104 results found
Kuimova M, Kubankova M, Chambers J, et al., 2019, Linker length affects photostability of protein-targeted sensor of cellular microviscosity, Methods and Applications in Fluorescence, Vol: 7, ISSN: 2050-6120
Viscosity sensitive fluorophores termed ‘molecular rotors’ represent a convenient and quantitative tool for measuring intracellular viscosity via Fluorescence Lifetime Imaging Microscopy (FLIM). We compare the FLIM performance of two BODIPY-based molecular rotors bound to HaloTag protein expressed in different subcellular locations. While both rotors are able to penetrate live cells and specifically label the desired intracellular location, we found that the rotor with a longer HaloTag protein recognition motif was significantly affected by photo-induced damage when bound to the HaloTag protein, while the other dye showed no changes upon irradiation. Molecular dynamics modelling indicates that the irradiation-induced electron transfer between the BODIPY moiety and the HaloTag protein is a plausible explanation for these photostability issues. Our results demonstrate that binding to the targeted protein may significantly alter the photophysical behaviour of a fluorescent probe and therefore its thorough characterisation in the protein bound form is essential prior to any in vitro and in cellulo applications.
Kubankova M, Summers P, Lopez-Duarte I, et al., 2019, Microscopic viscosity of neuronal plasma membranes measured using fluorescent molecular rotors: effects of oxidative stress and neuroprotection., ACS Applied Materials and Interfaces, Vol: 11, Pages: 36307-36315, ISSN: 1944-8244
Molecular mobility in neuronal plasma membranes is a crucial factor in brain function. Microscopic viscosity is an important parameter that determines molecular mobility. This study presents the first direct measurements of the microviscosity of plasma membranes of live neurons. Microviscosity maps were obtained using fluorescence lifetime imaging of environment-sensing dyes termed 'molecular rotors'. Neurons were investigated both in the basal state and following common neurodegenerative stimuli, excitotoxicity or oxidative stress. Both types of neurotoxic challenges induced microviscosity decrease in cultured neurons, and the oxidant-induced membrane fluidification was counteracted by the wide-spectrum neuroprotectant, the H3 peptide. These results provide new insights into molecular mobility in neuronal membranes, paramount for basic brain function, and suggest that preservation of membrane stability may be an important aspect of neuroprotection in brain insults and neurodegenerative disorders.
Perez MP, Kuimova MK, Brooks NJ, 2019, Reorganization of liquid ordered lipid domains dampens changes in membrane tension, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S232-S232, ISSN: 0175-7571
Kubánková M, Lin X, Albrecht T, et al., 2019, Rapid fragmentation during seeded lysozyme aggregation revealed at the single molecule level, Analytical Chemistry, ISSN: 0003-2700
Protein aggregation is associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The poorly understood pathogenic mechanism of amyloid diseases makes early stage diagnostics or therapeutic intervention a challenge. Seeded polymerization that reduces the duration of the lag phase and accelerates fibril growth is a widespread model to study amyloid formation. Seeding effects are hypothesized to be important in the "infectivity" of amyloids and are linked to the development of systemic amyloidosis in vivo. The exact mechanism of seeding is unclear yet critical to illuminating the propagation of amyloids. Here we report on the lateral and axial fragmentation of seed fibrils in the presence of lysozyme monomers at short time scales, followed by the generation of oligomers and growth of fibrils.
Woodcock EM, Girvan P, Eckert J, et al., Measuring intracellular viscosity in conditions of hypergravity, Biophysical Journal, ISSN: 0006-3495
Gravity-sensitive cellular responses are regularly observed in both specialised and non-specialised cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel viscosity-sensitive molecular rotor based on meso-substituted boron-dipyrrin (BODIPY) used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the Large Diameter Centrifuge at the European Space Agency Technology Centre ESA-ESTEC. Mouse osteoblastic (MC3T3-E1) and endothelial (HUVEC) cell lines were tested and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first quantitative data for cellular viscosity changes under hypergravity, up to 15 g.
Kuimova M, Kubankova M, Kiryushko D, et al., 2018, Molecular rotors report on changes of live cell plasma membrane microviscosity upon interaction with beta-amyloid aggregates, Soft Matter, Vol: 14, Pages: 9466-9474, ISSN: 1744-683X
Amyloid deposits of aggregated beta-amyloid Aβ(1–42) peptides are a pathological hallmark of Alzheimer's disease. Aβ(1–42) aggregates are known to induce biophysical alterations in cells, including disruption of plasma membranes. We investigated the microviscosity of plasma membranes upon interaction with oligomeric and fibrillar forms of Aβ(1–42). Viscosity-sensing fluorophores termed molecular rotors were utilised to directly measure the microviscosities of giant plasma membrane vesicles (GPMVs) and plasma membranes of live SH-SY5Y and HeLa cells. The fluorescence lifetimes of membrane-inserting BODIPY-based molecular rotors revealed a decrease in bilayer microviscosity upon incubation with Aβ(1–42) oligomers, while fibrillar Aβ(1–42) did not significantly affect the microviscosity of the bilayer. In addition, we demonstrate that the neuroprotective peptide H3 counteracts the microviscosity change induced by Aβ(1–42) oligomers, suggesting the utility of H3 as a neuroprotective therapeutic agent in neurodegenerative disorders and indicating that ligand-induced membrane stabilisation may be a possible mechanism of neuroprotection during neurodegenerative disorders such as Alzheimer's disease.
Vysniauskas A, Kuimova MK, 2018, A twisted tale: measuring viscosity and temperature of microenvironments using molecular rotors, International Reviews in Physical Chemistry, Vol: 37, Pages: 259-285, ISSN: 0144-235X
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.
Chambers JE, Kubánková M, Huber RG, et al., 2018, An Optical Technique for Mapping Microviscosity Dynamics in Cellular Organelles., ACS Nano, Vol: 12, Pages: 4398-4407, ISSN: 1936-0851
Microscopic viscosity (microviscosity) is a key determinant of diffusion in the cell and defines the rate of biological processes occurring at the nanoscale, including enzyme-driven metabolism and protein folding. Here we establish a rotor-based organelle viscosity imaging (ROVI) methodology that enables real-time quantitative mapping of cell microviscosity. This approach uses environment-sensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartment-specific microviscosity measurements via fluorescence lifetime imaging. ROVI visualized spatial and temporal dynamics of microviscosity with suborganellar resolution, reporting on a microviscosity difference of nearly an order of magnitude between subcellular compartments. In the mitochondrial matrix, ROVI revealed several striking findings: a broad heterogeneity of microviscosity among individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarization. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes.
Vysniauskas A, Lopez Duarte I, Thompson AJ, et al., 2018, Surface functionalisation with viscosity-sensitive BODIPY molecular rotor, Methods and Applications in Fluorescence, Vol: 6, ISSN: 2050-6120
Surface functionalisation with viscosity sensitive dyes termed ‘molecular rotors’ can potentially open up new opportunities in sensing, for example for non-invasive biological viscosity imaging, in studying the effect of shear stress on lipid membranes and in cells, and in imaging contacts between surfaces upon applied pressure. We have functionalised microscope slides with BODIPY-based molecular rotor capable of viscosity sensing via its fluorescence lifetime. We have optimised functionalisation conditions and prepared the slides with the BODIPY rotor attached directly to the surface of glass slides and through polymer linkers of 5 kDa and 40 kDa in mass. The slides were characterised for their sensitivity to viscosity, and used to measure viscosity of supported lipid bilayers during photooxidation, and of giant unilamellar vesicles lying on the surface of the slide. We conclude that our functionalised slides show promise for a variety of viscosity sensing applications.
Clark R, Edel J, Kirchner B, et al., 2018, Ion diffusion in ionic liquids in electric fields, 255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Chambers J, Kubankova M, Huber R, et al., 2018, Measuring Endoplasmic Reticulum Viscosity in Cell Models of alpha 1-Antitrypsin Deficiency, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Shirmanova MV, Shimolina LE, Lukina MM, et al., 2017, Live Cell Imaging of Viscosity in 3D Tumour Cell Models, MULTI-PARAMETRIC LIVE CELL MICROSCOPY OF 3D TISSUE MODELS, Vol: 1035, Pages: 143-153, ISSN: 0065-2598
Kuimova MK, Vysniauskas, Lopez Duarte, et al., 2017, Exploring viscosity, polarity and temperature sensitivity of BODIPY-based molecular rotors, Physical Chemistry Chemical Physics, Vol: 19, Pages: 25252-25259, ISSN: 1463-9084
Microviscosity is a key parameter controlling the rate of diffusion and reactions on the microscale. One of the most convenient tools for measuring microviscosity is by fluorescent viscosity sensors termed ‘molecular rotors’. BODIPY-based molecular rotors in particular proved extremely useful in combination with fluorescence lifetime imaging microscopy, for providing quantitative viscosity maps of living cells as well as measuring dynamic changes in viscosity over time. In this work, we investigate several new BODIPY-based molecular rotors with the aim of improving on the current viscosity sensing capabilities and understanding how the structure of the fluorophore is related to its function. We demonstrate that due to subtle structural changes, BODIPY-based molecular rotors may become sensitive to temperature and polarity of their environment, as well as to viscosity, and provide a photophysical model explaining the nature of this sensitivity. Our data suggests that a thorough understanding of the photophysics of any new molecular rotor, in environments of different viscosity, temperature and polarity, is a must before moving on to applications in viscosity sensing.
Kubankova M, Lopez-Duarte I, Bull JA, et al., 2017, Probing supramolecular protein assembly using fluorescent molecular rotors, Publisher: SPRINGER, Pages: S331-S331, ISSN: 0175-7571
Aron M, Pereno V, Carugo D, et al., 2017, Mechanisms of microbubble mediated drug delivery, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S91-S91, ISSN: 0175-7571
Kubankova M, Chambers JE, Marciniak SJ, et al., 2017, Rotor-based organelle viscosity imaging, Publisher: SPRINGER, Pages: S331-S331, ISSN: 0175-7571
Vysniauskas A, Ding D, Qurashi M, et al., 2017, Tuning the sensitivity of fluorescent porphyrin dimers to viscosity and temperature, Chemistry-A European Journal, Vol: 23, Pages: 11001-11010, ISSN: 1521-3765
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.
Kuimova MK, Kubankova M, Lopez Duarte, et al., 2017, Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors, Biomaterials, Vol: 139, Pages: 195-201, ISSN: 1878-5905
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.
Sherin PS, Lopez-Duarte I, Dent MR, et al., 2017, Visualising the membrane viscosity of porcine eye lens cells using molecular rotors, CHEMICAL SCIENCE, Vol: 8, Pages: 3523-3528, ISSN: 2041-6520
The plasma membranes of cells within the eye lens play an important role in metabolite transport within the avascular tissue of the lens, maintaining its transparency over the entire lifespan of an individual. Here we use viscosity-sensitive ‘molecular rotors’ to map the microscopic viscosity within these unusual cell membranes, establishing that they are characterised by an unprecedentedly high degree of lipid organisation.
Davidson N, Tong H-J, Kalberer M, et al., 2017, Measurement of the Raman spectra and hygroscopicity of four pharmaceutical aerosols as they travel from pressurised metered dose inhalers (pMDI) to a model lung, INTERNATIONAL JOURNAL OF PHARMACEUTICS, Vol: 520, Pages: 59-69, ISSN: 0378-5173
Particle inhalation is an effective and rapid delivery method for a variety of pharmaceuticals, particularly bronchodilation drugs used for treating asthma and COPD. Conditions of relative humidity and temperature inside the lungs are generally very different from the outside ambient air, with the lung typically being warmer and more humid. Changes in humidity, from inhaler to lung, can cause hygroscopic phase transitions and particle growth. Increasing particle size and mass can negatively affect particle deposition within the lung leading to inefficient treatment, while deliquescence prior to impaction is liable to accelerate drug uptake. To better understand the hygroscopic properties of four pharmaceutical aerosol particles; pharmaceutical particles from four commercially available pressurised metered dose inhalers (pMDIs) were stably captured in an optical trap, and their composition was examined online via Raman spectroscopy. Micron-sized particles of salbutamol sulfate, salmeterol xinafoate, fluticasone propionate and ciclesonide were levitated and examined over a range of relative humidity values inside a chamber designed to mimic conditions within the respiratory tract. The effect of temperature upon hygroscopicity was also investigated for salbutamol sulfate particles. Salbutamol sulfate was found to have significant hygroscopicity, salmeterol xinafoate showed some hygroscopic interactions, whilst fluticasone propionate and ciclesonide revealed no observable hygroscopicity. Thermodynamic and structural modelling is used to explain the observed experimental results.
Shimolina LE, Izquierdo MA, Lopez-Duarte I, et al., 2017, Imaging tumor microscopic viscosity in vivo using molecular rotors, Scientific Reports, Vol: 7, ISSN: 2045-2322
The microscopic viscosity plays an essential role in cellular biophysics by controlling the rates of diffusion and bimolecular reactions within the cell interior. While several approaches have emerged that have allowed the measurement of viscosity and diffusion on a single cell level in vitro, the in vivo viscosity monitoring has not yet been realized. Here we report the use of fluorescent molecular rotors in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) to image microscopic viscosity in vivo, both on a single cell level and in connecting tissues of subcutaneous tumors in mice. We find that viscosities recorded from single tumor cells in vivo correlate well with the in vitro values from the same cancer cell line. Importantly, our new method allows both imaging and dynamic monitoring of viscosity changes in real time in live animals and thus it is particularly suitable for diagnostics and monitoring of the progress of treatments that might be accompanied by changes in microscopic viscosity.
Shirmanova MV, Lukina MM, Shimolina LE, et al., 2017, Probing energy metabolism and microviscosity in cancer using FLIM, Conference on Clinical and Preclinical Optical Diagnostics, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Carugo D, Aron M, Sezgin E, et al., 2016, Modulation of the molecular arrangement in artificial and biological membranes by phospholipid-shelled microbubbles, Biomaterials, Vol: 113, Pages: 105-117, ISSN: 1878-5905
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.
Athanasiadis A, Fitzgerald C, Davidson NM, et al., 2016, Dynamic viscosity mapping of the oxidation of squalene aerosol particles., Physical Chemistry Chemical Physics, Vol: 18, Pages: 30385-30393, ISSN: 1463-9084
Organic aerosols (OAs) play important roles in multiple atmospheric processes, including climate change, and can impact human health. The physico-chemical properties of OAs are important for all these processes and can evolve through reactions with various atmospheric components, including oxidants. The dynamic nature of these reactions makes it challenging to obtain a true representation of their composition and surface chemistry. Here we investigate the microscopic viscosity of the model OA composed of squalene, undergoing chemical aging. We employ Fluorescent Lifetime Imaging Microscopy (FLIM) in conjunction with viscosity sensitive probes termed molecular rotors, in order to image the changes in microviscosity in real time during oxidation with ozone and hydroxyl radicals, which are two key oxidising species in the troposphere. We also recorded the Raman spectra of the levitated particles to follow the reactivity during particle ozonolysis. The levitation of droplets was achieved via optical trapping that enabled simultaneous levitation and measurement via FLIM or Raman spectroscopy and allowed the true aerosol phase to be probed. Our data revealed a very significant increase in viscosity of the levitated squalene droplets upon ozonolysis, following their transformation from the liquid to solid phase that was not observable when the oxidation was carried out on coverslip mounted droplets. FLIM imaging with sub-micron spatial resolution also revealed spatial heterogeneity in the viscosity distribution of oxidised droplets. Overall, a combination of molecular rotors, FLIM and optical trapping is able to provide powerful insights into OA chemistry and the microscopic structure that enables the dynamic monitoring of microscopic viscosity in aerosol particles in their true phase.
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.
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