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Background: Direct acting antivirals (DAAs) as a curative treatment of hepatitis C have been available for several years and have replaced interferon-containing therapies. However, treatment rates of people who inject drugs (PWID) are declining in Germany, putting the elimination of hepatitis C by 2030 at risk. This study aimed at elucidating the knowledge of, and attitude towards, hepatitis C treatment in a clinical sample of PWID., Methods: Participants were recruited between February 2019 and October 2020 at two opioid agonist therapy (OAT) clinics and two in-patient drug detoxification wards. Based on the European Addiction Severity Index (Europ-ASI), a standardized interview focusing on: sociodemographic data, drug history, risky behavior, infection with hepatitis C virus (HCV) and HIV, and previous experience with HCV treatment was carried out. In addition, participants filled in a questionnaire evaluating 13 statements relating to HCV treatment (right/wrong) and 15 statements on their personal 'pros and cons' views to start such a treatment assessed with the means of a 6-point Likert scale., Results: A total of 153 patients (average age 45 years, male 78%; 106 (69.3%) currently in opioid maintenance treatment, 47 (30.7%) currently admitted to an inpatient detoxification) with an opioid use disorder were investigated. All of them reported having injected drugs at least once in their lives; 97 participants (63.3%) stated that they had been previously diagnosed with HCV infection. Among them, 27/97 patients (27.8%) reported a previous treatment with interferon; 27/97 (27.8%) with DAAs; and 32/97 (33.0%) reported a currently active hepatitis C. Most patients knew about the availability and efficacy of DAAs. However, DAAs' low rate of side effects, their short treatment duration, and their replacement of interferon, were not correctly evaluated by up to 50.3% of patients. 25-40% of 32 patients with currently active hepatitis C prioritized handling of social and other medical issues, e.g., reduction of heroin use, over treatment of hepatitis C., Conclusions: Although current levels of risky behavior have reportedly been reduced by active PWID over the past few years, educational and motivational interventions to increase hepatitis C treatment uptake should address the gaps in patients' knowledge., (© 2024. The Author(s).)

Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus, centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys, the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species., (© 2024. The Author(s).)

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation., (© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Two decades after its introduction, optogenetics - a biological technique to control the activity of neurons or other cell types with light - remains a cutting edge and promising tool to study biological processes. Its increasing usage in research varies widely from causally exploring biological mechanisms and neural computations, to neurostimulation and sensory restauration. To stimulate neurons in the brain, a variety of approaches have been developed to generate precise spatiotemporal light patterns. Yet certain constrains still exists in the current optical techniques to activate a neuronal population with both cellular resolution and millisecond precision. Here, we describe an experimental setup allowing to stimulate a few tens of neurons in a plane at sub-millisecond rates using 2-photon activation. A liquid crystal on silicon spatial light modulator (LCoS-SLM) was used to generate spatial patterns in 2 dimensions. The image of the patterns was formed on the plane of a digital micromirror device (DMD) that was used as a fast temporal modulator of each region of interest. Using fluorescent microscopy and patch-clamp recording of neurons in culture expressing the light-gated ion channels, we characterized the temporal and spatial resolution of the microscope. We described the advantages of combining the LCoS-SLM with the DMD to maximize the temporal precision, modulate the illumination amplitude, and reduce background activation. Finally, we showed that this approach can be extended to patterns in 3 dimensions. We concluded that the methodology is well suited to address important questions about the role of temporal information in neuronal coding.

Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength., (© 2021. The Author(s).)

Fluorescence detection, either involving propagating or near-field emission, is widely being used in spectroscopy, sensing, and microscopy. Total internal reflection fluorescence (TIRF) confines fluorescence excitation by an evanescent (near) field, and it is a popular contrast generator for surface-selective fluorescence assays. Its emission equivalent, supercritical angle fluorescence (SAF), is comparably less established, although it achieves a similar optical sectioning as TIRF does. SAF emerges when a fluorescing molecule is located very close to an interface and its near-field emission couples to the higher refractive index medium (n 2 >n 1 ) and becomes propagative. Then, most fluorescence is detectable on the side of the higher-index substrate, and a large fraction of this fluorescence is emitted into angles forbidden by Snell's law. SAF, as well as the undercritical angle fluorescence (UAF; far-field emission) components, can be collected with microscope objectives having a high-enough detection aperture (numerical aperture >n 2 ) and be separated in the back focal plane by Fourier filtering. The back focal plane image encodes information about the fluorophore radiation pattern, and it can be analyzed to yield precise information about the refractive index in which the emitters are embedded, their nanometric distance from the interface, and their orientation. A SAF microscope can retrieve this near-field information through wide-field optics in a spatially resolved manner, and this functionality can be added to an existing inverted microscope. Here, we describe the potential underpinning of SAF microscopy and spectroscopy, particularly in comparison with TIRF. We review the challenges and opportunities that SAF presents from a biophysical perspective, and we discuss areas in which we see potential., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Roughly half of a cell's proteins are located at or near the plasma membrane. In this restricted space, the cell senses its environment, signals to its neighbors, and exchanges cargo through exo- and endocytotic mechanisms. Ligands bind to receptors, ions flow across channel pores, and transmitters and metabolites are transported against concentration gradients. Receptors, ion channels, pumps, and transporters are the molecular substrates of these biological processes, and they constitute important targets for drug discovery. Total internal reflection fluorescence (TIRF) microscopy suppresses the background from the cell's deeper layers and provides contrast for selectively imaging dynamic processes near the basal membrane of live cells. The optical sectioning of TIRF is based on the excitation confinement of the evanescent wave generated at the glass/cell interface. How deep the excitation light actually penetrates the sample is difficult to know, making the quantitative interpretation of TIRF data problematic. Nevertheless, many applications like superresolution microscopy, colocalization, Förster resonance energy transfer, near-membrane fluorescence recovery after photobleaching, uncaging or photoactivation/switching as well as single-particle tracking require the quantitative interpretation of evanescent-wave-excited images. Here, we review existing techniques for characterizing evanescent fields, and we provide a roadmap for comparing TIRF data across images, experiments, and laboratories., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that-in many aspects-recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that-unlike two- or three-lens light-sheet microscopes-it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petráň spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

Dipole radiation patterns change when a fluorescent molecule comes close to the boundary between media of different refractive indices. Near-interface molecules emit mostly into the higher-index medium, predominantly around the critical angle. The radiation pattern encodes information about the emitter distance, orientation, and the refractive index of the embedding medium. Analyses of the supercritical angle fluorescence on pupil plane images can retrieve this information and have been applied both for refractometry with subcellular resolution and for the detection of metabolically active cancerous cells. In this issue of ACS Nano, Ferdman et al. employ this strategy in a label-free assay for detecting single bacteria, based on measuring the refractive-index change produced by bacterial growth in a fluorophore-coated microfluidic channel.

Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating evanescent and far-field fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts and BON cells. By targeting various dyes and fluorescent-protein chimeras to vesicles, the plasma membrane, as well as mitochondria and the endoplasmic reticulum, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope, with a removable Bertrand lens as the only modification. Our technique has important applications for imaging axial vesicle dynamics and the mitochondrial energy state or detecting metabolically more active cancer cells., (Copyright © 2017. Published by Elsevier Inc.)

The advent and increasing availability of super-resolution microscopies has prompted re-searchers to re-investigate questions of co-localization and co-clustering in the hope of providing more precise and relevant data. Here, we focus on the problem of studying inter-organelle interfaces, a topic of growing interest in cell biology. We sought to identify mitochondria-associated membrane (MAM) candidate sites from dual-colour large-field super-resolution images. MAMs are specialized lipid microdomains of the endoplasmic reticulum (ER) in close apposition with mitochondria. Using total internal reflection fluorescence structured-illumination microscopy (TIRF-SIM, Brunstein et al., Optics Express, 2013), we achieved a three-dimensional spatial resolution down to ∼100 nm. Based on experimental and simulated data, we studied how the spatio-temporal resolution affects common descriptors of co-localization. The apparent overlap scaled inversely with spatial resolution (as expected for objects that are in close apposition and do not merge), independently of the precise metrics used. Important for live-cell imaging, organelle motility made measurements more uncertain, rendering statements of how physiological stimulations or pharmacologic manipulations affect co-localization less robust. Organelle density, or equivalent, the choice of different subcellular regions of interest (ROIs) had a marked effect on the amount of co-localization, as had the size of the ROI chosen. Our study calls for prudence when interpreting co-localization data and suggests that cell and organelle motility, the choice of the ROI analysed, the effective spatiotemporal resolution all impact on the result and hence should systematically be stated, particularly when co-localization arguments are used to assess the effect of drug application on cellular signalling pathways., (© 2016 Wiley Periodicals, Inc.)

Metallic and dielectric nanostructures can show sharp contrasted resonances, sensitive to the environment, and high field enhancement in sub-wavelength volumes. For this reason, these structures are commonly used as molecular sensors. Only few works have focused on their application in optical microscopy, in particular in superresolution. In this work we have designed, fabricated and optically tested a nanostructured TiO(2) substrate, fabricated by direct embossing of TiO(2) derived film, as a substrate for fluorescence microscopy. Moreover, using numerical simulations, we have compared the signal to background noise with respect to other metallo-dielectric structures. We show that the TiO(2) structure is a good candidate for reducing the thickness of the fluorescence excitation down to ~100 nm. Therefore, this substrate can be used to obtain Total Internal Reflection (TIRF) axial resolution without a TIRF-Microscopy system.

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2)., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)