109 results found
Dent MR, López-Duarte I, Dickson CJ, et al., 2016, Imaging plasma membrane phase behaviour in live cells using a thiophene-based molecular rotor, Chemical Communications, Vol: 52, Pages: 13269-13272, ISSN: 1364-548X
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.
Kuimova MK, Mika JT, Thompson AJ, et al., 2016, Measuring the viscosity of the Escherichia coli plasma membrane using molecular rotors, Biophysical Journal, Vol: 111, Pages: 1528-1540, ISSN: 1542-0086
The viscosity is a highly important parameter within the cell membrane, affecting the diffusion ofsmall molecules and, hence, controlling the rates of intra-cellular reactions. There is significantinterest in the direct, quantitative assessment of membrane viscosity. Here we report the use offluorescence lifetime imaging microscopy (FLIM) of the molecular rotor BODIPY C10 in themembranes of live Escherichia coli (E. coli) bacteria to permit direct quantification of the viscosity.Using this approach we investigated the viscosity in live E. coli cells, spheroplasts and liposomesmade from E. coli membrane extracts. For live cells and spheroplasts the viscosity was measured atboth room temperature (23o C) and the E. coli growth temperature (37o C), while the membraneextract liposomes were studied over a range of measurement temperatures (5-40o C). At 37o C werecorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than thatpreviously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillusvegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid orderingwithin their liquid-phase plasma membranes.
Vysniauskas A, Qurashi M, Kuimova MK, 2016, A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress, Chemistry-A European Journal, Vol: 22, Pages: 13210-13217, ISSN: 1521-3765
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.
Fitzgerald C, Hosny NA, Tong H, et al., 2016, Fluorescence lifetime imaging of optically levitated aerosol: a technique to quantitatively map the viscosity of suspended aerosol particles, Physical Chemistry Chemical Physics, Vol: 18, Pages: 21710-21719, ISSN: 1463-9084
We describe a technique to measure the viscosity of stably levitated single micron-sized aerosol particles. Particle levitation allows the aerosol phase to be probed in the absence of potentially artefact-causing surfaces. To achieve this feat, we combined two laser based techniques: optical trapping for aerosol particle levitation, using a counter-propagating laser beam configuration, and fluorescent lifetime imaging microscopy (FLIM) of molecular rotors for the measurement of viscosity within the particle. Unlike other techniques used to measure aerosol particle viscosity, this allows for the non-destructive probing of viscosity of aerosol particles without interference from surfaces. The well-described viscosity of sucrose aerosol, under a range of relative humidity conditions, is used to validate the technique. Furthermore we investigate a pharmaceutically-relevant mixture of sodium chloride and salbutamol sulphate under humidities representative of in vivo drug inhalation. Finally, we provide a methodology for incorporating molecular rotors into already levitated particles, thereby making the FLIM/optical trapping technique applicable to real world aerosol systems, such as atmospheric aerosols and those generated by pharmaceutical inhalers.
Loison P, Gervais P, Perrier-Cornet JM, et al., 2016, Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores., Biochimica et Biophysica Acta, Vol: 1858, Pages: 2060-2069, ISSN: 0006-3002
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-C12 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 1000cP to 860cP 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-C12 to measure changes in the microviscosity of the spores upon perturbation.
Davidson N, Tong H-J, Kalberer M, et al., 2016, Measurement of the physico-chemical properties of pharmaceutical aerosols as they travel from pressurised metered dose inhalers (PMDI) to a model lung, Journal of Aerosol Medicine and Pulmonary Drug Delivery, Vol: 29, Pages: A25-A25, ISSN: 1941-2711
Thanh TV, Meallet-Renault R, Clavier G, et al., 2016, Tuning BODIPY molecular rotors into the red: sensitivity to viscosity vs. temperature, Journal of Materials Chemistry C, Vol: 4, Pages: 2828-2833, ISSN: 2050-7534
Viscosity variations in the microscopic world are of paramount importance for diffusion and reactions. In the last decade a new class of fluorescent probes for measuring viscosity has emerged termed ‘molecular rotors’, which allows quantitative mapping of viscosity in microscopically heterogeneous environments. Here we attempt to tune the absorption and emission of one such ‘molecular rotor’ based on the BODIPY fluorescent core into the red region of the spectrum, to allow better compatibility with the ‘tissue optical window’ and imaging of cells and tissues. We consequently find that our red-emitting BODIPY fluorophores are sensitive to environmental temperature rather than to viscosity, thus suggesting a new prototype for a ‘molecular thermometer’.
Vilar Compte R, Shivalingam A, Vysniauskas A, et al., 2016, Trianguleniums as optical probes for G-quadruplexes: A photophysical, electrochemical and computational study, Chemistry - A European Journal, Vol: 22, Pages: 4129-4139, ISSN: 0947-6539
Nucleic acids can adopt non-duplex topologies such as G-quadruplexes in vitro. Yet it hasbeen challenging to establish their existence and function in vivo due to a lack of suitabletools. Recently, we identified the triangulenium compound DAOTA-M2 as a uniquefluorescence probe for such studies. This probe’s emission lifetime is highly dependent onthe topology of the DNA it interacts with opening up the possibility of carrying out live cellimaging studies. Herein we describe the origin of its fluorescence selectivity for Gquadruplexes.Cyclic voltammetry predicts that the appended morpholino groups can act asintra-molecular photo-induced electron transfer (PET) quenchers. Photophysical studies showthat a delicate balance between this effect and inter-molecular PET with nucleobases is key tothe overall fluorescence enhancement observed upon nucleic acid binding. We utilisedcomputational modelling to demonstrate a conformational dependence of intra-molecularPET. Finally, we performed orthogonal studies with a triangulenium compound where themorpholino groups were removed and demonstrate that this change inverts trianguleniumfluorescence selectivity from G-quadruplex to duplex DNA, thus highlighting the importanceof fine-tuning the molecular structure not only for target affinity but also for fluorescenceresponse.
Hosny NA, Fitzgerald C, Vysniauskas A, et al., 2016, Direct imaging of changes in aerosol particle viscosity upon hydration and chemical aging, Chemical Science, Vol: 7, Pages: 1357-1367, ISSN: 2041-6539
Organic aerosol particles (OA) play major roles in atmospheric chemistry, climate, and public health. Aerosol particle viscosity is highly important since it can determine the ability of chemical species such as oxidants, organics or water to diffuse into the particle bulk. Recent measurements indicate that OA may be present in highly viscous states, however, diffusion rates of small molecules such as water are not limited by these high viscosities. Direct observational evidence of kinetic barriers caused by high viscosity and low diffusivity in aerosol particles were not available until recently; and techniques that are able to dynamically quantify and track viscosity changes during atmospherically relevant processes are still unavailable for atmospheric aerosols. Here we report quantitative, real-time, online observations of microscopic viscosity changes in aerosol particles of atmospherically relevant composition, using fluorescence lifetime imaging (FLIM) of viscosity. We show that microviscosity in ozonated oleic acid droplets and secondary organic aerosol (SOA) particles formed by ozonolysis of myrcene increases substantially with decreasing humidity and atmospheric oxidative aging processes. Furthermore, we found unexpected heterogeneities of microviscosity inside individual aerosol particles. The results of this study enhance our understanding of organic aerosol processes on microscopic scales and may have important implications for the modeling of atmospheric aerosol growth, composition and interactions with trace gases and clouds.
Shivalingam A, Izquierdo MA, Le Marois A, et al., 2015, The interactions between a small molecule and G-quadruplexes are visualised by fluorescence lifetime imaging microscopy, Nature Communications, Vol: 6, ISSN: 2041-1723
Guanine-rich oligonucleotides can fold into quadruple-stranded helical structures known as G-quadruplexes. Mounting experimental evidence has gathered suggesting that these non-canonical nucleic acid structures form in vivo and play essential biological roles. However, to date, there are no small-molecule optical probes to image G-quadruplexes in live cells. Herein, we report the design and development of a small fluorescent molecule, which can be used as an optical probe for G-quadruplexes. We demonstrate that the fluorescence lifetime of this new probe changes considerably upon interaction with different nucleic acid topologies. Specifically, longer fluorescence lifetimes are observed in vitro for G-quadruplexes than for double- and single-stranded nucleic acids. Cellular studies confirm that this molecule is cell permeable, has low cytotoxicity and localizes primarily in the cell nucleus. Furthermore, using fluorescence lifetime imaging microscopy, live-cell imaging suggests that the probe can be used to study the interaction of small molecules with G-quadruplexes in vivo.
Thompson AJ, Herling TW, Kubankova M, et al., 2015, Molecular Rotors Provide Insights into Microscopic Structural Changes During Protein Aggregation., Journal of Physical Chemistry B, Vol: 119, Pages: 10170-10179, ISSN: 1520-6106
Changes in microscopic viscosity represent an important characteristic of structural transitions in soft matter systems. Here we demonstrate the use of molecular rotors to explore the changes in microrheology accompanying the transition of proteins from their soluble states into a gel phase composed of amyloid fibrils. The formation of beta-sheet rich protein aggregates, including amyloid fibrils, is a hallmark of a number of neurodegenerative disorders, and as such, the mechanistic details of this process are actively sought after. In our experiments, molecular rotors report an increase in rigidity of approximately three orders of magnitude during the aggregation reaction. Moreover, phasor analysis of the fluorescence decay signal from the molecular rotors suggests the presence of multiple distinct mechanistic stages during the aggregation process. Our results show that molecular rotors can reveal key microrheological features of protein systems not observable through classical fluorescent probes operating in light switch mode.
Vysniauskas A, Qurashi M, Gallop N, et al., 2015, Unravelling the effect of temperature on viscosity-sensitive fluorescent molecular rotors, CHEMICAL SCIENCE, Vol: 6, Pages: 5773-5778, ISSN: 2041-6520
Viscosity and temperature variations in the microscopic world are of paramount importance for diffusion and reactions. Consequently, a plethora of fluorescent probes have evolved over the years to enable fluorescent imaging of both parameters in biological cells. However, the simultaneous effect of both temperature and viscosity on the photophysical behavior of fluorophores is rarely considered, yet unavoidable variations in temperature can lead to significant errors in the readout of viscosity and vice versa. Here we examine the effect of temperature on the photophysical behavior of three classes of viscosity-sensitive fluorophores termed ‘molecular rotors’. For each of the fluorophores we decouple the effect of temperature from the effect of viscosity. In the case of the conjugated porphyrin dimer, we demonstrate that, uniquely, simultaneous dual-mode lifetime and intensity measurements of this fluorophore can be used for measuring both viscosity and temperature concurrently.
Dent M, Lopez-Duarte I, Bull J, et al., 2015, Imaging patterns in lipid membranes through the use of molecular rotors, EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, Vol: 44, Pages: S107-S107, ISSN: 0175-7571
Kubankova M, Kuimova M, 2015, Molecular rotors as novel probes of protein aggregation, EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, Vol: 44, Pages: S80-S80, ISSN: 0175-7571
Mika JT, Thompson AJ, Hofkens J, et al., 2015, Measurement of the Viscosity of E. coli Membranes using Molecular Rotors and Flim, 59th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 542A-542A, ISSN: 0006-3495
Dent MR, Lopez-Duarte I, Dickson CJ, et al., 2015, Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors, PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol: 17, Pages: 18393-18402, ISSN: 1463-9076
Vysniauskas A, Balaz M, Anderson HL, et al., 2015, Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer, PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol: 17, Pages: 7548-7554, ISSN: 1463-9076
Izquierdo MA, Vyšniauskas A, Lermontova SA, et al., 2015, Dual use of porphyrazines as sensitizers and viscosity markers in photodynamic therapy, J. Mater. Chem. B, Vol: 3, Pages: 1089-1096, ISSN: 2050-750X
Richardson RD, Baud MGJ, Weston CE, et al., 2015, Dual wavelength asymmetric photochemical synthesis with circularly polarized light, Chemical Science, Vol: 6, Pages: 3853-3862, ISSN: 2041-6539
Tang T-YD, Hak CRC, Thompson AJ, et al., 2014, Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model, NATURE CHEMISTRY, Vol: 6, Pages: 527-533, ISSN: 1755-4330
Lopez-Duarte I, Thanh TV, Izquierdo MA, et al., 2014, A molecular rotor for measuring viscosity in plasma membranes of live cells, CHEMICAL COMMUNICATIONS, Vol: 50, Pages: 5282-5284, ISSN: 1359-7345
Thompson AJ, Tang T-YD, Herling TW, et al., 2014, Quantitative sensing of microviscosity in protocells and amyloid materials using fluorescence lifetime imaging of molecular rotors, Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XII, Publisher: SPIE- Society of Photo-optical Instrumentation Engineers, ISSN: 0277-786X
Molecular rotors are fluorophores that have a fluorescence quantum yield that depends upon intermolecular rotation. The fluorescence quantum yield, intensity and lifetime of molecular rotors all vary as functions of viscosity, as high viscosities inhibit intermolecular rotation and cause an increase in the non-radiative decay rate. As such, molecular rotors can be used to probe viscosity on microscopic scales. Here, we apply fluorescence lifetime imaging microscopy (FLIM) to measure the fluorescence lifetimes of three different molecular rotors, in order to determine the microscopic viscosity in two model systems with significant biological interest. First, the constituents of a novel protocell – a model of a prebiotic cell – were studied using the molecular rotors BODIPY C10 and kiton red. Second, amyloid formation was investigated using the molecular rotor Cy3. © (2014) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Karim NHA, Mendoza O, Shivalingam A, et al., 2014, Salphen metal complexes as tunable G-quadruplex binders and optical probes, RSC ADVANCES, Vol: 4, Pages: 3355-3363, ISSN: 2046-2069
Tong H-J, Fitzgerald C, Gallimore PJ, et al., 2014, Rapid interrogation of the physical and chemical characteristics of salbutamol sulphate aerosol from a pressurised metered-dose inhaler (pMDI), CHEMICAL COMMUNICATIONS, Vol: 50, Pages: 15499-15502, ISSN: 1359-7345
Loison P, Hosny NA, Gervais P, et al., 2013, Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: A molecular rotor/FLIM study, BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, Vol: 1828, Pages: 2436-2443, ISSN: 0005-2736
Kuimova MK, Phillips D, 2013, Photomedicine, Applied Photochemistry, Pages: 331-347, ISBN: 9789048138296
© 2013 Springer Science+Business Media Dordrecht. All rights reserved. This chapter discusses the various modalities of photomedicine, an interdisciplinary branch of medicine that involves the study and application of light with respect to health and disease. The following main concepts are covered: Photodynamic Therapy (PDT) for the treatment of cancer, PDT for bacterial infections, vascular PDT, photochemical internalisation, photochemical tissue bonding and the use of lasers in medicine.
Hosny NA, Mohamedi G, Rademeyer P, et al., 2013, Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: 9225-9230, ISSN: 0027-8424
Mohamedi G, Hosny NA, Rademeyer P, et al., 2013, Investigating the effect of fabrication method on the stability and acoustic response of microbubble agents., J Acoust Soc Am, Vol: 133
Microbubbles stabilized by a surfactant or polymer coating are already in clinical use as ultrasound imaging contrast agents. They have also been widely investigated as vehicles for drug delivery and gene therapy that can be tracked and triggered using ultrasound. Extensive studies have been made of the effects of the coating material and gas core on microbubble characteristics, but the influence of the fabrication method has received less attention. The aim of this study was to compare the behavior of microbubbles prepared using different techniques. Phospholipid-coated microbubbles were produced using sonication, electrospraying, or in a specially designed microfluidic device. The microbubbles were observed using optical, electron, and fluorescence lifetime imaging microscopy (FLIM) to interrogate their surface microstructure and stability over time. Their acoustic response was then determined in a flow chamber by detecting the pressure scattered from individual microbubbles as they passed through the focal region of a transducer (center frequencies 1, 2.25, and 3.5 MHz; peak negative pressures 50-300 kPa). The method of bubble generation was found to significantly affect the bubble surface characteristics, stability, and acoustic response. The results demonstrate that the processing method affects not only the bubble size distribution but other characteristics important for biomedical applications.
Mohamedi G, Hosny NA, Rademeyer P, et al., 2013, Influence of fabrication method on functional properties of microbubble agents for theranostic applications, Symp on Physicochemical Characterization of Organic Matter: Past, Present, Future, and Role of Environment / 245th Natl Spring Meeting of the Amer-Chem-Soc (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
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