MINFLUX fluorescence nanoscopy in biological tissue.

Optical imaging access to nanometer-level protein distributions in intact tissue is a highly sought-after goal, as it would provide visualization in physiologically relevant contexts. Under the unfavorable signal-to-background conditions of increased absorption and scattering of the excitation and fluorescence light in the complex tissue sample, superresolution fluorescence microscopy methods are severely challenged in attaining precise localization of molecules. We reasoned that the typical use of a confocal detection pinhole in MINFLUX nanoscopy, suppressing background and providing optical sectioning, should facilitate the detection and resolution of single fluorophores even amid scattering and optically challenging tissue environments.

Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site.

Auditory orientation and distance estimation of sighted humans using virtual echolocation with artificial and self-generated sounds.

Active echolocation of sighted humans using predefined synthetic and self-emitted sounds, as habitually used by blind individuals, was investigated. Using virtual acoustics, distance estimation and directional localization of a wall in different rooms were assessed. A virtual source was attached to either the head or hand with realistic or increased source directivity.

Acoustically driven orientation and navigation in enclosed spaces.

Awareness of space, and subsequent orientation and navigation in rooms, is dominated by the visual system. However, humans are able to extract auditory information about their surroundings from early reflections and reverberation in enclosed spaces. To better understand orientation and navigation based on acoustic cues only, three virtual corridor layouts (I-, U-, and Z-shaped) were presented using real-time virtual acoustics in a three-dimensional 86-channel loudspeaker array.

Copper-Mediated Single-Electron Approach to Indoline Amination: Scope, Mechanism, and Total Synthesis of Asperazine A.

Pyrroloindolines bearing a C3-N linkage comprise the core of many biologically active natural products, but many methods toward their synthesis are limited by the sterics or electronics of the product. We report a single electron-based approach for the synthesis of this scaffold and demonstrate high-yielding aminations, regardless of electronic or steric demands. The transformation uses copper wire and isopropanol to promote the reaction.

Environmental enrichment enhances patterning and remodeling of synaptic nanoarchitecture as revealed by STED nanoscopy.

Synaptic plasticity underlies long-lasting structural and functional changes to brain circuitry and its experience-dependent remodeling can be fundamentally enhanced by environmental enrichment. It is however unknown, whether and how the environmental enrichment alters the morphology and dynamics of individual synapses. Here, we present a virtually crosstalk-free two-color in vivo stimulated emission depletion (STED) microscope to simultaneously superresolve the dynamics of endogenous PSD95 of the post-synaptic density and spine geometry in the mouse cortex.

Novel Reagent Space: Identifying Unorderable but Readily Synthesizable Building Blocks.

Drug discovery building blocks available commercially or within an internal inventory cover a diverse range of chemical space and yet describe only a tiny fraction of all chemically feasible reagents. Vendors will eagerly provide tools to search the former; there is no straightforward method of mining the latter. We describe a procedure and use case in assembling chemical structures not available for purchase but that could likely be synthesized in one robust chemical transformation starting from readily available building blocks.

Ketone Incorporation Extends the Emission Properties of the Xanthene Scaffold Beyond 1000 nm.

Imaging in the shortwave-infrared region (SWIR, λ = 1000-2500 nm) has the potential to enable deep tissue imaging with high resolution. Critical to the development of these methods is the identification of low molecular weight, biologically compatible fluorescent probes that emit beyond 1000 nm. Exchanging the bridging oxygen atom on the xanthene scaffold (C10' position) with electron withdrawing groups has been shown to lead to significant redshifts in absorbance and emission.

Stable but not rigid: Chronic in vivo STED nanoscopy reveals extensive remodeling of spines, indicating multiple drivers of plasticity.

Excitatory synapses on dendritic spines of pyramidal neurons are considered a central memory locus. To foster both continuous adaption and the storage of long-term information, spines need to be plastic and stable at the same time. Here, we advanced in vivo STED nanoscopy to superresolve distinct features of spines (head size and neck length/width) in mouse neocortex for up to 1 month.

Multi-label in vivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins.

Despite the tremendous success of super-resolution microscopy, multi-color in vivo applications are still rare. Here we present live-cell multi-label STED microscopy in vivo and in vitro by combining spectrally separated excitation and detection with temporal sequential imaging of reversibly switchable fluorescent proteins (RSFPs). Triple-label STED microscopy resolves pre- and postsynaptic nano-organizations in vivo in mouse visual cortex employing EGFP, Citrine, and the RSFP rsEGP2.

The role of early and late reflections on perception of source orientation.

Sound radiation of most natural sources, like human speakers or musical instruments, typically exhibits a spatial directivity pattern. This directivity contributes to the perception of sound sources in rooms, affecting the spatial energy distribution of early reflections and late diffuse reverberation. Thus, for convincing sound field reproduction and acoustics simulation, source directivity has to be considered.

Low self-reported stress despite immune-physiological changes in paramedics during rescue operations.

Despite the high stress levels, paramedics seem to ignore or even negate the stress. This can be detrimental and lead to stress-related diseases. Therefore, we investigated the divergence between physiological and psychological stress responses of paramedics.

Modulation of cognition and neuronal plasticity in gain- and loss-of-function mouse models of the schizophrenia risk gene Tcf4.

The transcription factor TCF4 was confirmed in several large genome-wide association studies as one of the most significant schizophrenia (SZ) susceptibility genes. Transgenic mice moderately overexpressing Tcf4 in forebrain (Tcf4tg) display deficits in fear memory and sensorimotor gating. As second hit, we exposed Tcf4tg animals to isolation rearing (IR), chronic social defeat (SD), enriched environment (EE), or handling control (HC) conditions and examined mice with heterozygous deletion of the exon 4 (Tcf4Ex4δ) to unravel gene-dosage effects.

Enantioselective PCCP Brønsted acid-catalyzed aza-Piancatelli rearrangement.

An enantioselective aza-Piancatelli rearrangement has been developed using a chiral Brønsted acid based on pentacarboxycyclopentadiene (PCCP). This reaction provides rapid access to valuable chiral 4-amino-2-cyclopentenone building blocks from readily available starting material and is operationally simple.

In vivo STED microscopy: A roadmap to nanoscale imaging in the living mouse.

Superresolution microscopy techniques are now widely used, but their application in living animals remains a challenging task. The first superresolution imaging in a live vertebrate was demonstrated with STED microscopy in the visual cortex of an anaesthetized mouse. Here, we explain the requirements for a simple but robust in vivo STED microscope as well as the surgical preparation of the cranial window and the mounting of the mouse in detail.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio.

In vivo STED microscopy visualizes PSD95 sub-structures and morphological changes over several hours in the mouse visual cortex.

The post-synaptic density (PSD) is an electron dense region consisting of ~1000 proteins, found at the postsynaptic membrane of excitatory synapses, which varies in size depending upon synaptic strength. PSD95 is an abundant scaffolding protein in the PSD and assembles a family of supercomplexes comprised of neurotransmitter receptors, ion channels, as well as signalling and structural proteins. We use superresolution STED (STimulated Emission Depletion) nanoscopy to determine the size and shape of PSD95 in the anaesthetised mouse visual cortex.

In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins.

The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse.

Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism.

Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function.

Contractile characteristics of gastrocnemius-soleus muscle in the SOD1G93A ALS mouse model.

Objectives: In the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS), a selective degeneration of fast-fatigable motor units and consequently an early decline of contractile force in individual fast-twitch muscles have been observed in the preclinical stage. However, most human muscles include fast and slow motor units. Gastrocnemius-soleus group (GS) contains such a mixture of units.

Long lasting activity of nociceptive muscular afferents facilitates bilateral flexion reflex pattern in the feline spinal cord.

Chronic muscular limb pain requires the adoption of motor patterns distinct from the classic ipsilateral flexion, crossed extension and corresponding reciprocal inhibitions to acute exteroceptive stimulation. Using selective chemical activation of group III/IV afferents in gastrocnemius-soleus (GS) muscles we investigated bilaterally their reflex responses conditioned by (a) acute 'myositis' induced by intramuscular carrageenan; and (b) sub-acute 'myositis' induced by infusion of complete Freund's adjuvant (CFA). Reflex transmission was detected by monosynaptic testing and c-fos staining used to identify increased neuronal activity.

Predictive factors for sustained virological response after treatment with pegylated interferon α-2a and ribavirin in patients infected with HCV genotypes 2 and 3.

Background: Previous trials have often defined genotype 2 and 3 patients as an "easy to treat" group and guidelines recommend similar management.

Aims: The present study looks for differences between the two genotypes and analyzes predictive factors for SVR.

Methods: Prospective, community-based cohort study involving 421 physicians throughout Germany.

Dysregulated expression of neuregulin-1 by cortical pyramidal neurons disrupts synaptic plasticity.

Neuregulin-1 (NRG1) gene variants are associated with increased genetic risk for schizophrenia. It is unclear whether risk haplotypes cause elevated or decreased expression of NRG1 in the brains of schizophrenia patients, given that both findings have been reported from autopsy studies. To study NRG1 functions in vivo, we generated mouse mutants with reduced and elevated NRG1 levels and analyzed the impact on cortical functions.

Nanoscopy of filamentous actin in cortical dendrites of a living mouse.

We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 μm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43-70 nm resolution reveal dynamical changes in spine morphology.