| 1. |
- Alvelid, Jonatan
(författare)
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Automating STED microscopy for functional and structural live-cell imaging
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Optical microscopy imaging methods are today invaluable tools for studies in life sciences as they allow visualization of biological systems, tissues, cells, and sub-cellular compartments from millimetres down to nanometres. The invention and development of nanoscopy in the past 20 years has pushed fluorescence microscopy down to the nanoscale, reaching beyond the natural diffraction limit of light that does not allow focusing of visible light below sizes of around 200 nm, and into the realm of what was previously only thought possible with electron microscopy. The superior spatial resolution does however come at a price, including complex sample preparation, prolonged recording times, increased illumination doses, and limited fields of view. Stimulated emission depletion (STED) microscopy is one of the techniques that can deliver nanoscale resolution in a range of biological systems, but with all the above-mentioned costs. However, with the right sample the technique can deliver single nanometre spatial resolution, and with the right considerations live-cell imaging is more than possible.In this thesis I present the development of a flexible STED microscope with methodological advancements in a range of directions that aim at facilitating the use of STED microscopy in life sciences and optimising the information extraction from the image data. The developments firstly focused on automation of the data acquisition, to allow the recording of imaging data both with a higher throughput and correlated with fast dynamic processes. I also implemented improved image analysis, both in terms of high throughput and precision as well as in connection with the data acquisition. Furthermore, I worked on control software development, with novel strategies to unify the control software of microscopes and to allow development and implementation of novel acquisition schemes. I also utilized novel fluorophores, to improve live-cell and multicolour possibilities and allow a wider range of applications in STED microscopy. Lastly, I developed a novel concept that takes advantage of STED. Additionally, I present applications of the microscope and image analysis in diverse biological samples such as mammalian cells, tissue sections, and bacteria. Altogether, this work aims at presenting new tools for an imaging technique that is already well-established, to contribute to further development, facilitation of novel experiments, and expansion of the range of applications.
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| 2. |
- Alvelid, Jonatan, et al.
(författare)
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Event-triggered STED imaging
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The observation of protein organization during cellular signalling calls for imaging methods with increased spatial and temporal resolution. STED nanoscopy can access dynamics of nanoscale structures in living cells. However, the available number of recordable frames is often limited by photo-bleaching. Here, we present an automated method, event-triggered STED, which instantly (< 40 ms) images synaptic proteins with high spatial and temporal resolution (~30 nm, 2.5 Hz) in small regions upon and at the site of local calcium sensing.
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| 3. |
- Alvelid, Jonatan, et al.
(författare)
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Event-triggered STED imaging
- 2022
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Ingår i: Nature Methods. - : Springer Nature. - 1548-7091 .- 1548-7105. ; 19:10, s. 1268-1275
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Tidskriftsartikel (refereegranskat)abstract
- Monitoring the proteins and lipids that mediate all cellular processes requires imaging methods with increased spatial and temporal resolution. STED (stimulated emission depletion) nanoscopy enables fast imaging of nanoscale structures in living cells but is limited by photobleaching. Here, we present event-triggered STED, an automated multiscale method capable of rapidly initiating two-dimensional (2D) and 3D STED imaging after detecting cellular events such as protein recruitment, vesicle trafficking and second messengers activity using biosensors. STED is applied in the vicinity of detected events to maximize the temporal resolution. We imaged synaptic vesicle dynamics at up to 24 Hz, 40 ms after local calcium activity; endocytosis and exocytosis events at up to 11 Hz, 40 ms after local protein recruitment or pH changes; and the interaction between endosomal vesicles at up to 3 Hz, 70 ms after approaching one another. Event-triggered STED extends the capabilities of live nanoscale imaging, enabling novel biological observations in real time.
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| 4. |
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| 5. |
- Alvelid, Jonatan, et al.
(författare)
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Far Red‐Shifted CdTe Quantum Dots for Multicolour Stimulated Emission Depletion Nanoscopy
- 2022
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235 .- 1439-7641. ; 24:3
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Tidskriftsartikel (refereegranskat)abstract
- Stimulated emission depletion (STED) nanoscopy is a widely used nanoscopy technique. Two-colour STED imaging in fixed and living cells is standardised today utilising both fluorescent dyes and fluorescent proteins. Solutions to image additional colours have been demonstrated using spectral unmixing, photobleaching steps, or long-Stokes-shift dyes. However, these approaches often compromise speed, spatial resolution, and image quality, and increase complexity. Here, we present multicolour STED nanoscopy with far red-shifted semiconductor CdTe quantum dots (QDs). STED imaging of the QDs is optimized to minimize blinking effects and maximize the number of detected photons. The far-red and compact emission spectra of the investigated QDs free spectral space for the simultaneous use of fluorescent dyes, enabling straightforward three-colour STED imaging with a single depletion beam. We use our method to study the internalization of QDs in cells, opening up the way for future super-resolution studies of particle uptake and internalization.
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| 6. |
- Alvelid, Jonatan, et al.
(författare)
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Fluorescence microscopy at the molecular scale
- 2019
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Ingår i: Current Opinion in Biomedical Engineering. - : Elsevier. - 2468-4511. ; 12, s. 34-42
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Forskningsöversikt (refereegranskat)abstract
- The diffraction limit is no longer a concept that stands as a true constant in imaging, with fluorescence switching–based methods having made the breakthrough to circumvent this limit. Multiple ingenious solutions have been presented over the last decades and continue to be explored. The techniques used today have undergone constant development both conceptually and technically, which has enabled an increased number of biological studies at the molecular scale. Here we review recent developments to stimulated emission depletion microscopy, reversible saturable optical fluorescence transitions microscopy, single-molecule localization microscopy, and MINFLUX and mention key applications of these methods. Finally, we present our view on what the future holds for super-resolution imaging, especially in terms of even more challenging live-cell imaging in different biological model systems.
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| 7. |
- Alvelid, Jonatan, et al.
(författare)
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Stable stimulated emission depletion imaging of extended sample regions
- 2020
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Ingår i: Journal of Physics D. - : IOP PUBLISHING LTD. - 0022-3727 .- 1361-6463. ; 53:2
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Tidskriftsartikel (refereegranskat)abstract
- Stimulated emission depletion (STED) nanoscopy has become one of the most used nanoscopy techniques over the last decade. However, most recordings are done in specimen regions no larger than 10–30 × 10–30 μm2 due to aberrations, instability and manual mechanical stages. Here, we demonstrate automated 2D and 3D STED nanoscopy of extended sample regions up to 0.5 × 0.5 mm2 by using a scanning system that maintains stationary beams in the back focal plane. The setup allows up to 80–100 × 80–100 μm2 field of view (FOV) with uniform spatial resolution, a mechanical stage allowing sequential tiling to record larger sample areas, and a feedback system keeping the sample in focus at all times. Taken together, this allows automated recording of theoretically unlimited-sized sample areas and volumes, without compromising the achievable spatial resolution and image quality.
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| 8. |
- Battista, Simone, et al.
(författare)
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Living with migraine : A meta-synthesis of qualitative studies
- 2023
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Ingår i: Frontiers in Psychology. - : Frontiers Media SA. - 1664-1078. ; 14, s. 1-20
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Forskningsöversikt (refereegranskat)abstract
- INTRODUCTION: Migraine is one of the top ten causes of disability worldwide. However, migraine is still underrated in society, and the quality of care for this disease is scant. Qualitative research allows for giving voice to people and understanding the impact of their disease through their experience of it. This study aims at synthesising the state of the art of qualitative studies focused on how people with migraine experience their life and pathology.METHODS: MEDLINE via PubMed, EMBASE, CINAHL, PsycINFO, and Cochrane Library were consulted up to November 2021 for qualitative studies. Studies to be eligible had to focus on adults (age > 18 years) with a diagnosis of primary episodic or chronic migraine following the International Classification of Headache. The quality of the study was analysed using the CASP (Critical Appraisal Skills Programme) tool. The synthesis was done through a thematic analysis. CERQual (Confidence in Evidence from Reviews of Qualitative research) approach was used to assess the confidence in retrieved evidence. RESULTS: Ten studies were included, counting 262 people with migraine. Our synthesis produced four main themes. (1) "Negative impact of migraine symptoms on overall life" as migraine negatively impacts people's whole life. (2) "Impact of migraine on family, work and social relationship" as migraine reduces the possibility to focus at work and interact with people. (3) "Impact of migraine on emotional health" as people with migraine experience psychological distress. (4) "Coping strategies to deal with migraine" such as keep on living one's own life, no matter the symptoms.CONCLUSIONS: Migraine negatively impacts people's whole life, from private to social and work sphere. People with migraine feel stigmatised as others struggle with understanding their condition. Hence, it is necessary to improve awareness among society of this disabling condition, and the quality of care of these people, tackling this disease from a social and health-policy point of view.
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| 9. |
- Battista, Simone, et al.
(författare)
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Which lecturers’ characteristics facilitate the learning process? A qualitative study on students’ perceptions in the rehabilitation sciences
- 2023
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Ingår i: BMC Medical Education. - 1472-6920. ; 23, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- Background: In education, lecturers play a crucial role in facilitating students’ learning process. However, only a few studies explored which lecturers’ characteristics can facilitate this process in higher education for rehabilitation healthcare professionals. Starting from students’ perspectives, our qualitative study investigated the lecturers’ characteristics that facilitate students’ learning process in the rehabilitation sciences. Methods: A qualitative interview study. We enrolled students attending the 2nd year of the Master of Science (MSc) degree in ‘Rehabilitation Sciences of Healthcare Professions’. Different themes were generated following a ‘Reflexive Thematic Analysis’. Results: Thirteen students completed the interviews. From their analysis, we generated five themes. Specifically, a lecturer that facilitates students’ learning process should be: 1) ‘A Performer who Interacts with the Classroom’, 2) A Flexible Planner who Adopts Innovative Teaching Skills’, 3) ‘A Motivator who Embraces Transformational Leadership’, 4) ‘A Facilitator Who Encourages a Constructive Learning Context’ and 5) ‘A Coach who Devises Strategies to Reach Shared Learning Goals’. Conclusions: The results of this study underscore the importance for lecturers in rehabilitation to cultivate a diverse set of skills drawn from the arts and performance, education, team building and leadership to facilitate students’ learning process. By developing these skills, lecturers can design lessons that are worth attending not only for their relevant content but also for their value in human experience.
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| 10. |
- Bodén, Andreas, 1990-
(författare)
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3D super-resolution microscopy of living cells using reversibly switchable fluorophores
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Traditional optical microscopy techniques are limited in spatial resolution due to the wave nature of light. This means that neighboring objects separated by a distance smaller than about 200 nm cannot be distinguished. Super‑resolution microscopy techniques overcome this limitation by utilizing specific light-matter interactions of fluorescent labels to encode finer spatial detail into the recorded data. Regrettably, current super‑resolution approaches often increase the complexity of sample preparation as well as the energy, time, and invasiveness of the imaging scheme compared to conventional imaging techniques. This makes many of these techniques ill‑suited for imaging the dynamics of living cells. Since many biological studies rely on highly spatially resolved data containing three‑dimensional and temporally dynamic information, developing super‑resolution techniques toward the goal of acquiring such data is vital. With this work, we take several important steps in this direction by utilizing reversibly switchable fluorescence proteins (RSFPs) together with new illumination patterns that allow for a parallelized data acquisition scheme. Even low intensity illumination patterns can induce photo‑switching of the RSFPs and generate specific patterns of fluorescent emission that carry high‑resolution spatial information in all three dimensions. By using RSFPs in a parallelized acquisition scheme, temporally extended recordings can be acquired with low illumination intensities and at high speed. In addition to the imaging schemes, we present a theoretical framework for modelling the impact that RSFP properties on image formation and show how different imaging parameters affect the final image quality. We predict and explore the effect of labelling density and photobleaching on single and timelapse recordings, taking into consideration the stochasticity of labelling and fluorophore fatigue. We also present a new family of red‑shifted RSFPs that can be imaged without the need for near‑UV illumination, allowing even less invasive live‑cell imaging. This work aims to not only provide new tools for imaging, but also to contribute to a better understanding of the underlying concepts and to facilitate future developments of super-resolution microscopy for bio-imaging applications.
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| 11. |
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| 12. |
- Bodén, Andreas, et al.
(författare)
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Predicting resolution and image quality in RESOLFT and other point scanning microscopes [Invited]
- 2020
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Ingår i: Biomedical Optics Express. - : The Optical Society. - 2156-7085. ; 11:5, s. 2313-2327
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Tidskriftsartikel (refereegranskat)abstract
- The performance of fluorescence microscopy and nanoscopy is often discussed by the effective point spread function and the optical transfer function. However, due to the complexity of the fluorophore properties such as photobleaching or other forms of photoswitching, which introduce a variance in photon emission, it is not trivial to choose optimal imaging parameters and to predict the spatial resolution. In this paper, we analytically derive a theoretical framework for estimating the achievable resolution of a microscope depending on parameters such as photoswitching, labeling densities, exposure time and sampling. We developed a numerical simulation software to analyze the impact of reversibly switchable probes in RESOLFT imaging.
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| 13. |
- Bodén, Andreas, et al.
(författare)
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Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy
- 2021
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Ingår i: Nature Biotechnology. - : Springer Nature. - 1087-0156 .- 1546-1696. ; 39:5, s. 609-618
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Tidskriftsartikel (refereegranskat)abstract
- Elucidating the volumetric architecture of organelles and molecules inside cells requires microscopy methods with a sufficiently high spatial resolution in all three dimensions. Current methods are limited by insufficient resolving power along the optical axis, long recording times and photobleaching when applied to live cell imaging. Here, we present a 3D, parallelized, reversible, saturable/switchable optical fluorescence transition (3D pRESOLFT) microscope capable of delivering sub-80-nm 3D resolution in whole living cells. We achieved rapid (1-2 Hz) acquisition of large fields of view (similar to 40 x 40 mu m(2)) by highly parallelized image acquisition with an interference pattern that creates an array of 3D-confined and equally spaced intensity minima. This allowed us to reversibly turn switchable fluorescent proteins to dark states, leading to a targeted 3D confinement of fluorescence. We visualized the 3D organization and dynamics of organelles in living cells and volumetric structural alterations of synapses during plasticity in cultured hippocampal neurons.
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| 14. |
- Caceres, Rodrigo, et al.
(författare)
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Forces drive basement membrane invasion in Caenorhabditis elegans
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:45, s. 11537-11542
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Tidskriftsartikel (refereegranskat)abstract
- During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-inkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.
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| 15. |
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| 16. |
- Casas Moreno, Xavier, 1994-
(författare)
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Automated super-resolution microscopy for high-throughput imaging
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fluorescence microscopes enable the visualization of biological samples with high contrast by labeling specific structures with fluorescent molecules. However, the spatial resolution of widely used microscopy techniques, such as widefield and confocal microscopy, is limited by the size of a focused spot of light, which roughly corresponds to half the wavelength of the illumination. To overcome this limitation, optical fluorescence nanoscopy techniques were developed, which achieve a higher spatial resolution by switching the fluorescent molecules within the sample between bright and dark states. Optical fluorescence nanoscopy techniques can be divided into two categories. The first, namely coordinate-targeted nanoscopy, switches the fluorescent molecules in a spatially annotated way, where it is known where and when the switching is induced. Instead, in stochastic approaches, the emitting molecules appear randomly in the sample and their location can be annotated with high spatial precision. This thesis focuses on reversible saturable optical fluorescence transitions (RESOLFT), a coordinate-targeted nanoscopy technique that initially relied on a beam of light that is moved across the sample (i.e., point scanning). Beams of different shapes and wavelengths are synchronized in time to generate super-resolution images. However, this approach creates a trade-off between imaging speed and the field of view. While it can acquire small fields of view at a fast speed, imaging larger areas can take up to several minutes. To overcome this limitation, the molecular nanoscale live imaging with sectioning ability (MoNaLISA) microscope employs patterns of light to parallelize RESOLFT imaging, collecting the fluorescence at different points simultaneously.Throughput in microscopy is characterized as the number of cells per unit of time and area that a microscope can image. Achieving high throughput enables capturing fast cell dynamics and understanding how they correlate over large fields of view, providing insights into biological processes. Therefore, in this thesis I developed strategies to increase the throughput of coordinate-targeted nanoscopy methods. Firstly, I was involved in the mathematical formulation of fluorophore switching and its relationship to image resolution, in order to provide a framework to relate different parameters to image quality (Paper I). Secondly, I developed ImSwitch, an open-source software for microscope control. It implements a software architecture that enables flexibility and adaptability between different microscopy modalities (Paper II). Thirdly, I built a setup that increases the field of view by more than four times than previous implementations of MoNaLISA (Paper III). Finally, I combined MoNaLISA and ImSwitch to provide a framework to parallelize image acquisition, reconstruction, and visualization using multiple computational units (Paper IV).
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| 17. |
- Casas Moreno, Xavier, et al.
(författare)
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ImSwitch: Generalizing microscope control in Python
- 2021
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Ingår i: Journal of Open Source Software. - : The Open Journal. - 2475-9066. ; 6:64
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Tidskriftsartikel (refereegranskat)abstract
- The constant development of novel microscopy methods with an increased number of dedicated hardware devices poses significant challenges to software development. On the onehand, software should control complex instruments, provide flexibility to adapt between different microscope modalities, and be open and resilient to modification and extension byusers and developers. On the other hand, the community needs software that can satisfy therequirements of the users, such as a user-friendly interface and robustness of the code. In this context, we present ImSwitch, based on the model-view-presenter (MVP) design pattern (Potel, 1996), with an architecture that uses polymorphism to provide a generalized solutionto microscope control. Consequently, ImSwitch makes it possible to adapt between different modalities and aims at satisfying the needs of both users and developers. We have alsoincluded a scripting module for microscope automation applications and a structure to efficiently share data between different modules, such as hardware control and image processing. Currently, ImSwitch provides support for light microscopy techniques but could be extendedto other microscopy modalities requiring multiple hardware synchronization.
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| 18. |
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| 19. |
- Damenti, Martina
(författare)
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Investigation of Neuronal Protein Trafficking at the Molecular Scale
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neurons are polarized cells that encode information in the nervous system viaelectrochemical connections named synapses. The tuning of synapticconnections is enabled by a plastic protein trafficking system which operates atthe nanoscale to finely tweak the neuronal ultrastructure. Our understanding ofthe neuronal biology has certainly benefited from the advent of live-cellcompatible fluorescence techniques able to reach the molecular level. However,the neuronal trafficking system involves molecular complexes, from organelles tosynaptic modulators, which act with varying dynamics at differentspatiotemporal scales. A single technique struggles to portrait these complexphenomena since it is hard to combine molecular resolution, speed, and lowphototoxicity. Hence, their investigation often demands, together with technicaladvancements, the combination of advanced fluorescence methods withcomplementary features. In this thesis, I explore the neuronal protein traffickingsystem at the molecular scale applying cutting-edge fluorescence microscopy andspectroscopy techniques.The relationship between the geometry and dynamics of the tubular endoplasmicnetwork and the sub-compartment size of neurons is investigated using acombination of STED and parallelized RESOLFT microscopy. In addition, thethree-dimensional dynamic interaction between tubular endoplasmic reticulumand mitochondria is described.The basal activity-driven recycling of synaptic vesicles is, for the first time,monitored via event-triggered STED, an automated method able to initiate STEDimaging upon detection of events such as calcium spikes.Insights into the post-synaptic reorganization of scaffolding and skeletal proteinsupon stimulation is gained by extending the live-cell super-resolution throughputto all the dimensions with multi-foci and 3D parallelized RESOLFT.Lastly, the molecular states of Activity-Regulated Cytoskeleton-Associatedprotein (Arc) involved in distinct aspects of neuronal protein trafficking arestudied. Our observations, obtained combining distinct advanced methods asDNA-PAINT and STARSS, support a previously unexplored Arc mechanism ofaction.
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| 20. |
- Damenti, Martina, et al.
(författare)
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Quantitative and functional assessment of Arc n-meric states in membrane interaction and AMPA receptor endocytosis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Arc (or Arg3.1), Activity Regulated-Cytoskeleton associated-protein is pivotal to mediate plastic responses in neuronal cells. In vitro and in vivo studies suggest its ability to form high- and low-order oligomers which are potentially involved in neuronal trafficking. Despite its important function, no direct observation of Arc oligomers in cells has been presented due to its highly regulated spatiotemporal expression, the small size of the structures, the lack of appropriate labelling strategies and the background associated to free diffusing cytosolic proteins. Here, we take advantage of several complementary advanced fluorescence microscopy and spectroscopy techniques to observe and quantify Arc oligomeric states in cellular environment especially in the synapses. In cells, we uncovered Arc-Arc intermolecular interactions, Arc tendency to form liquid condensates and to interact with lipid bilayers. High-order oligomers are found to localize at the excitatory synaptic compartment and to directly affects AMPA receptor surface levels. Together, our observations support the model by which Arc oligomerization mediates plasma- membrane negative inward curvature favoring AMPA receptors endocytosis.
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| 21. |
- Damenti, Martina, et al.
(författare)
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STED and parallelized RESOLFT optical nanoscopy of the tubular endoplasmic reticulum and its mitochondrial contacts in neuronal cells
- 2021
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Ingår i: Neurobiology of Disease. - : Elsevier BV. - 0969-9961 .- 1095-953X. ; 155
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Tidskriftsartikel (refereegranskat)abstract
- The classic view of organelle cell biology is undergoing a constant revision fueled by the new insights unraveled by fluorescence nanoscopy, which enable sensitive, faster and gentler observation of specific proteins in situ. The endoplasmic reticulum (ER) is one of the most challenging structure to capture due the rapid and constant restructuring of fine sheets and tubules across the full 3D cell volume. Here we apply STED and parallelized 2D and 3D RESOLFT live imaging to uncover the tubular ER organization in the fine processes of neuronal cells with focus on mitochondria-ER contacts, which recently gained medical attention due to their role in neurodegeneration. Multi-color STED nanoscopy enables the simultaneous visualization of small transversal ER tubules crossing and constricting mitochondria all along axons and dendrites. Parallelized RESOLFT allows for dynamic studies of multiple contact sites within seconds and minutes with prolonged time-lapse imaging at similar to 50 nm spatial resolution. When operated in 3D super resolution mode it enables a new isotropic visualization of such contacts extending our understanding of the three-dimensional architecture of these packed structures in axons and dendrites.
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| 22. |
- Dreier, Jes, et al.
(författare)
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Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo
- 2019
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70-90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20-30 mu m inside a living Caenorhabditis elegans worm.
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| 23. |
- Fang, Trixy, et al.
(författare)
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Spatial Regulation of T-Cell Signaling by Programmed Death-Ligand 1 on Wireframe DNA Origami Flat Sheets
- 2021
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Ingår i: ACS Nano. - : AMER CHEMICAL SOC. - 1936-0851 .- 1936-086X. ; 15:2, s. 3441-3452
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Tidskriftsartikel (refereegranskat)abstract
- Programmed Death-1 (PD-1) is a coinhibitory receptor expressed on activated T cells that suppresses T-cell signaling and effector functions. It has been previously shown that binding to its ligand PD-L1 induces a spatial reorganization of PD-1 receptors into microclusters on the cell membrane. However, the roles of the spatial organization of PD-L1 on PD-1 clustering and T-cell signaling have not been elucidated. Here, we used DNA origami flat sheets to display PD-L1 ligands at defined nanoscale distances and investigated their ability to inhibit T-cell activation in vitro. We found that DNA origami flat sheets modified with CD3 and CD28 activating antibodies (FS-alpha-CD3-CD28) induced robust T-cell activation. Co-treatment with flat sheets presenting PD-L1 ligands separated by similar to 200 nm (FS-PD-L1-200), but not 13 nm (FS-PD-L1-13) or 40 nm (FS-PD-L1-40), caused an inhibition of T-cell signaling, which increased with increasing molar ratio of FS-PD-L1-200 to FS-alpha-CD3-CD28. Furthermore, FS-PD-L1-200 induced the formation of smaller PD-1 nanoclusters and caused a larger reduction in IL-2 expression compared to FS-PD-L1-13. Together, these findings suggest that the spatial organization of PD-L1 determines its ability to regulate T-cell signaling and may guide the development of future nanomedicine-based immunomodulatory therapies.
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| 24. |
- Fuhrmann, Martin, et al.
(författare)
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Super-Resolution Microscopy Opens New Doors to Life at the Nanoscale
- 2022
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Ingår i: Journal of Neuroscience. - : Society for Neuroscience. - 0270-6474 .- 1529-2401. ; 42:45, s. 8488-8497
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Tidskriftsartikel (refereegranskat)abstract
- Super-resolution fluorescence microscopy holds tremendous potential for discovery in neuroscience. Much of the molecular machinery and anatomic specializations that give rise to the unique and bewildering electrochemical activity of neurons are nanoscale by design, ranging somewhere between 1 nm and 1 lm. It is at this scale where most of the unknown and exciting action is and where cell biolo-gists flock to in their dreams, but it was off limits for light microscopy until recently. While the optical principles of super-resolution microscopy are firmly established by now, the technology continues to advance rapidly in many crucial areas, enhancing its perform-ance and reliability, and making it more accessible and user-friendly, which is sorely needed. Indeed, super-resolution microscopy tech-niques are nowadays widely used for visualizing immunolabeled protein distributions in fixed or living cells. However, a great potential of super-resolution microscopy for neuroscience lies in shining light on the nanoscale structures and biochemical activities in live-tissue settings, which should be developed and harnessed much more fully. In this review, we will present several vivid examples based on STED and RESOLFT super-resolution microscopy, illustrating the possibilities and challenges of nano-imaging in vivo to pique the interest of tech-developers and neurobiologists alike. We will cover recent technical progress that is facilitating in vivo applications, and share new biological insights into the nanoscale mechanisms of cellular communication between neurons and glia.
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| 25. |
- Hell, Stefan W., et al.
(författare)
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The 2015 super-resolution microscopy roadmap
- 2015
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Ingår i: Journal of Physics D. - : Institute of Physics (IOP). - 0022-3727 .- 1361-6463. ; 48:44
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Forskningsöversikt (refereegranskat)abstract
- Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio) physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of 'super-resolution' farfield optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact.
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| 26. |
- Jalalvand, Elham, et al.
(författare)
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ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the central canal
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The spatial location of cerebrospinal fluid contacting (CSF-c) neurons enables important regulatory homeostatic functions regarding pH and motion control. Their intricate organization, facing the central canal and extending across the spinal cord, in relation to specific subtypes is poorly understood. This calls for imaging methods with a high spatial resolution (5-10 nm) to resolve the synaptic and ciliary compartments of each individual cell to elucidate their signalling pathways and enough throughput to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine and somatostatin CSF-c neuronal types.The number of somatostatin-containing dense core vesicles, resolved by STED microscopy, was shown to be markedly reduced upon each exposure to alkaline or acidic pH inhibiting any movement as part of a homeostatic response. Their cilia symmetry was unravelled by ExSTED as sensory in contrast with the motile one found in the dopaminergic ph insensitive neurons. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell type classification.
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| 27. |
- Jalalvand, Elham, 1971-, et al.
(författare)
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ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal
- 2022
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Ingår i: eLIFE. - : eLIFE SCIENCES PUBL LTD. - 2050-084X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid-contacting (CSF-c) neurons line the central canal of the spinal cord and a subtype of CSF-c neurons expressing somatostatin, forms a homeostatic pH regulating system. Despite their importance, their intricate spatial organization is poorly understood. The function of another subtype of CSF-c neurons expressing dopamine is also investigated. Imaging methods with a high spatial resolution (5-10 nm) are used to resolve the synaptic and ciliary compartments of each individual cell in the spinal cord of the lamprey to elucidate their signalling pathways and to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine- and somatostatin-expressing CSF-c neuronal subtypes. The density of somatostatin-containing dense-core vesicles, resolved by stimulated emission depletion microscopy, was shown to be markedly reduced upon each exposure to either alkaline or acidic pH and being part of a homeostatic response inhibiting movements. Their cilia symmetry was unravelled by stimulated emission depletion microscopy in expanded tissues as sensory with 9 + 0 microtubule duplets. The dopaminergic CSF-c neurons on the other hand have a motile cilium with the characteristic 9 + 2 duplets and are insensitive to pH changes. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell-type classification.
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| 28. |
- Mahecic, Dora, et al.
(författare)
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Strategies for increasing the throughput of super-resolution microscopies
- 2019
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Ingår i: Current opinion in chemical biology. - : ELSEVIER SCI LTD. - 1367-5931 .- 1879-0402. ; 51, s. 84-91
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Forskningsöversikt (refereegranskat)abstract
- Fluorescence microscopy methods have been developed to circumvent the diffraction limit by exploiting nonlinearities in the interactions between light and fluorophores. Initially, these methods were up to orders of magnitude slower than standard microscopies. In recent years, a wide array of technological advances have increased the throughput of super-resolution microscopies, through parallelization, smart scanning or data processing, and sample expansion. Here, we review re cent innovations for increased throughput, some applications that have benefitted from them, and how they could be improved in the future.
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| 29. |
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| 30. |
- Masullo, Luciano A., et al.
(författare)
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Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems
- 2018
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45-65 nm within the entire cell volume at low light intensities (W-kW cm(-2)). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined to gain fluorescence ON-OFF switching cycles and image contrast. By maximizing the detected photon flux, MoNaLISA enables prolonged (40-50 frames) and large (50 x 50 mu m(2)) recordings at 0.3-1.3 Hz with enhanced optical sectioning ability. We demonstrate the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.
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| 31. |
- Matlashov, M. E., et al.
(författare)
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A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales
- 2020
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Ingår i: Nature Communications. - : Nature Research. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.
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| 32. |
- Mishra, K., et al.
(författare)
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Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging
- 2022
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Ingår i: Nature Biotechnology. - : Springer Nature. - 1087-0156 .- 1546-1696. ; 40:4, s. 598-605
-
Tidskriftsartikel (refereegranskat)abstract
- Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.
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| 33. |
- Moreno, Xavier Casas, et al.
(författare)
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An open-source microscopy framework for simultaneous control of image acquisition, reconstruction, and analysis
- 2023
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Ingår i: HardwareX. - : Elsevier BV. - 2468-0672. ; 13, s. e00400-e00400
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Tidskriftsartikel (refereegranskat)abstract
- We present a computational framework to simultaneously perform image acquisition, reconstruction, and analysis in the context of open-source microscopy automation. The setup features multiple computer units intersecting software with hardware devices and achieves automation using python scripts. In practice, script files are executed in the acquisition computer and can perform any experiment by modifying the state of the hardware devices and accessing experimental data. The presented framework achieves concurrency by using multiple instances of ImSwitch and napari working simultaneously. ImSwitch is a flexible and modular open-source software package for microscope control, and napari is a multidimensional image viewer for scientific image analysis. The presented framework implements a system based on file watching, where multiple units monitor a filesystem that acts as the synchronization primitive. The proposed solution is valid for any microscope setup, supporting various biological applications. The only necessary element is a shared filesystem, common in any standard laboratory, even in resource-constrained settings. The file watcher functionality in Python can be easily integrated into other python-based software. We demonstrate the proposed solution by performing tiling experiments using the molecular nanoscale live imaging with sectioning ability (MoNaLISA) microscope, a high-throughput super-resolution microscope based on reversible saturable optical fluorescence transitions (RESOLFT).
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| 34. |
- Moreno, Xavier Casas, et al.
(författare)
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Multi‐foci parallelised RESOLFT nanoscopy in an extended field‐of‐view
- 2022
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Ingår i: Journal of Microscopy. - : Wiley. - 0022-2720 .- 1365-2818.
-
Tidskriftsartikel (refereegranskat)abstract
- Live-cell imaging of biological structures at high resolution poses challenges in the microscope throughput regarding area and speed. For this reason, different parallelisation strategies have been implemented in coordinate- and stochastictargeted switching super-resolution microscopy techniques. In this line, the molecular nanoscale live imaging with sectioning ability (MoNaLISA), based on reversible saturable optical fluorescence transitions (RESOLFT), offers 45 - 65 nm resolution of large fields of view in a few seconds. In MoNaLISA, engineered light patterns strategically confine the fluorescence to sub-diffracted volumes in a large area and provide optical sectioning, thus enabling volumetric imaging at high speeds. The optical setup presented in this paper extends the degree of parallelisation of the MoNaLISA microscope by more than four times, reaching a field-of-view of (100 - 130 mu m)(2). We set up the periodicity and the optical scheme of the illumination patterns to be power-efficient and homogeneous. In a single recording, this new configuration enables super-resolution imaging of an extended population of the post- synaptic density protein Homer1c in living hippocampal neurons.
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| 35. |
- Nevskyi, Oleksii, et al.
(författare)
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Fluorescent Diarylethene Photoswitches-A Universal Tool for Super-Resolution Microscopy in Nanostructured Materials
- 2018
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Ingår i: Small. - : Wiley-VCH Verlagsgesellschaft. - 1613-6810 .- 1613-6829. ; 14:10
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Tidskriftsartikel (refereegranskat)abstract
- Super-resolution fluorescence microscopy allows for unprecedented in situ visualization of biological structures, but its application to materials science has so far been comparatively limited. One of the main reasons is the lack of powerful dyes that allow for labeling and photoswitching in materials science systems. In this study it is shown that appropriate substitution of diarylethenes bearing a fluorescent closed and dark open form paves the way for imaging nanostructured materials with three of the most popular super-resolution fluorescence microscopy methods that are based on different concepts to achieve imaging beyond the diffraction limit of light. The key to obtain optimal resolution lies in a proper control over the photochemistry of the photoswitches and its adaption to the system to be imaged. It is hoped that the present work will provide researchers with a guide to choose the best photoswitch derivative for super-resolution microscopy in materials science, just like the correct choice of a Swiss Army Knife's tool is essential to fulfill a given task.
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| 36. |
- Pennacchietti, Francesca, et al.
(författare)
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Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms
- 2023
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400–650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.
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| 37. |
- Pennacchietti, Francesca, et al.
(författare)
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Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy
- 2018
-
Ingår i: Nature Methods. - : Nature Publishing Group. - 1548-7091 .- 1548-7105. ; 15:8, s. 601-
-
Tidskriftsartikel (refereegranskat)abstract
- Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.
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| 38. |
- Rems, Lea, et al.
(författare)
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Identification of electroporation sites in the complex lipid organization of the plasma membrane
- 2022
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon termed electroporation is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrhythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study, we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. Additionally, by analysing poration kinetics with Bayesian survival analysis we show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.
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| 39. |
- Rems, Lea, et al.
(författare)
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Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated Ion Channels
- 2020
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 119:1, s. 190-205
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Tidskriftsartikel (refereegranskat)abstract
- Pulsed electric fields are increasingly used in medicine to transiently increase the cell membrane permeability via electroporation to deliver therapeutic molecules into the cell. One type of event that contributes to this increase in membrane permeability is the formation of pores in the membrane lipid bilayer. However, electrophysiological measurements suggest that membrane proteins are affected as well, particularly voltage-gated ion channels (VGICs). The molecular mechanisms by which the electric field could affects these molecules remain unidentified. In this study, we used molecular dynamics simulations to unravel the molecular events that take place in different VGICs when exposing them to electric fields mimicking electroporation conditions. We show that electric fields can induce pores in the voltage-sensor domains (VSDs) of different VGICs and that these pores form more easily in some channels than in others. We demonstrate that poration is more likely in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions. We further show that pores in VSDs can expand into socalled complex pores, which become stabilized by lipid headgroups. Our results suggest that such complex pores are considerably more stable than conventional lipid pores, and their formation can lead to severe unfolding of VSDs from the channel. We anticipate that such VSDs become dysfunctional and unable to respond to changes in transmembrane voltage, which is in agreement with previous electrophysiological measurements showing a decrease in the voltage-dependent transmembrane ionic currents after pulse treatment. Finally, we discuss the possibility of activation of VGICs by submicrosecond-duration pulses. Overall, our study reveals a new, to our knowledge, mechanism of electroporation through membranes containing VGICs.
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| 40. |
- Richter, Katharina N., et al.
(författare)
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Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy
- 2018
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Ingår i: EMBO Journal. - : WILEY. - 0261-4189 .- 1460-2075. ; 37:1, s. 139-159
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Tidskriftsartikel (refereegranskat)abstract
- Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.
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| 41. |
- Schramm, Frederic D., 1989-, et al.
(författare)
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Growth‐driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus
- 2019
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 111:6, s. 1430-1448
-
Tidskriftsartikel (refereegranskat)abstract
- All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein collects at the cell poles and is retained throughout consecutive cell divisions only in old pole‐inheriting daughter cells, resulting in aggregation‐free progeny within a few generations. In this study we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium protein aggregates form as multiple distributed foci located throughout the cell volume. Time‐lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short‐lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long‐lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.
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| 42. |
- Smoler, M., et al.
(författare)
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Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers
- 2020
-
Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1867:8
-
Tidskriftsartikel (refereegranskat)abstract
- The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.
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| 43. |
- Storti, Barbara, et al.
(författare)
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Role of Gln222 in Photoswitching of Aequorea Fluorescent Proteins : A Twisting and H-Bonding Affair?
- 2018
-
Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 13:8, s. 2082-2093
-
Tidskriftsartikel (refereegranskat)abstract
- Reversibly photoswitchable fluorescent proteins (RSFPs) admirably combine the genetic encoding of fluorescence with the ability to repeatedly toggle between a bright and dark state, adding a new,temporal dimension to the fluorescence signal. Accordingly, in recent years RSFPs have paved the way to novel applications in cell imaging that rely on their reversible photoswitching, including many super-resolution techniques such as F-PALM, RESOLFT, and SOFI that provide nanoscale pictures of the living matter. Yet many RSFPs have been engineered by a rational approach only to a limited extent, in the absence of clear structure property relationships that in most cases make anecdotic the emergence of the photoswitching. We reported [Bizzarri et al. J. Am Chem Soc. 2010, 102, 85] how the E222Q replacement is a single photoswitching mutation, since it restores the intrinsic cis-trans photo-isomerization properties of the chromophore in otherwise nonswitchable Aequorea proteins of different color and mutation pattern (Q-RSFPs). We here investigate the subtle role of Q222 on the excited-state photophysics of the two simplest Q-RSFPs by a combined experimental and theoretical approach, using their nonswitchable anacestor EGFP as benchmark. Our findings link indissolubly photoswitching and Q222 presence, by a simple yet elegant scenario: largely twisted chromophore structures around the double bond (including hula-twist configurations) are uniquely stabilized by Q222 via H-bonds. Likely, these H-bonds subtly modulate the electronic properties of the chromophore, enabling the conical intersection that connects the excited cis to ground trans chromophore. Thus, Q222 belongs to a restricted family of single mutations that change dramatically the functional phenotype of a protein. The capability to distinguish quantitatively T65S/E222Q EGFP ("WildQ", wQ) from the spectrally identical EGFP by quantitative Optical Lock-In Detection (qOLID) witnesses the relevance of this mutation for cell imaging.
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| 44. |
- Terriac, Emmanuel, et al.
(författare)
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Vimentin Levels and Serine 71 Phosphorylation in the Control of Cell-Matrix Adhesions, Migration Speed, and Shape of Transformed Human Fibroblasts
- 2017
-
Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 6:1
-
Tidskriftsartikel (refereegranskat)abstract
- Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30-50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix adhesions. Long vimentin filaments were found in the proximity of large FAs. Vimentin expression in these cells caused a reduction in FAs size and an elongated cell shape, but did not affect FA lifetime, or the speed or directionality of cell migration. Expression of a phospho-mimicking mutant (S71D) of vimentin increased the speed of cell migration. Taken together, our results suggest that in highly migratory, transformed mesenchymal cells, vimentin levels control the cell shape and FA size, but not cell migration, which instead is linked to the phosphorylation status of S71 vimentin. These observations are consistent with the possibility that not only levels, but also the assembly status of vimentin control cell migration.
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| 45. |
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| 46. |
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| 47. |
- Testa, Ilaria
(författare)
-
RESOLFT super resolution microscopy in neuronal cells
- 2017
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Ingår i: Optics InfoBase Conference Papers. - : Optics Info Base, Optical Society of America. - 9781557528209
-
Konferensbidrag (refereegranskat)abstract
- We show that emergent RESOLFT fluorescence nanoscopy enables fast and continuous imaging of living hippocampal cells and tissues with details at the nanoscale by producing raw data images using only ultralow levels of light.
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| 48. |
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| 49. |
- Volpato, Andrea, et al.
(författare)
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Extending fluorescence anisotropy to large complexes using reversibly switchable proteins
- 2023
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Ingår i: Nature Biotechnology. - : Springer Nature. - 1087-0156 .- 1546-1696. ; 41:4, s. 552-559
-
Tidskriftsartikel (refereegranskat)abstract
- The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome.
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| 50. |
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