High-density volumetric super-resolution microscopy.

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs µm-2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs µm-2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs µm-2) in dense cytosolic tubulin datasets.

Scanless two-photon voltage imaging.

Two-photon voltage imaging has long been heralded as a transformative approach capable of answering many long-standing questions in modern neuroscience. However, exploiting its full potential requires the development of novel imaging approaches well suited to the photophysical properties of genetically encoded voltage indicators. We demonstrate that parallel excitation approaches developed for scanless two-photon photostimulation enable high-SNR two-photon voltage imaging. We use whole-cell patch-clamp electrophysiology to perform a thorough characterization of scanless two-photon voltage imaging using three parallel illumination approaches and lasers with different repetition rates and wavelengths. We demonstrate voltage recordings of high-frequency spike trains and sub-threshold depolarizations from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-Kv. Using a low repetition-rate laser, we perform multi-cell recordings from up to fifteen targets simultaneously. We co-express JEDI-2P-Kv and the channelrhodopsin ChroME-ST and capitalize on their overlapping two-photon absorption spectra to simultaneously evoke and image action potentials using a single laser source. We also demonstrate in vivo scanless two-photon imaging of multiple cells simultaneously up to 250 µm deep in the barrel cortex of head-fixed, anaesthetised mice.

Scanless two-photon voltage imaging.

Parallel light-sculpting methods have been used to perform scanless two-photon photostimulation of multiple neurons simultaneously during all-optical neurophysiology experiments. We demonstrate that scanless two-photon excitation also enables high-resolution, high-contrast, voltage imaging by efficiently exciting fluorescence in a large fraction of the cellular soma. We present a thorough characterisation of scanless two-photon voltage imaging using existing parallel approaches and lasers with different repetition rates. We demonstrate voltage recordings of high frequency spike trains and sub-threshold depolarizations in intact brain tissue from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-kv. Using a low repetition-rate laser, we perform recordings from up to ten neurons simultaneously. Finally, by co-expressing JEDI-2P-kv and the channelrhodopsin ChroME-ST in neurons of hippocampal organotypic slices, we perform single-beam, simultaneous, two-photon voltage imaging and photostimulation. This enables in-situ validation of the precise number and timing of light evoked action potentials and will pave the way for rapid and scalable identification of functional brain connections in intact neural circuits.

Design and manufacture of one-size-fits-all healthcare face shields for the NHS during the COVID-19 pandemic.

During the COVID-19 pandemic, there was a shortage of personal protective equipment, PPE, which resulted in non-certified PPE being used by healthcare staffs. These would not provide the appropriate protection against the SARS-CoV-2 virus. Together with the local NHS Trust (University Hospitals of Derby and Burton (UHDB) NHS Foundation Trust) and a local small and medium enterprise (SME), Riverside Medical Packaging Ltd, the University of Derby (UoD) developed test protocols for PPE with a one-size-fits-all concept. Building on best practice in reviewing the literature and current design requirements, key design parameters were identified such as a minimum strap width and comfort level for healthcare related Face Shield. Two strap headbands made from fabric and elastomer with linear stiffness of 44.1 ± 0.3 N/m and 149.1 ± 3.1 N/m respectively were tested with respect to fit and comfort on small and large arc-shaped models. There was an exponential change in pressure from the side to the middle of the strap headbands. The high stiffness of the elastomer in a radial set-up influenced the pressure exerted on a wearer's head when the elastomer strap was used. Meanwhile the coefficient of friction between the fabric strap and arc-shaped model influenced the pressure exerted when a fabric strap was used. The ergonomics of the designed Face Shields supported the one-size-fits-all concept, whereby various gender and head circumferences were considered. The findings in this paper will promote new standards in the design of PPE with a one-size-fits-all target.

Prominent in vivo influence of single interneurons in the developing barrel cortex.

Spontaneous synchronous activity is a hallmark of developing brain circuits and promotes their formation. Ex vivo, synchronous activity was shown to be orchestrated by a sparse population of highly connected GABAergic 'hub' neurons. The recent development of all-optical methods to record and manipulate neuronal activity in vivo now offers the unprecedented opportunity to probe the existence and function of hub cells in vivo. Using calcium imaging, connectivity analysis and holographic optical stimulation, we show that single GABAergic, but not glutamatergic, neurons influence population dynamics in the barrel cortex of non-anaesthetized mouse pups. Single GABAergic cells mainly exert an inhibitory influence on both spontaneous and sensory-evoked population bursts. Their network influence scales with their functional connectivity, with highly connected hub neurons displaying the strongest impact. We propose that hub neurons function in tailoring intrinsic cortical dynamics to external sensory inputs.

Optogenetics for light control of biological systems.

Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms.

Improving Equine Welfare through Human Habit Formation.

This paper explores the potential for interventions to develop pro-animal welfare habitual behaviours (PAWHBs) in people to improve the lives of animals. Human behavioural research indicates that opportunities exist to deliver lasting change through developing positive habitual behaviours. The routine nature of many equine care and management practices lends itself to habit formation and maintenance. This proof-of-concept paper aims to evaluate a theory-based intervention of developing and maintaining a PAWHB in people caring for equines. Qualitative research methods were used. A 30 day PAWHB intervention (PAWHBInt) of providing enrichment to an equine by scratching them in a consistent context linked to an existing routine behaviour was undertaken. Participants (n = 9) then engaged in semi-structured interviews that were analysed using thematic analysis, where the participants self-reported the outcomes they observed during the intervention. The study findings suggest that the PAWHBInt had a positive impact on human behaviour and habit formation. The research helps to address the dearth of evidence regarding the application of habit theory to equine welfare interventions and emphasised linking a desired new behaviour to an existing routine behaviour when developing PAWHBs. The research also highlights the role of mutual benefit for human and equine, and emotion in providing feedback and potential reward, supporting the link to the cue-routine-reward principle of habit theory.

BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.

The HADRIAN approach to accessible transport.

This paper describes research carried out at Loughborough University in the UK into the areas of 'design for all' and accessible transport. The research addresses two common needs for designers and ergonomists working towards developing more inclusive products and environments, namely data on users that is accessible, valid, and applicable and a means of utilising the data to assess the accessibility of designs during the early stages of development. HADRIAN is a computer-based inclusive design tool that has been developed to support designers in their efforts to develop products that meet the needs of a broader range of users. Currently HADRIAN is being expanded to support transport design. This includes data on an individual's ability to undertake a variety of transport-related tasks, such as vehicle ingress/egress, coping with uneven surfaces, steps, street furniture and complex pedestrian environments. The subsequent use of this data will be supported either through a task analysis system that will allow a designer to evaluate a design for a part of the transport infrastructure (ticket barrier, train carriage etc.), or alternatively allow the designer or an end user to evaluate a whole journey. The 'journey planner' feature of the HADRIAN tool will compare an individual's physical, cognitive and emotional abilities with the demands placed upon that individual by the mode(s) of transport available and the route options selected. It is envisaged that these developments will prove extremely useful to users, designers, planners and all those involved with transport use and implementation.

Do office workers adjust their chairs? End-user knowledge, use and barriers to chair adjustment.

A quantitative field study measured end-user availability, knowledge and use levels of adjustable office chair functions in Korea-based office workers, together with their perceived barriers towards making adjustments. Fifty-one English-speaking workers were interviewed and surveyed in a related design. Results showed that of the number of adjustable functions available on their office chair (M = 5.39, SD = 2.3), participants knew fewer than half of them (M = 2.51, SD = 1.52) and used even less (M = 1.86, SD = 1.21). Fifty-three percent of participants knew two or less and 73% had used only two or less. Ten percent had used none. Results suggested physical needs (such as increased comfort or postural change) were a strong driver for previous chair adjustment behaviour. Perceived cognitive barriers played a more significant role in limiting chair adjustment knowledge and use than physical or organizational barriers. Highly adjustable office chairs have the possibility of satisfying the adjustment needs of most end-users. However, adjustable chair functions need to be both available and known in order to be used.

At Grandma's Home. Interview by Charlotte Alderman.

Among African countries, Uganda has been help up as an example in tackling HIV/AIDS. Ruth Sims has pioneered care and treatment for many in need.

Design and evaluation: end users, user datasets and personas.

Understanding the needs and aspirations of a suitable range of users during the product design process is an extremely difficult task. Methods such as ethnographic studies can be used to gain a better understanding of users needs, but they are inherently time consuming and expensive. The time pressures that are evident in the work performed by design consultancies often make these techniques impractical. This paper contains a discussion about the use of 'personas', a method used by designers to overcome these issues. Personas are descriptive models of archetypal users derived from user research. The discussion focuses on two case studies, the first of which examines the use of personas in the car design process. The second examines the use of personas in the field of 'inclusive design', as demonstrated by the HADRIAN system. These case studies exemplify the benefits 'data rich' personas contribute as opposed to 'assumption based' personas.

Senior Volunteers Programme: a visit to Zambia.

The Board of International Affairs of the Royal College of Psychiatrists initiated the Senior Volunteers Programme in 2004 following discussion at the Board and planning in a subgroup over the preceding year. The aim was to match requests from other countries for specific psychiatric teaching with the services of senior psychiatrists in Britain. The College has acted as intermediary, inviting requests from countries, Members and Fellows overseas, and setting up a database of psychiatrists offering a range of skills. The concept is simple but all the skill and hard work lies in the detail!