| 1. |
- Abariute, Laura, et al.
(författare)
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Uptake of nanowires by human lung adenocarcinoma cells
- 2019
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14:6
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Tidskriftsartikel (refereegranskat)abstract
- Semiconductor nanowires are increasingly used in optoelectronic devices. However, their effects on human health have not been assessed fully. Here, we investigate the effects of gallium phosphide nanowires on human lung adenocarcinoma cells. Four different geometries of nanowires were suspended in the cell culture for 48 hours. We show that cells internalize the nanowires and that the nanowires have no effect on cell proliferation rate, motility, viability and intracellular ROS levels. By blocking specific internalization pathways, we demonstrate that the nanowire uptake is the result of a combination of processes, requiring dynamin and actin polymerization, which suggests an internalization through macropinocytosis and phagocytosis.
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| 2. |
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| 3. |
- Hebisch, Elke, et al.
(författare)
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Nanostraw-Assisted Cellular Injection of Fluorescent Nanodiamonds via Direct Membrane Opening
- 2021
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 17:7
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Tidskriftsartikel (refereegranskat)abstract
- Due to their stable fluorescence, biocompatibility, and amenability to functionalization, fluorescent nanodiamonds (FND) are promising materials for long term cell labeling and tracking. However, transporting them to the cytosol remains a major challenge, due to low internalization efficiencies and endosomal entrapment. Here, nanostraws in combination with low voltage electroporation pulses are used to achieve direct delivery of FND to the cytosol. The nanostraw delivery leads to efficient and rapid FND transport into cells compared to when incubating cells in a FND-containing medium. Moreover, whereas all internalized FND delivered by incubation end up in lysosomes, a significantly larger proportion of nanostraw-injected FND are in the cytosol, which opens up for using FND as cellular probes. Furthermore, in order to answer the long-standing question in the field of nano-biology regarding the state of the cell membrane on hollow nanostructures, live cell stimulated emission depletion (STED) microscopy is performed to image directly the state of the membrane on nanostraws. The time-lapse STED images reveal that the cell membrane opens entirely on top of nanostraws upon application of gentle electrical pulses, which supports the hypothesis that many FND are delivered directly to the cytosol, avoiding endocytosis and lysosomal entrapment.
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| 4. |
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| 5. |
- Hebisch, Elke, et al.
(författare)
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STED Nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- STED Nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells The specific arrangement of membrane lipids and proteins in a living cell at the interface to high-aspect ratio nanostructures (nanowires and nanostraws) is still unknown – as are the dynamic structural adaptations and molecular rearrangements of living cells in the vicinity of such nanostructures. Whether the nanostructures actually pierce through the cell membrane or how introduced changes in membrane curvature change the biophysical properties of the cell membrane is of particular interest for investigations of the efficacy and safety of nano-sized tissue implants and for studying the delivery of substances into living cells via hollow nanostraws. To elucidate these questions, STimulated Emission Depletion (STED) nanoscopy is the ideal technique because it is live-cell compatible, target-specific, and offers a lateral resolution on the protein level (<30 nm). Here we present STED based investigations of the live-cell membrane and the cytoskeletal Actin signal in the presence of hollow Alumina nanostraws with diameter of 100 nm. As cellular model system we chose the lung-cancer derived A549 culture cell line. The cells were incubated on the nanostraws and subsequently fluorescence-tagged with live-cell compatible labels targeting the cell membrane and filamentous Actin, respectively. We find that the cellular membrane forms ring structures of about 100 nm in diameter, wrapping tightly around the nanostraws. On the other hand, the Actin cytoskeleton forms intricate, coil-like nanometric structures around the nanostraws; these structures strongly vary in diameters between 250-600 nm and appear to widen with increasing distance from the nanostraw substrate. In addition, STED images of living cells stained for both membrane and Actin signal reveal a significant degree of co-localization at the apical cell membrane, i.e. further away from the nanostraws. This co-localization is almost entirely lost at the basal membrane close to the nanostraws which is due to a strongly reduced Actin signal on that side of the cell. In conclusion, our sub-diffraction STED imaging based investigations of the behavior of single living cells cultured on nanostraws reveals a strong response of the cellular membrane and the Actin cytoskeleton – two of the main structure-giving features of the cell. In a next step, we will extend our studies to additional scaffolding proteins to arrive at a more detailed map of the topology of living cells at the interface to nanostructures of different geometries.
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| 6. |
- Hebisch, Elke, et al.
(författare)
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STED nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- STED Nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells The specific arrangement of membrane lipids and proteins in a living cell at the interface to high-aspect ratio nanostructures (nanowires and nanostraws) is still unknown – as are the dynamic structural adaptations and molecular rearrangements of living cells in the vicinity of such nanostructures. Whether the nanostructures actually pierce through the cell membrane or how introduced changes in membrane curvature change the biophysical properties of the cell membrane is of particular interest for investigations of the efficacy and safety of nano-sized tissue implants and for studying the delivery of substances into living cells via hollow nanostraws. To elucidate these questions, STimulated Emission Depletion (STED) nanoscopy is the ideal technique because it is live-cell compatible, target-specific, and offers a lateral resolution on the protein level (<30 nm). Here we present STED based investigations of the live-cell membrane and the cytoskeletal Actin signal in the presence of hollow Alumina nanostraws with diameter of 100 nm. As cellular model system we chose the lung-cancer derived A549 culture cell line. The cells were incubated on the nanostraws and subsequently fluorescence-tagged with live-cell compatible labels targeting the cell membrane and filamentous Actin, respectively. We find that the cellular membrane forms ring structures of about 100 nm in diameter, wrapping tightly around the nanostraws. On the other hand, the Actin cytoskeleton forms intricate, coil-like nanometric structures around the nanostraws; these structures strongly vary in diameters between 250-600 nm and appear to widen with increasing distance from the nanostraw substrate. In addition, STED images of living cells stained for both membrane and Actin signal reveal a significant degree of co-localization at the apical cell membrane, i.e. further away from the nanostraws. This co-localization is almost entirely lost at the basal membrane close to the nanostraws which is due to a strongly reduced Actin signal on that side of the cell. In conclusion, our sub-diffraction STED imaging based investigations of the behavior of single living cells cultured on nanostraws reveals a strong response of the cellular membrane and the Actin cytoskeleton – two of the main structure-giving features of the cell. In a next step, we will extend our studies to additional scaffolding proteins to arrive at a more detailed map of the topology of living cells at the interface to nanostructures of different geometries.
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| 7. |
- O. Freitas, Raul, et al.
(författare)
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Nano-Infrared Imaging of Primary Neurons
- 2021
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 10:10, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease (AD) accounts for about 70% of neurodegenerative diseases and is a cause of cognitive decline and death for one-third of seniors. AD is currently underdiagnosed, and it cannot be effectively prevented. Aggregation of amyloid-β (Aβ) proteins has been linked to the development of AD, and it has been established that, under pathological conditions, Aβ proteins undergo structural changes to form β-sheet structures that are considered neurotoxic. Numerous intensive in vitro studies have provided detailed information about amyloid polymorphs; however, little is known on how amyloid β-sheet-enriched aggregates can cause neurotoxicity in relevant settings. We used scattering-type scanning near-field optical microscopy (s-SNOM) to study amyloid structures at the nanoscale, in individual neurons. Specifically, we show that in well-validated systems, s-SNOM can detect amyloid β-sheet structures with nanometer spatial resolution in individual neurons. This is a proof-of-concept study to demonstrate that s-SNOM can be used to detect Aβ-sheet structures on cell surfaces at the nanoscale. Furthermore, this study is intended to raise neurobiologists’ awareness of the potential of s-SNOM as a tool for analyzing amyloid β-sheet structures at the nanoscale in neurons without the need for immunolabeling
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| 8. |
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| 9. |
- Van Der Velde, Jasper H.M., et al.
(författare)
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Self-healing dyes for super-resolution fluorescence microscopy
- 2019
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Ingår i: Journal of Physics D: Applied Physics. - : IOP Publishing. - 0022-3727 .- 1361-6463. ; 52:3
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, optical microscopy techniques have emerged that allow optical imaging at unprecedented resolution beyond the diffraction limit. These techniques exploit photostabilizing buffers to enable photoswitching and/or the enhancement of fluorophore brightness and stability. A major drawback with the use of photostabilizing buffers, however, is that they cannot be used in live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores, which undergo intramolecular photostabilization, in super-resolution microscopy examining both targeted (stimulated emission depletion (STED) microscopy) and stochastic readout (stochastic optical reconstruction microscopy (STORM)). The overall goal of the study was to identify dyes and conditions that lead to improved spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. As a result of previously shown superior performance, we identified an ATTO647N-photostabilizer conjugate as a potential candidate for STED microscopy. We have here characterized the photostability and resulting performance of this nitrophenylalanine (NPA) conjugate of ATTO647N on oligonucleotides in STED microscopy. We found that the superior photophysical performance resulted in optimal STED imaging and demonstrated that single-molecule fluorescent transients of individual fluorophores can be obtained with both the excitation- and STED-laser. In similar experiments, we also tested a nitrophenylacetic acid conjugate of STAR635P, another frequently used dye in STED microscopy, and present a characterization of its photophysical properties. Finally, we performed an analysis of the photoswitching kinetics of self-healing Cy5 dyes (containing trolox, cyclooctatetraene and NPA-based stabilizers) in the presence of Tris(2-carboxyethyl)phosphine and cysteamine, which are typically used in STORM microscopy. In line with previous work, we found that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.
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