Nanoscale imaging of restricted cell membrane receptor diffusion

In der vorliegenden Arbeit wurde eine neue bildgebende Methode zur Untersuchung der Diffusion in heterogenen Medien auf der Nanometerskala entwickelt (TNIM - Thermal Noise Imaging Microscopy). Die TNI-Mikroskopie wurde gezielt benutzt, um zu erforschen, ob die Beweglichkeit von Zellmembranrezeptoren durch laterale Nanostrukturen in der Membran beeinflusst wird. Bei der TNI-Mikroskopie wird die Diffusion eines Sondenpartikels mit Hilfe einer optischen Falle auf einen submikroskopischen Raumbereich limitiert und dort mit Nanometer räumlicher und Mikrosekunden zeitlicher Auflösung verfolgt. Damit kann die Position von diffusionsbehindernden Nanostrukturen bestimmt werden. Gleichzeitig wird erfasst wie die Mobilität des Partikels durch hydrodynamische Kopplung zu den beobachteten Strukturen beeinflusst wird. Mit Hilfe der TNI-Mikroskopie konnten existierende hydrodynamische Theorien, die die dreidimensionale Mobilität einer Kugel für unterschiedliche Abstände zu einer Grenzfläche beschreiben, mit bisher unerreichter Genauigkeit bestätigt werden. Für die zweidimensionalen Diffusionsmessungen in der Zellmembran wurde ein Nanopartikel an den epidermalen Wachstumsfaktorrezeptor (EGFR - Epidermal Growth Factor Receptor) gebunden. Die Analyse der Partikelbewegung zeigte nanoskopische Areale, die die Diffusion des Rezeptors stark beschränken, wobei sich die Position und Größe der unzugänglichen Areale im Sekundenbereich verändern kann. Der Vergleich mit einem lipidverankertem Protein ergab, dass die Areale vom beobachteten Protein abhängig sind. Des Weiteren wurden deutliche Hinweise darauf gefunden, dass die Diffusion des Rezeptors auf der Nanometerskala von der Lipiddoppelschichtstruktur der Zellmembran dominiert, auf Mikrometerskala jedoch durch die unzugänglichen Areale stark verlangsamt wird. Im letzten Teil der Arbeit wird analysiert wie die Beschränkung der Rezeptormobilität dessen Aktivierungskinetik und die laterale Informationsausbreitung in der Membran beeinflusst.In the work presented, a novel imaging technique was developed for the purpose of studying diffusive motion in heterogeneous media on the nanometer scale (TNIM - Thermal Noise Imaging Microscopy). TNI-Microscopy was specifically used to investigate if there exist lateral nanostructures that restrict the mobility of receptor proteins in cell membranes. In TNI-Microscopy, the diffusion of a nanoparticle is limited by an optical trap to a small region. Within this region the diffusion of the particle is tracked with nanometer spatial and microsecond temporal resolution. Thus, the position of nanoscopic structures that restrict diffusion can be determined. Furthermore, it is also recorded how the mobility of the particle is influenced by hydrodynamic coupling to the sampled structures. Using TNI-Microscopy, existing hydrodynamic theories that describe the three-dimensional mobility of a sphere at nanoscopic distances to an interface could be validated with unsurpassed accuracy. For two-dimensional diffusion measurements in the cellular plasma membrane, a nanoparticle was coupled to the epidermal growth factor receptor (EGFR). The analysis of the particle’s motion revealed nanoscopic membrane areas that strongly restrict the diffusion of the receptor. Furthermore, it was possible to observe that these inaccessible areas change shape and position on the second time scale. The comparison with a lipid anchored protein showed that the characteristics of these areas depend on the observed protein. Evidence is presented that the diffusion of the receptor on the nanometer scale is dominated by the lipid bilayer structure of the cell membrane, whereas its mobility on the micrometer scale is severely slowed by the observed nanoscopic membrane heterogeneities. In the last part of this work it is analyzed how the restricted receptor mobility influences its activation kinetics and the lateral spreading of a signal within the plane of the plasma membrane

Direct measurement of the nonconservative force field generated by optical tweezers

The force field of optical tweezers is commonly assumed to be conservative, neglecting the complex action of the scattering force. Using a novel method that extracts local forces from trajectories of an optically trapped particle, we measure the three dimensional force field experienced by a Rayleigh particle with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is nonconservative with the nonconservative component increasing radially away from the optical axis, in agreement with the Gaussian beam model of the optical trap. Together with thermal position fluctuations of the trapped particle, the presence of the nonconservative force can cause a complex flux of energy into the optical trap depending on the experimental conditions

Measurement properties of quality-of-life outcome measures for children and adults with eczema: an updated systematic review

Objective The aim of this updated systematic review was to systematically assess the measurement properties of previously discussed and new quality‐of‐life patient‐reported outcome measures (PROMs) in children and adults with eczema using the new COSMIN guideline. Methods A systematic literature search was conducted in PubMed and EMBASE. Eligible studies reported on measurement properties of quality‐of‐life PROMs for children and adults with eczema. The methodological quality of selected already known PROMs and new evidence identified through the literature search was assessed with the COSMIN Risk of Bias checklist. The adequacy of included PROMs was judged with updated quality criteria, and the quality of evidence of the summarized results was graded. Finally, PROMs were placed in a recommendation category (A‐C). Results In total, 133 measurement properties of nine different PROMs were assessed. No PROM could be placed in category A due to a lack of validation studies. Only the DLQI fulfilled the criteria for category C and therefore should not be recommended for use. All other PROMs were placed in category B, that is, they still have the opportunity to be recommended, but need further validation. Conclusions Currently, no PROM for quality of life can be recommended for use in children and adults with eczema. Further validation is needed. The DLQI cannot be recommended for future use

Customer journey management capability in business‑to‑business markets: Its bright and dark sides and overall impact on firm performance

Business-to-business (B2B) practitioners are increasingly interested in capabilities to holistically manage touchpoints along B2B customer journeys (CJs) to remain competitive. Research in the B2B context, however, has investigated neither what constitutes such a customer journey management capability (CJMC) nor how, whether, or when it creates value. Taking a mixed-methods approach, we conceptualize and operationalize B2B CJMC as a supplier's ability to achieve superior customer value along the B2B CJ by strategically creating value-anchored customer touchpoints characterized through the implementation of consistent resource usage across internal organizational boundaries and by continuously monitoring value creation toward the individual members of the buying center. Analyzing a multisource dataset, we provide evidence that B2B CJMC has an indirect effect on firm performance (i.e., return on sales) through two opposing mechanisms (i.e., customer loyalty and customer-related coordination costs). Importantly, using survey and archival data, we show that, overall, B2B CJMC has a significant and positive impact on firm performance through the two mechanisms. Finally, these underlying mechanisms are also prevalent when testing for the moderating factors switching costs, number of touchpoints, and product versus service

The German RECAP questionnaire: linguistic validation and cognitive debriefing in German adults with self-reported atopic eczema and parents of affected children

BackgroundRecap of atopic eczema (RECAP) is a patient-reported outcome measure (PROM) assessing eczema control. Long-term control of eczema is one of the four core outcome domains for atopic eczema trials. This instrument has been recently developed in the UK.ObjectiveThis study aimed to translate the English RECAP into German and test its content validity in a German population with self-reported atopic eczema.MethodsA six-step procedure including two forward and one backward translations, two consensus decisions and an expert review was performed to obtain a German version of RECAP. We conducted semi-standardized cognitive interviews with adults with atopic eczema (n = 7) and parents having children affected by this disease (n = 5). A “think-aloud” method was used and aspects of comprehensibility, comprehensiveness and relevance according to the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) criteria were examined. Interviews were coded using qualitative content analysis.ResultsNo particular linguistic problems were encountered during forward-backward translation. Minor wording changes were made as required. The title was adjusted to a more familiar German term of the disease (which is ‘Neurodermitis’). The recall period was rephrased from ‘over the last week’ to ‘over the last seven days’ since there was a different cultural understanding of the time frame. Regarding content validity, the items of the German RECAP were considered to be comprehensible, comprehensive and relevant for the participants and parents of affected children. The participants understood the instruction and considered the one-week recall period and the response options as appropriate.ConclusionsA German version of RECAP that is linguistically equivalent to the original version is now available but further assessment of its measurement properties is needed

Measurement properties of patient-reported outcome measures (PROMs) in hyperhidrosis: a systematic review

Purpose To critically appraise, compare and summarize the quality of all existing PROMs that have been validated in hyperhidrosis to at least some extend by applying the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) methodology. Thereby, we aim to give a recommendation for the use of PROMs in future clinical trials in hyperhidrosis. Methods We considered studies evaluating, describing or comparing measurement properties of PROMs as eligible. A systematic literature search in three big databases (MEDLINE, EMBASE and Web of Science) was performed. We assessed the methodological quality of each included study using the COSMIN Risk of Bias checklist. Furthermore, we applied predefined quality criteria for good measurement properties and finally, graded the quality of the evidence. Results Twenty-four articles reporting on 13 patient-reported outcome measures were included. Three instruments can be further recommended for use. They showed evidence for sufficient content validity and moderate- to high-quality evidence for sufficient internal consistency. The methodological assessment showed existing evidence gaps for eight other PROMs, which therefore require further validation studies to make an adequate decision on their recommendation. The Hyperhidrosis Disease Severity Measure-Axillary (HDSM-Ax) and the short-form health survey with 36 items (SF-36) were the only questionnaires not recommended for use in patients with hyperhidrosis due to moderate- to high-quality evidence for insufficient measurement properties. Conclusion Three PROMs, the Hyperhidrosis Quality of Life Index (HidroQoL), the Hyperhidrosis Questionnaire (HQ) and the Sweating Cognitions Inventory (SCI), can be recommended for use in future clinical trials in hyperhidrosis. Results obtained with these three instruments can be seen as trustworthy. Nevertheless, further validation of all three PROMs is desirable. Systematic review registration PROSPERO CRD4202017024

A Hitchhiker's guide through the bio-image analysis software universe

Modern research in the life sciences is unthinkable without computational methods for extracting, quantifying and visualising information derived from microscopy imaging data of biological samples. In the past decade, we observed a dramatic increase in available software packages for these purposes. As it is increasingly difficult to keep track of the number of available image analysis platforms, tool collections, components and emerging technologies, we provide a conservative overview of software that we use in daily routine and give insights into emerging new tools. We give guidance on which aspects to consider when choosing the platform that best suits the user's needs, including aspects such as image data type, skills of the team, infrastructure and community at the institute and availability of time and budget.Peer reviewe

The Image Data Explorer: Interactive exploration of image-derived data

International audienceMany bioimage analysis projects produce quantitative descriptors of regions of interest in images. Associating these descriptors with visual characteristics of the objects they describe is a key step in understanding the data at hand. However, as many bioimage data and their analysis workflows are moving to the cloud, addressing interactive data exploration in remote environments has become a pressing issue. To address it, we developed the Image Data Explorer (IDE) as a web application that integrates interactive linked visualization of images and derived data points with exploratory data analysis methods, annotation, classification and feature selection functionalities. The IDE is written in R using the shiny framework. It can be easily deployed on a remote server or on a local computer. The IDE is available at https://git.embl.de/heriche/image-data-explorer and a cloud deployment is accessible at https://shiny-portal.embl.de/shinyapps/app/01_image-data-explorer

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM

This work was supported by EMBL funds and by by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project number 240245660 – SFB 1129 (project Z2).In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope’s field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.Publisher PDFPeer reviewe