1,721,332 research outputs found
Orientational distribution of DPH in lipid membranes: a comparison of molecular dynamics calculations and experimental time-resolved anisotropy experiments
No full textCharacterization of the membrane phases is a crucial task in cell biology. Cells differ in composition of the lipids and consequently in adopted phases. The phases can be discriminated based upon lipid ordering and molecular diffusion and their identification could be used for characterization of cell membranes. Here we used molecular dynamics (MD) simulations to study the behavior of the fluorescent reporter molecule diphenylhexatriene (DPH) in different lipid phases - liquid disordered (L-d), liquid ordered (L-o), and solid ordered (S-o) composed of phosphatidylcholines (L-d and S-o) or a sphingomyelin/cholesterol (SM/Chol) mixture (L-o). To the best of our knowledge, this is the first simulation of DPH in L-o SM/Chol and S-o DPPC membranes. For the considered membrane compositions DPH is mostly oriented parallel to lipid tails. In the L-o phase we observed a significant fraction of DPH positioned in between membrane leaflets, which agrees with experimental findings, but which has not been observed in previous MD simulations of DPH in phosphatidylcholine membranes. Further, we calculated rotational autocorrelation functions (ROTACF) from our MD simulations in order to model the time-resolved fluorescence anisotropy decay. We observed that order parameters P-2 and P-4 are sufficient to fully describe the orientation distribution of DPH. We analyzed the ROTACFs by a so-called general model for the time-resolved fluorescence anisotropy [W. van der Meer et al., Biophys. J., 1984, 46, 515] and observed an overestimation of P-4. We suggest a rescaling of the recovered P-4 yielding an orientation distribution of DPH close to the one observed in our MD simulations.The Swedish Infrastructure Committee (SNIC) is acknowledged for the computational time granted through the medium allocations 3-397, 3-156, 3-396, 3-23 which were active in 2018. Gratitude goes also to the VSC (Flemish Supercomputer Center) for providing computational time on the Breniac cluster. M. P. acknowledges the Department of Theoretical Chemistry and Biology at KTH for her post-doctoral scholarship and is grateful for the Special Research Fund (BOF) of Hasselt University
Fluorescence lifetime microscopy of the sodium indicators SBFI and sodium green in HeLa cells.
No full textFluorescence lifetime microscopy of the sodium indicators SBFI and sodium green in HeLa cells.
No full textGeneralized polarization and time-resolved fluorescence provide evidence for different populations of Laurdan in lipid vesicles
No full textThe solvatochromic dye Laurdan is widely used in sensing the lipid packing of both model and biological membranes. The fluorescence emission maximum shifts from about 440 nm (blue channel) in condensed membranes (So) to about 490 nm (green channel) in the liquid-crystalline phase (Lα). Although the fluorescence intensity based generalized polarization (GP) is widely used to characterize lipid membranes, the fluorescence lifetime of Laurdan, in the blue and the green channel, is less used for that purpose. Here we explore the correlation between GP and fluorescence lifetimes by spectroscopic measurements on the So and Lα phases of large unilamellar vesicles of DMPC and DPPC. A positive correlation between GP and the lifetimes is observed in each of the optical channels for the two lipid phases. Microfluorimetric determinations on giant unilamellar vesicles of DPPC and DOPC at room temperature are performed under linearly polarized two-photon excitation to disentangle possible subpopulations of Laurdan at a scale below the optical resolution. Fluorescence intensities, GP and fluorescence lifetimes depend on the angle between the orientation of the linear polarization of the excitation light and the local normal to the membrane of the optical cross-section. This angular variation depends on the lipid phase and the emission channel. GP and fluorescence intensities in the blue and green channel in So and in the blue channel in Lα exhibit a minimum near 90o. Surprisingly, the intensity in the green channel in Lα reaches a maximum near 90o. The fluorescence lifetimes in the two optical channels also reach a pronounced minimum near 90o in So and Lα, apart from the lifetime in the blue channel in Lα where the lifetime is short with minimal angular variation. To our knowledge, these experimental observations are the first to demonstrate the existence of a bent conformation of Laurdan in lipid membranes, as previously suggested by molecular dynamics calculations.The authors are grateful to prof. L. Bagatolli for discussions at some point of this work, to Dr. N. Smisdom for advices regarding the use of the confocal microscope and data analysis of the confocal images and to Dr. R. Paesen for the home-made automated polarization controller device. The authors thank prof. M. Roeffaers for the ITO coated coverslips that were essential in the generation of GUV. S. O. is grateful to the National Science Centre, Poland for funding (grant no. UMO-2018/31/D/ST4/ 01475 and UMO/2020/39/I/ST4/01446). Computational time was at the Polish side provided by the Interdisciplinary Centre for Mathematical and Computational Modelling at the University of Warsaw (ICM UW) under grants no. G83-28 and GB80-24, while in Belgium the Flemish Supercomputer Centre (VSC) and the Herculesstichting are acknowledged
Coherent intensity fluctuation model for autocorrelation imaging spectroscopy with higher harmonic generating point scatterers - a comprehensive theoretical study
No full textWe present a general analytical model for the intensity fluctuation autocorrelation function for second and third harmonic generating point scatterers. Expressions are derived for a stationary laser beam and for scanning beam configurations for specific correlation methodologies. We discuss free translational diffusion in both three and two dimensions. At low particle concentrations, the expressions for fluorescence are retrieved, while at high particle concentrations a rescaling of the function parameters is required for a stationary illumination beam, provided that the phase shift per unit length of the beam equals zero.This work has been funded by the Interuniversity Attraction
Poles Program (IAP FS2 P7/05, Functional Supramolecular
Systems) initiated by the Belgian Science Policy Office
Label-free detection of uptake, accumulation, and translocation of diesel exhaust particles in ex vivo perfused human placenta
No full textBackgroundPregnant women and developing fetuses comprise a particularly vulnerable population as multiple studies have shown associations between prenatal air pollution exposure and adverse pregnancy outcomes. However, the mechanisms underlying the observed developmental toxicity are mostly unknown, in particular, if pollution particles can cross the human placenta to reach the fetal circulation.ResultsHere, we investigated the accumulation and translocation of diesel exhaust particles (DEPs), as a model particle for combustion-derived pollution, in human perfused placentae using label-free detection by femtosecond pulsed laser illumination. The results do not reveal a significant particle transfer across term placentae within 6 h of perfusion. However, DEPs accumulate in placental tissue, especially in the syncytiotrophoblast layer that mediates a wealth of essential functions to support and maintain a successful pregnancy. Furthermore, DEPs are found in placental macrophages and fetal endothelial cells, showing that some particles can overcome the syncytiotrophoblasts to reach the fetal capillaries. Few particles are also observed inside fetal microvessels.ConclusionsOverall, we show that DEPs accumulate in key cell types of the placental tissue and can cross the human placenta, although in limited amounts. These findings are crucial for risk assessment and protection of pregnant women and highlight the urgent need for further research on the direct and indirect placenta-mediated developmental toxicity of ambient particulates.This work received fnancial support from the Flemish Scientifc Research Foundation (Grant no 1150920N, G082317N, and 12P6819N) and the Swiss National Science Foundation (Grant no 31003A_179337 and IZSEZ0_193948). The detection equipment was funded by the Interuniversity Attraction Poles Program (P7/05) initiated by the Belgian Science Policy Ofce and the INCALO project (ERC-PoC).
Acknowledgements
The authors would like to acknowledge all participants of the study. In addition, we owe special thanks to the nurses, midwives, and doctors involved in placenta collection.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request
Investigating the effect of poly-l-lactic acid nanoparticles carrying hypericin on the flow-biased diffusive motion of HeLa cell organelles
No full textObjectives In this study, we investigate in human cervical epithelial HeLa cells the intracellular dynamics and the mutual interaction with the organelles of the poly-l-lactic acid nanoparticles (PLLA NPs) carrying the naturally occurring hydrophobic photosensitizer hypericin. Methods Temporal and spatiotemporal image correlation spectroscopy was used for the assessment of the intracellular diffusion and directed motion of the nanocarriers by tracking the hypericin fluorescence. Using image cross-correlation spectroscopy and specific fluorescent labelling of endosomes, lysosomes and mitochondria, the NPs dynamics in association with the cell organelles was studied. Static colocalization experiments were interpreted according to the Manders' overlap coefficient. Key findings Nanoparticles associate with a small fraction of the whole-organelle population. The organelles moving with NPs exhibit higher directed motion compared to those moving without them. The rate of the directed motion drops substantially after the application of nocodazole. The random component of the organelle motions is not influenced by the NPs. Conclusions Image correlation and cross-correlation spectroscopy are most appropriate to unravel the motion of the PLLA nanocarrier and to demonstrate that the rate of the directed motion of organelles is influenced by their interaction with the nanocarriers. Not all PLLA-hypericin NPs are associated with organelles.</p
Rapid and label-free optical detection of individual carbon air pollutant nanoparticulates in biomedical samples
No full textCarbonaceous particle exposure and air pollution in general lead to a multitude of adverse human health effects and pose multiple challenges in terms of exposure, risk and safety assessment. Highly desirable for fast screening are label-free approaches for detecting these particle types in biological or medical context. We report a powerful approach for detecting carbonaceous particles using photothermal pump-probe microscopy, which directly probes their strong light absorption. The principle and reliability of this approach is demonstrated by examining 4 different carbon black (CB) species modeling soot with diameters ranging from 13 to 500 nm. Our results show that the proposed approach is applicable to a large number of CB types as well as black carbon. As the particles show a strong absorption over a wide spectral range as compared to other absorbing species, we can image CB particles almost background free. Our pump-probe approach allows label-free optical detection and unambiguous localization of CB particles in (bio)fluids and 3D cellular environments. In combination with fluorescence microscopy, this method allows for simultaneous colocalization of CB with different cellular components using fluorophores as shown here for human lung fibroblasts. We further demonstrate the versatility of pump-probe detection in a flow cell.Fonds Wetenschappelijk Onderzoek, Grant/Award numbers: 11ZB115N, 12R6315N, G082317; Onderzoeksraad, KU Leuven, Grant/Award number: C14/15/053, OT/12/059; H2020 European Research Council, Grant/Award number: ERC-2012-StG 31089
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