144 research outputs found
Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans
No full textFluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.sponsorship: This work was supported by the Research Foundation Flanders (FWO Vlaanderen) (1S01817N to W.V.G. and G062616N to P.V.D. and P.D.) and ERC starting grant 714688 NanoCellActivity. (Research Foundation Flanders (FWO Vlaanderen)|1S01817N, Research Foundation Flanders (FWO Vlaanderen)|G062616N, ERC|714688, European Research Council (ERC)|714688)status: Accepte
Wind-tunnel study of flow through STS shuttle launch site exhaust vents, Vandenberg Air Force Base
No full textIncludes bibliographical references.September 1982.CER82-83JAP-JEC13.For Martin Marietta Corporation, Vandenberg Operations.CSU Project 2-95400
Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization
No full textReversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties.sponsorship: Peter Dedecker acknowledges support by the European Research Council (ERC) via ERC Starting Grant 714688 and from the Research Foundation-Flanders via grants G062616N, G0B8817N, G0A5817N, and VS.003.16N. Thijs Roebroek and Sam Duwe thank the Research Foundation Flanders (FWO-Vlaanderen) for a doctoral- and post-doctoral fellowship. Wim Vandenberg acknowledges support by the Horizons 2020 program via grant 686271. (European Research Council (ERC)|714688, Research Foundation-Flanders|G062616N, Research Foundation-Flanders|G0B8817N, Research Foundation-Flanders|G0A5817N, Research Foundation-Flanders|VS.003.16N, Research Foundation Flanders (FWO-Vlaanderen), Horizons program|686271)status: Publishe
Galaxy Zoo:chiral correlation function of galaxy spins
No full textGalaxy Zoo is the first study of nearby galaxies that contains reliable information about the spiral sense of rotation of galaxy arms for a sizeable number of galaxies. We measure the correlation function of spin chirality (the sense in which galaxies appear to be spinning) of face-on spiral galaxies in angular, real and projected spaces. Our results indicate a hint of positive correlation at separations less than ~0.5 Mpc at a statistical significance of 2-3 sigma. This is the first experimental evidence for chiral correlation of spins. Within tidal torque theory it indicates that the inertia tensors of nearby galaxies are correlated. This is complementary to the studies of nearby spin axis correlations that probe the correlations of the tidal field. Theoretical interpretation is made difficult by the small distances at which the correlations are detected, implying that substructure might play a significant role, and our necessary selection of face-on spiral galaxies, rather than a general volume-limited sample
Quantitative comparison of camera technologies for cost-effective super-resolution optical fluctuation imaging (SOFI)
No full textVan den Eynde R, Sandmeyer A, Vandenberg W, et al. Quantitative comparison of camera technologies for cost-effective super-resolution optical fluctuation imaging (SOFI). Journal of Physics: Photonics. 2019;1(4): 044001
Het evalueren en verbeteren van de kwaliteit van geavanceerde microscopie in levende cellen
No full textstatus: Publishe
Effect of probe diffusion on the SOFI imaging accuracy
No full textAbstractLive-cell super-resolution fluorescence imaging is becoming commonplace for exploring biological systems, though sample dynamics can affect the imaging quality. In this work we evaluate the effect of probe diffusion on super-resolution optical fluctuation imaging (SOFI), using a theoretical model and numerical simulations based on the imaging of live cells labelled with photochromic fluorescent proteins. We find that, over a range of physiological conditions, fluorophore diffusion results in a change in the amplitude of the SOFI signal. The magnitude of this change is approximately proportional to the on-time ratio of the fluorophores. However, for photochromic fluorescent proteins this effect is unlikely to present a significant distortion in practical experiments in biological systems. Due to this lack of distortions, probe diffusion strongly enhances the SOFI imaging by avoiding spatial undersampling caused by the limited labeling density.</jats:p
NASA Launch Services Program Small Satellite Processing Capabilities at Kennedy Space Center and Vandenberg Air Force Base
No full textThe NASA Launch Services Program (LSP) Launch Site Integration Branch (LSIB) conducted a study to identify options for accommodating launch site support requirements for the next generation of NASA small satellites and secondary payloads launched from the Kennedy Space Center (KSC) and Vandenberg Air Force Base (VAFB). The anticipated needs range from cleanroom facilities, to long-term secure hardware storage, to propellant loading/unloading, to stacking and fairing encapsulation. Existing commercial and government payload processing facilities (PPF) are highly capable and commonly used to support satellite processing, while alternate options may be better tailored for the needs and budgets of small satellites. These include peripheral facilities such as PPF airlocks or partitioned spaces, existing facilities which can be refurbished or upgraded, and portable facilities such as cleanroom trailers. The LSP LSIB continues to evaluate its capability to meet the needs of small satellites and secondary payloads. The author requests input from the small satellite community, to help identify any emerging launch site requirements for future NASA small satellite and secondary payload missions
Self-contained and modular structured illumination microscope
No full textVan den Eynde R, Vandenberg W, Hugelier S, et al. Self-contained and modular structured illumination microscope. Biomedical Optics Express. 2021;12(7):4414-4422.We present a modular implementation of structured illumination microscopy (SIM) that is fast, largely self-contained and that can be added onto existing fluorescence microscopes. Our strategy, which we call HIT-SIM, can theoretically deliver well over 50 super-resolved images per second and is readily compatible with existing acquisition software packages. We provide a full technical package consisting of schematics, a list of components and an alignment scheme that provides detailed specifications and assembly instructions. We illustrate the performance of the instrument by imaging optically large samples containing sequence-specifically stained DNA fragments. (c) 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen
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