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- ItemNanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer(Washington, DC : ACS Publ., 2018-7-24) Urbančič, Iztok; Garvas, Maja; Kokot, Boštjan; Majaron, Hana; Umek, Polona; Cassidy, Hilary; Škarabot, Miha; Schneider, Falk; Galiani, Silvia; Arsov, Zoran; Koklic, Tilen; Matallanas, David; Čeh, Miran; Muševič, Igor; Eggeling, Christian; Štrancar, JanezAlthough the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane’s disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.
- ItemNanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging(Washington, DC : ACS Publ., 2018-6-12) Schneider, Falk; Waithe, Dominic; Galiani, Silvia; Bernardino de la Serna, Jorge; Sezgin, Erdinc; Eggeling, ChristianThe diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.
- ItemIntracellular Photophysics of an Osmium Complex bearing an Oligothiophene Extended Ligand(Weinheim : Wiley-VCH, 2020) Schneider, Kilian R.A.; Chettri, Avinash; Cole, Houston D.; Reglinski, Katharina; Breckmann, Jannik; Roque, John A. III; Stumper, Anne; Nauroozi, Djawed; Schmid, Sylvia; Lagerholm, Christoffer B.; Rau, Sven; Bäuerle, Peter; Eggeling, Christian; Cameron, Colin G.; McFarland, Sherri A.; Dietzek, BenjaminThis contribution describes the excited-state properties of an Osmium-complex when taken up into human cells. The complex 1 [Os(bpy)2(IP-4T)](PF6)2 with bpy=2,2′-bipyridine and IP-4T=2-{5′-[3′,4′-diethyl-(2,2′-bithien-5-yl)]-3,4-diethyl-2,2′-bithiophene}imidazo[4,5-f][1,10]phenanthroline) can be discussed as a candidate for photodynamic therapy in the biological red/NIR window. The complex is taken up by MCF7 cells and localizes rather homogeneously within in the cytoplasm. To detail the sub-ns photophysics of 1, comparative transient absorption measurements were carried out in different solvents to derive a model of the photoinduced processes. Key to rationalize the excited-state relaxation is a long-lived 3ILCT state associated with the oligothiophene chain. This model was then tested with the complex internalized into MCF7 cells, since the intracellular environment has long been suspected to take big influence on the excited state properties. In our study of 1 in cells, we were able to show that, though the overall model remained the same, the excited-state dynamics are affected strongly by the intracellular environment. Our study represents the first in depth correlation towards ex-vivo and in vivo ultrafast spectroscopy for a possible photodrug. © 2020 The Authors. Published by Wiley-VCH GmbH
- ItemExcited-State Dynamics in Borylated Arylisoquinoline Complexes in Solution and in cellulo(Weinheim : Wiley-VCH, 2023) Yang, Tingxiang; Valavalkar, Abha; Romero‐Arenas, Antonio; Dasgupta, Anindita; Then, Patrick; Chettri, Avinash; Eggeling, Christian; Ros, Abel; Pischel, Uwe; Dietzek‐Ivanšić, BenjaminTwo four-coordinate organoboron N,C-chelate complexes with different functional terminals on the PEG chains are studied with respect to their photophysical properties within human MCF-7 cells. Their excited-state properties are characterized by time-resolved pump-probe spectroscopy and fluorescence lifetime microscopy. The excited-state relaxation dynamics of the two complexes are similar when studied in DMSO. Aggregation of the complexes with the carboxylate terminal group is observed in water. When studying the light-driven excited-state dynamics of both complexes in cellulo, i. e., after being taken up into human MCF-7 cells, both complexes show different features depending on the nature of the anchoring PEG chains. The lifetime of a characteristic intramolecular charge-transfer state is significantly shorter when studied in cellulo (360±170 ps) as compared to in DMSO (∼960 ps) at 600 nm for the complexes with an amino group. However, the kinetics of the complexes with the carboxylate group are in line with those recorded in DMSO. On the other hand, the lifetimes of the fluorescent state are almost identical for both complexes in cellulo. These findings underline the importance to evaluate the excited-state properties of fluorophores in a complex biological environment in order to fully account for intra- and intermolecular effects governing the light-induced processes in functional dyes.