Search

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events approximately five-fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific backgrounds., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Introduction/background: The aim of this educational perspective is to describe a program of learning activities developed to prepare radiography students for final year clinical placements. Authors' reflections are integrated with student feedback to discuss the educational value and feasibility of implementing the activities presented., Methods: A program of six components was developed: (1) Simulation-based learning, (2) clinical supervision, (3) tutorials and workshops, (4) medical imaging facility learning tours (5) lecture series and (6) journal club. Student volunteers provided anonymous and voluntary feedback on each activity's engagement, effectiveness, promotion of interaction and collaboration, role in future placement or professional role, and the perceived value to their learning needs., Results: Activities with clear links to practice were rated highest by students, including clinical supervision, 2-day simulation program, CT tutorials and interprofessional simulation. All students reported that half of all activities (6/12, 50%) were either very valuable or valuable, with only a minority of students indicating three activities to not be valuable to their learning needs., Discussion: Student feedback highlighted activities considered to support skill development, stemming from active learning opportunities. Overall characteristics considered to support students' clinical skill development were interprofessional teaching team, mixture of activities including simulation, and collaboration with clinical and industry partners., Conclusion: Based on student feedback and academics' perceptions of suitability of activities for preparing students for final year clinical placement, faculty have shifted their focus towards dedicated resources to support scale-up and sustainability pertaining to integration into curriculum. The goal is to embed these activities into the core curriculum, offering students consistent, up-to-date learning, while expanding preparatory activities with diverse, specialised clinical scenarios to better prepare them for practice., (Copyright © 2024. Published by Elsevier Inc.)

Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K + concentration. In combination with microfluidics, we employed our DNA-based K + sensor for extraction of the permeation coefficient of potassium ions. We measured K + permeability coefficients at least 1 order of magnitude larger than previously reported values from bulk experiments and show that permeation rates across the lipid bilayer increase in the presence of octanol. In addition, an analysis of the K + flux in different concentration gradients allows us to estimate the complementary H + flux that dissipates the charge imbalance across the GUV membrane. Subsequently, we show that our sensor can quantify the K + transport across prototypical cation-selective ion channels, gramicidin A and OmpF, revealing their relative H + /K + selectivity. Our results show that gramicidin A is much more selective to protons than OmpF with a H + /K + permeability ratio of ∼10 4 .

Cell membranes regulate the distribution of biological machinery between phase-separated lipid domains to facilitate key processes including signaling and transport, which are among the life-like functionalities that bottom-up synthetic biology aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in coexisting lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes and by changing nanostructure size and topology. We demonstrate the functionality of our strategy with a bioinspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral redistribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.