited cytotoxicity within osteosarcoma cells compared to the free drug. Light activation leads to a dramatic increase in cytotoxicity, arising from the translocation of PtII‐salphen to the nucleus and its binding to DNA. [ABSTRACT FROM AUTHOR]

The efficacy of many drugs can be limited by undesirable properties, such as poor aqueous solubility, low bioavailability, and “off‐target” interactions. To combat this, various drug carriers have been investigated to enhance the pharmacological profile of therapeutic agents. In this work, we demonstrate the use of mechanical protection to “cage” a DNA‐targeting metallodrug within a photodegradable rotaxane. More specifically, we report the synthesis of rotaxanes incorporating as a stoppering unit a known G‐quadruplex DNA binder, namely a PtII‐salphen complex. This compound cannot interact with DNA when it is part of the mechanically interlocked assembly. The second rotaxane stopper can be cleaved by either light or an esterase, releasing the PtII‐salphen complex. This system shows enhanced cell permeability and limited cytotoxicity within osteosarcoma cells compared to the free drug. Light activation leads to a dramatic increase in cytotoxicity, arising from the translocation of PtII‐salphen to the nucleus and its binding to DNA. [ABSTRACT FROM AUTHOR]

The biophysical properties of cells described at the level of whole cells or their membranes have many consequences for their biological behavior. However, our understanding of the relationships between mechanical parameters at the level of cell (stiffness, viscoelasticity) and at the level of the plasma membrane (fluidity) remains quite limited, especially in the context of pathologies, such as cancer. Here, we investigated the correlations between cells' stiffness and viscoelastic parameters, mainly determined via the actin cortex, and plasma membrane microviscosity, mainly determined via its lipid profile, in cancer cells, as these are the keys to their migratory capacity. The mechanical properties of cells were assessed using atomic force microscopy (AFM). The microviscosity of membranes was visualized using fluorescence-lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY 2. Measurements were performed for five human colorectal cancer cell lines that have different migratory activity (HT29, Caco-2, HCT116, SW 837, and SW 480) and their chemoresistant counterparts. The actin cytoskeleton and the membrane lipid composition were also analyzed to verify the results. The cell stiffness (Young's modulus), measured via AFM, correlated well (Pearson r = 0.93) with membrane microviscosity, measured via FLIM, and both metrics were elevated in more motile cells. The associations between stiffness and microviscosity were preserved upon acquisition of chemoresistance to one of two chemotherapeutic drugs. These data clearly indicate that mechanical parameters, determined by two different cellular structures, are interconnected in cells and play a role in their intrinsic migratory potential. [ABSTRACT FROM AUTHOR]

G‐quadruplex DNA is a non‐canonical structure that forms in guanine‐rich regions of the genome. There is increasing evidence showing that G‐quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G‐quadruplex DNA, display an increase in their phosphorescence, acting as switch‐on probes. More importantly, upon binding to G‐quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM). [ABSTRACT FROM AUTHOR]

G‐quadruplex DNA is a non‐canonical structure that forms in guanine‐rich regions of the genome. There is increasing evidence showing that G‐quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G‐quadruplex DNA, display an increase in their phosphorescence, acting as switch‐on probes. More importantly, upon binding to G‐quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM). [ABSTRACT FROM AUTHOR]

The cell membrane is an important regulator for the cytotoxicity of chemotherapeutic agents. However, the biochemical and biophysical effects that occur in the membrane under the action of chemotherapy drugs are not fully described. In the present study, changes in the microviscosity of membranes of living HeLa–Kyoto tumor cells were studied during chemotherapy with paclitaxel, a widely used antimicrotubule agent. To visualize the microviscosity of the membranes, fluorescence lifetime imaging microscopy (FLIM) with a BODIPY 2 fluorescent molecular rotor was used. The lipid profile of the membranes was assessed using time-of-flight secondary ion mass spectrometry ToF-SIMS. A significant, steady-state decrease in the microviscosity of membranes, both in cell monolayers and in tumor spheroids, was revealed after the treatment. Mass spectrometry showed an increase in the unsaturated fatty acid content in treated cell membranes, which may explain, at least partially, their low microviscosity. These results indicate the involvement of membrane microviscosity in the response of tumor cells to paclitaxel treatment. [ABSTRACT FROM AUTHOR]

The article presents a study which demonstrates that the use of wavelength light to excite conjugated porphyrin dimers is reflected in the quantum yield of oxygen production. It mentions that the wavelength-dependent effect offers an additional degree of control in the photosensitized production of oxygen. It relates that over the course of the study, the researchers have found out that oxygen production depends on irradiation wavelength.

The article presents a study on molecular rotor measures viscosity of live cells through fluorescence lifetime imaging. It forwards that viscosity is one of the major parameters identifying the diffusion rate of species in condensed media. It also forwards that in biosystems alternations in viscosity have been associated to disease and malfunction of the cellular level. A new approach to image local microviscosity using the fluorescence lifetime of a molecular rotor is also discussed.

Oxidation of cellular structures is typically an undesirable process that can be a hallmark of certain diseases. On the other hand, photooxidation is a necessary step of photodynamic therapy (PDT), a cancer treatment causing cell death upon light irradiation. Here, the effect of photooxidation on the microscopic viscosity of model lipid bilayers constructed of 1,2-dioleoyl- sn-glycero-3-phosphocholine has been studied. A molecular rotor has been employed that displays a viscosity-dependent fluorescence lifetime as a quantitative probe of the bilayer's viscosity. Thus, spatially-resolved viscosity maps of lipid photooxidation in giant unilamellar vesicles (GUVs) were obtained, testing the effect of the positioning of the oxidant relative to the rotor in the bilayer. It was found that PDT has a strong impact on viscoelastic properties of lipid bilayers, which 'travels' through the bilayer to areas that have not been irradiated directly. A dramatic difference in viscoelastic properties of oxidized GUVs by Type I (electron transfer) and Type II (singlet oxygen-based) photosensitisers was also detected. [ABSTRACT FROM AUTHOR]

Lipid peroxidation is a process which is key in cell signaling and disease, it is exploited in cancer therapy in the form of photodynamic therapy. The appearance of hydrophilic moieties within the bilayer's hydrocarbon core will dramatically alter the structure and mechanical behavior of membranes. Here, we combine viscosity sensitive fluorophores, advanced microscopy, and X-ray diffraction and molecular simulations to directly and quantitatively measure the bilayer's structural and viscoelastic properties, and correlate these with atomistic molecular modelling. Our results indicate an increase in microviscosity and a decrease in the bending rigidity upon peroxidation of the membranes, contrary to the trend observed with non-oxidized lipids. Fluorescence lifetime imaging microscopy and MD simulations give evidence for the presence of membrane regions of different local order in the oxidized membranes. We hypothesize that oxidation promotes stronger lipid-lipid interactions, which lead to an increase in the lateral heterogeneity within the bilayer and the creation of lipid clusters of higher order. Lipid peroxidation plays an important role in cell signaling and disease as well as therapy, however, the effect on the membrane structure and mechanical behavior remains poorly understood. Here, the authors report the direct and quantitative measurement of the bilayer's structure and viscoelastic properties upon peroxidation of lipid membranes. [ABSTRACT FROM AUTHOR]

Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch‐on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM). [ABSTRACT FROM AUTHOR]

Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch‐on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM). [ABSTRACT FROM AUTHOR]

Four‐stranded G‐quadruplex (G4) DNA is a non‐canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA‐M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo‐bridged and isopropyl‐bridged diazatriangulenium dyes, BDATA‐M2 and CDATA‐M2 respectively, featuring ethyl‐morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA‐M2 and CDATA‐M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium‐based G4 probes. [ABSTRACT FROM AUTHOR]

A new family of robust, non‐toxic, water‐compatible ruthenium(II) vinyl probes allows the rapid, selective and sensitive detection of endogenous carbon monoxide (CO) in live mammalian cells under normoxic and hypoxic conditions. Uniquely, these probes incorporate a viscosity‐sensitive BODIPY fluorophore that allows the measurement of microscopic viscosity in live cells via fluorescence lifetime imaging microscopy (FLIM) while also monitoring CO levels. This is the first example of a probe that can simultaneously detect CO alongside small viscosity changes in organelles of live cells. [ABSTRACT FROM AUTHOR]

A new family of robust, non‐toxic, water‐compatible ruthenium(II) vinyl probes allows the rapid, selective and sensitive detection of endogenous carbon monoxide (CO) in live mammalian cells under normoxic and hypoxic conditions. Uniquely, these probes incorporate a viscosity‐sensitive BODIPY fluorophore that allows the measurement of microscopic viscosity in live cells via fluorescence lifetime imaging microscopy (FLIM) while also monitoring CO levels. This is the first example of a probe that can simultaneously detect CO alongside small viscosity changes in organelles of live cells. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported. [ABSTRACT FROM AUTHOR]

The fluorescent protein aptly named “Killer Red” (KRed) is capable of killing transfected cells and inactivating fused proteins upon exposure to visible light in the presence of oxygen. We have investigated the source of the bioactive species through a variety of photophysical and photochemical techniques. Our results indicate a Type I (electron transfer mediated) photosensitizing mechanism. [ABSTRACT FROM AUTHOR]

Changes in microscopic viscosity and macromolecular crowding accompany the transition of proteins from their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated that viscosity sensitive fluorophores termed ‘molecular rotors’, when freely mixed with monomers of interest, are able to report on changes in microrheology accompanying amyloid formation, and measured an increase in rigidity of approximately three orders of magnitude during aggregation of lysozyme and insulin. Here we extend this strategy by covalently attaching molecular rotors to several proteins capable of assembly into fibrils, namely lysozyme, fibrinogen and amyloid-β peptide (Aβ(1–42)). We demonstrate that upon covalent attachment the molecular rotors can successfully probe supramolecular assembly in vitro . Importantly, our new strategy has wider applications in cellulo and in vivo , since covalently attached molecular rotors can be successfully delivered in situ and will colocalise with the aggregating protein, for example inside live cells. This important advantage allowed us to follow the microscopic viscosity changes accompanying blood clotting and during Aβ(1–42) aggregation in live SH-SY5Y cells. Our results demonstrate that covalently attached molecular rotors are a widely applicable tool to study supramolecular protein assembly and can reveal microrheological features of aggregating protein systems both in vitro and in cellulo not observable through classical fluorescent probes operating in light switch mode. [ABSTRACT FROM AUTHOR]

Conjugated porphyrin dimers have emerged as versatile viscosity-sensitive fluorophores that are suitable for quantitative measurements of microscopic viscosity by ratiometric and fluorescence lifetime-based methods, in a concentration-independent manner. Here, we investigate the effect of extended conjugation in a porphyrin-dimer structure on their ability to sense viscosity and temperature. We show that the sensitivity of the fluorescence lifetime to temperature is a unique property of only a few porphyrin dimers. [ABSTRACT FROM AUTHOR]

The transfer of material from phospholipid-coated microbubbles to cell membranes has been hypothesized to play a role in ultrasound-mediated drug delivery. In this study, we employed quantitative fluorescence microscopy techniques to investigate this phenomenon in both artificial and biological membrane bilayers in an acoustofluidic system. The results of the present study provide strong evidence for the transfer of material from microbubble coatings into cell membranes. Our results indicate that transfer of phospholipids alters the organization of molecules in cell membranes, specifically the lipid ordering or packing, which is known to be a key determinant of membrane mechanical properties, protein dynamics, and permeability. We further show that polyethylene-glycol, used in many clinical microbubble formulations, also has a major impact on both membrane lipid ordering and the extent of lipid transfer, and that this occurs even in the absence of ultrasound exposure. [ABSTRACT FROM AUTHOR]

Molecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscosity-sensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes. [ABSTRACT FROM AUTHOR]

In this work, we investigated how a combination of ethanol and high temperature (70 °C), affect the properties of the inner membrane of Bacillus subtilis spores. We observed membrane permeabilization for ethanol concentrations ≥ 50%, as indicated by the staining of the spores' DNA by the cell impermeable dye Propidium Iodide. The loss of membrane integrity was also confirmed by a decrease in the peak corresponding to dipicolinic acid using infrared spectroscopy. Finally, the spore refractivity (as measured by phase contrast microscopy) was decreased after the ethanol-heat treatment, suggesting a partial rehydration of the protoplast. Previously we have used fluorescent lifetime imaging microscopy (FLIM) combined with the fluorescent molecular rotor Bodipy-C 12 to study the microscopic viscosity in the inner membrane of B. subtilis spores, and showed that at normal conditions it is characterized by a very high viscosity. Here we demonstrate that the ethanol/high temperature treatment led to a decrease of the viscosity of the inner membrane, from 1000 cP to 860 cP for wild type spores at 50% of ethanol. Altogether, our present work confirms the deleterious effect of ethanol on the structure of B. subtilis spores, as well as demonstrates the ability of FLIM — Bodipy-C 12 to measure changes in the microviscosity of the spores upon perturbation. [ABSTRACT FROM AUTHOR]

Nucleic acids can adopt non-duplex topologies, such as G-quadruplexes in vitro. Yet it has been challenging to establish their existence and function in vivo due to a lack of suitable tools. Recently, we identified the triangulenium compound DAOTA-M2 as a unique fluorescence probe for such studies. This probe's emission lifetime is highly dependent on the topology of the DNA it interacts with opening up the possibility of carrying out live-cell imaging studies. Herein, we describe the origin of its fluorescence selectivity for G-quadruplexes. Cyclic voltammetry predicts that the appended morpholino groups can act as intra- molecular photo-induced electron transfer (PET) quenchers. Photophysical studies show that a delicate balance between this effect and inter-molecular PET with nucleobases is key to the overall fluorescence enhancement observed upon nucleic acid binding. We utilised computational modelling to demonstrate a conformational dependence of intra-molecular PET. Finally, we performed orthogonal studies with a triangulenium compound, in which the morpholino groups were removed, and demonstrated that this change inverts triangulenium fluorescence selectivity from G-quadruplex to duplex DNA, thus highlighting the importance of fine tuning the molecular structure not only for target affinity, but also for fluorescence response. [ABSTRACT FROM AUTHOR]

Mechanisms of prebiotic compartmentalization are central to providing insights into how protocellular systems emerged on the early Earth. Protocell models are based predominantly on the membrane self-assembly of fatty-acid vesicles, although membrane-free scenarios that involve liquid-liquid microphase separation (coacervation) have also been considered. Here we integrate these alternative models of prebiotic compartmentalization and develop a hybrid protocell model based on the spontaneous self-assembly of a continuous fatty-acid membrane at the surface of preformed coacervate microdroplets prepared from cationic peptides/polyelectrolytes and adenosine triphosphate or oligo/polyribonucleotides. We show that the coacervate-supported membrane is multilamellar, and mediates the selective uptake or exclusion of small and large molecules. The coacervate interior can be disassembled without loss of membrane integrity, and fusion and growth of the hybrid protocells can be induced under conditions of high ionic strength. Our results highlight how notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration can be integrated in protocell models via simple chemical and physical processes. [ABSTRACT FROM AUTHOR]

Abstract: We utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress. [Copyright &y& Elsevier]

Simple Summary: Understanding the role of the plasma membrane in the responses of cancer cells to chemotherapy is important because the cell membrane is directly involved in drug transport and the regulation of numerous biological processes. However, the role of the plasma membrane in cell resistance to platinum drugs like oxaliplatin is not fully understood. In this study we identified the changes to plasma membrane viscosity and lipid composition induced by oxaliplatin in responsive, cultured cancer cells and in mouse tumors. It was also found that the acquisition of chemoresistance is accompanied by modification of membrane lipids in ways that preserve the viscous properties unchanged upon further treatment. Therefore, new therapeutic approaches could be developed to reverse chemoresistance based on membrane lipid modifications and the de-stabilisation of membrane viscosity. Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer. [ABSTRACT FROM AUTHOR]

An eye lens is constantly exposed to the solar UV radiation, which is considered the most important external source of age-related changes to eye lens constituents. The accumulation of modifications of proteins and lipids with age can eventually lead to the development of progressive lens opacifications, such as cataracts. Though the impact of solar UV radiation on the structure and function of proteins is actively studied, little is known about the effect of photodamage on plasma membranes of lens cells. In this work we exploit Fluorescence Lifetime Imaging Microscopy (FLIM), together with viscosity-sensitive fluorophores termed molecular rotors, to study the changes in viscosity of plasma membranes of porcine eye lens resulting from two different types of photodamage: Type I (electron transfer) and Type II (singlet oxygen) reactions. We demonstrate that these two types of photodamage result in clearly distinct changes in viscosity – a decrease in the case of Type I damage and an increase in the case of Type II processes. Finally, to simulate age-related changes that occur in vivo , we expose an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts. [Display omitted] • Type I damage leads to a decrease of viscosity within membranes. • Type II damage results in an increase of viscosity within membranes. • Photosensitizer within intracellular milieu mediates Type I damage to both proteins and membranes. • Simulation of age-related impact of UV-A radiation revealed Type I damage to membranes. • Changes in membrane viscosity may alter the transport of metabolites and predispose lens to cataract development. [ABSTRACT FROM AUTHOR]

Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of micro- bubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a "molecular rotor" embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component. [ABSTRACT FROM AUTHOR]

Singlet oxygen, O2(a1Δg), was produced upon pulsed-laser irradiation of an intracellular photosensitizer and detected by its 1275 nm O2(a1Δg) → O2(X3Σg–) phosphorescence in time-resolved experiments using (1) individual mammalian cells on the stage of a microscope and (2) suspensions of mammalian cells in a 1 cm cuvette. Data were recorded using hydrophilic and, independently, hydrophobic sensitizers. The microscope-based single cell results are consistent with a model in which the behavior of singlet oxygen reflects the environment in which it is produced; nevertheless, the data also indicate that a significant fraction of a given singlet oxygen population readily crosses barriers between phase-separated intracellular domains. The singlet oxygen phosphorescence signals reflect the effects of singlet-oxygen-mediated damage on cell components which, at the limit, mean that data were collected from dead cells and, in some cases, reflect contributions from both intracellular and extracellular populations of singlet oxygen. Despite the irradiation-induced changes in the environment to which singlet oxygen is exposed, the “inherent” intracellular lifetime of singlet oxygen does not appear to change appreciably as the cell progresses toward death. The results obtained from cell suspensions reflect key features that differentiate cell ensemble from single cell experiments (e.g., the ensemble experiment is more susceptible to the effects of sensitizer that has leaked out of the cell). Overall, the data clearly indicate that measuring the intracellular lifetime of singlet oxygen in a O2(a1Δg) → O2(X3Σg–) phosphorescence experiment is a challenging endeavor that involves working with a dynamic system that is perturbed during the measurement. The most important aspect of this study is that it establishes a useful framework through which future singlet oxygen data from cells can be interpreted. [ABSTRACT FROM AUTHOR]

Oxidation of DNA represents a major pathway of genetic mutation. We have applied infrared spectroscopy in 77 K glass with supporting density functional theory (DFT) calculations (EDF1/6-31+G*) to provide an IR signature of the guanine radical cation G+•, formed as a result of 193 nm photoionization of DNA. Deprotonation of this species to produce the neutral radical G(−H)• does not occur in 77 K glass. DFT calculations indicate that the formation of G+• within the double helix does not significantly perturb the geometry of the G/C pair, even though there is a significant movement of the N1 proton away from G toward C. However, this is in stark contrast to drastic changes that are expected if full deprotonation of G/C occurs, producing the G(−H)•/C pair. These results are discussed in light of solution-phase time-resolved IR spectroscopic studies and demonstrate the power of IR to follow dynamics of DNA damage in natural environments. [ABSTRACT FROM AUTHOR]

In the title compound, [Re(C10H8N2)(C6H15O3P)2(CO)2]PF6, the carbonyl ligands are mutually cis and lie trans to the N donors of the 2,2′-bipyridyl ligand, while the triethoxy­phosphine ligands are mutually trans. The ReI centres exhibit approximate octa­hedral coordination, the major distortions from which arise from the narrow bite angle of the 2,2′-bipyridyl ligand which is expressed in the N—Re—N angles of 74.0 (3) and 74.3 (3)°. There are two cations and two anions in the asymmetric unit. The title compound is of inter­est because it has been shown to be a photocatalyst for CO2 reduction [Ishitani, George, Ibusuki, Johnson, Koike, Nozaki, Pac, Turner & Westwell (1994). Inorg. Chem. 33, 4712–4717]. [ABSTRACT FROM AUTHOR]

The spatial control of optical absorption provided by two-photon excitation has led to tremendous advances in microscopy and microfabrication. Medical applications of two-photon excitation in photodynamic therapy have been widely suggested, but thus far have been rendered impractical by the low two-photon cross-sections of photosensitizer drugs (which are compounds taken up by living tissues that become toxic on absorption of light). The invention of efficient two-photon activated drugs will allow precise three-dimensional manipulation of treatment volumes, providing a level of targeting unattainable with current therapeutic techniques. Here we present a new family of photodynamic therapy drugs designed for efficient two-photon excitation and use one of them to demonstrate selective closure of blood vessels through two-photon excitation photodynamic therapy in vivo. These conjugated porphyrin dimers have two-photon cross-sections that are more than two orders of magnitude greater than those of standard clinical photosensitizers. This is the first demonstration of in vivo photodynamic therapy using a photosensitizer engineered for efficient two-photon excitation. [ABSTRACT FROM AUTHOR]

Molecular rotors have emerged as versatile probes for microscopic viscosity in live cells, however, the exclusive localisation of rotors in the plasma membrane has remained elusive. We report the synthesis, spectroscopic characterisation and live cell imaging of a new BODIPY-based molecular rotor suitable for mapping viscosity in the cell plasma membrane. [ABSTRACT FROM AUTHOR]

Significance: Despite the importance of the cell membrane in regulation of drug activity, the influence of drug treatments on its physical properties is still poorly understood. The combination of fluorescence lifetime imaging microscopy (FLIM) with specific viscosity-sensitive fluorescent molecular rotors allows the quantification of membrane viscosity with high spatiotemporal resolution, down to the individual cell organelles. Aim: The aim of our work was to analyze microviscosity of the plasma membrane of living cancer cells during chemotherapy with cisplatin using FLIM and correlate the observed changes with lipid composition and cell's response to treatment. Approach: FLIM together with viscosity-sensitive boron dipyrromethene-based fluorescent molecular rotor was used to map the fluidity of the cell's membrane. Chemical analysis of membrane lipid composition was performed with time-of-flight secondary ion mass spectrometry (ToF-SIMS). Results: We detected a significant steady increase in membrane viscosity in viable cancer cells, both in cell monolayers and tumor spheroids, upon prolonged treatment with cisplatin, as well as in cisplatin-adapted cell line. ToF-SIMS revealed correlative changes in lipid profile of cisplatin-treated cells. Conclusions: These results suggest an involvement of membrane viscosity in the cell adaptation to the drug and in the acquisition of drug resistance. [ABSTRACT FROM AUTHOR]

Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed "molecular rotors", in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation. In order to correlate the viscosity values with membrane lipid composition, the detailed analysis of the corresponding membrane lipid composition of differentiated cells was performed by time-of-flight secondary ion mass spectrometry. Our results directly demonstrate for the first time that differentiation of MSCs results in distinct membrane viscosities, that reflect the change in lipidome of the cells following differentiation. [ABSTRACT FROM AUTHOR]