Designing chromatic optical retarder stacks for segmented next-generation easySTED phase plates

Fluorescence nanoscopy methods based on the RESOLFT principle, such as beam-scanning STED nanoscopy, require the co-alignment of optical beams for molecular state (on/off) switching and fluorescence excitation. The complexity and stability of the beam alignment can be drastically simplified and improved by using a single-mode fibre as the sole light source for all required laser beams. This in turn then requires a chromatic optical element for shaping the off-switching beam into a focal-plane donut while simultaneously leaving the focal intensity distributions at other wavelengths shaped as regular focal spots. Here we describe novel designs of such so-called ‘easySTED phase plates’ and provide a rationale how to find the desired spectral signature for combinations of multiple wavelengths

Synaptic activity and strength are reflected by changes in the post-synaptic secretory pathway

Neurons are highly asymmetric cells that span long distances and need to react promptly to local demands. Consequently, neuronal secretory pathway elements are distributed throughout neurites, specifically in post-synaptic compartments, to enable local protein synthesis and delivery. Whether and how changes in local synaptic activity correlate to post-synaptic secretory elements is still unclear. To assess this, we used STED nanoscopy and automated quantitative image analysis of post-synaptic markers of the endoplasmic reticulum, ER-Golgi intermediate compartment, trans-Golgi network, and spine apparatus. We found that the distribution of these proteins was dependent on pre-synaptic activity, measured as the amount of recycling vesicles. Moreover, their abundance correlated to both pre- and post-synaptic markers of synaptic strength. Overall, the results suggest that in small, low-activity synapses the secretory pathway components are tightly clustered in the synaptic area, presumably to enable rapid local responses, while bigger synapses utilise secretory machinery components from larger, more diffuse areas

Fluorescent and bioluminescent calcium indicators with tuneable colors and affinities

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum (ER). Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput-compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays

Ternary Phases (Heusler) in the System Ti-Co-Sn

acceptedVersio

Supplementary Material for: Activin-A Is a Pro-Inflammatory Regulator in Type-2-Driven Upper Airway Disease

<b><i>Background:</i></b> Allergic upper airway disease involves pro-inflammatory type-2 cytokines such as IL-5 and regulatory tissue repair mediators, in particular transforming growth factor (TGF)-β₁. The TGF-β-superfamily member activin-A displays multiple biological functions and shares certain signalling pathways with TGF-β₁. We aimed to examine the coregulation of mucosal activin-A and TGF-β₁ in acute allergic and chronic Th2-driven upper airway disease. <b><i>Methods:</i></b> We investigated mucosal cytokine expression profiles and kinetics using RT-PCR after nasal allergen challenges in patients with seasonal allergic rhinitis. Furthermore, we analysed mucosal specimens from patients with chronic upper airway disease with nasal polyps using ELISPOTs and confocal microscopy. In addition, we stimulated nasal mucosa ex vivo from patients with nasal polyps as well as primary nasal cell cultures from healthy donors. <b><i>Results:</i></b> Mucosal activin-A expression revealed increasing correlation with IL-5 and TGF-β₁ at 0.25, 6, and 24 h, respectively, and was significantly upregulated at 6 h after allergen challenge. The correlated expression was found to be more pronounced in chronic disease with nasal polyps, showing substantially (48-fold) increased activin-A-producing cells in nasal polyps by ELISPOT, while submucosal downstream signalling as determined by confocal microscopy was decreased. Ex vivo stimulations of nasal tissue suggested that activin-A and TGF-β₁ mutually regulate each other’s expression at the mRNA level and, when combined, enhance IL-5 expression. <b><i>Conclusion:</i></b> Activin-A in allergic upper airway disease acts as a pro-inflammatory mediator and TGF-β₁ modifier. Our data in the upper airways oppose the view of potentially anti-inflammatory properties in contrast to lymphatic compartments