High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

blique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo-4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure

Single-Step Fabrication of Multispectral Filter Arrays Using Grayscale Lithography and Metal-Insulator-Metal Geometry

© 2018 OSA. Metal-insulator-metal geometries can provide optical transmission filtering, with peak wavelength dependent on insulator thickness. Using grayscale electron beam lithography to control insulator thickness, we fabricate multispectral filter arrays, whereby dose determines wavelength

Coherent Imaging through Multicore Fibres with Applications in Endoscopy

Imaging through optical fibres has recently emerged as a promising method of micro-scale optical imaging within a hair-thin form factor. This has significant applications in endoscopy and may enable minimally invasive imaging deep within live tissue for improved diagnosis of disease. Multi-mode fibres (MMF) are the most common choice because of their high resolution but multicore fibres (MCF) offer a number of advantages such as widespread clinical use, ability to form approximate images without correction and an inherently sparse transmission matrix (TM) enabling simple and fast characterisation. We present a novel experimental investigation into properties of MCF important for imaging, specifically: a new method to upsample and downsample measured TMs with minimal information loss, the first experimental measurement of MCF spatial eigenmodes, a novel statistical treatment of behaviour under bending based on a wireless fading model, and an experimental observation of TM drift due to self-heating effects and discussion of how to compensate this. We next present practical techniques for imaging through MCFs, including alignment, how to parallelise TM characterisation measurements to improve speed and how to use non-interferometric phase and polarisation recovery for improved stability. Finally, we present two recent applications of MCF imaging: polarimetric imaging using a robust Bayesian inference approach, and entropic imaging for imaging early-stage tumours

Wide-field phase imaging for the endoscopic detection of dysplasia and early-stage esophageal cancer

© 2018 SPIE. Esophageal cancer has a 5-year survival rate below 20%, but can be curatively resected if it is detected early. At present, poor contrast for early lesions in white light imaging leads to a high miss rate in standard-of-care endoscopic surveillance. Early lesions in the esophagus, referred to as dysplasia, are characterized by an abundance of abnormal cells with enlarged nuclei. This tissue has a different refractive index profile to healthy tissue, which results in different light scattering properties and provides a source of endogenous contrast that can be exploited for advanced endoscopic imaging. For example, point measurements of such contrast can be made with scattering spectroscopy, while optical coherence tomography generates volumetric data. However, both require specialist interpretation for diagnostic decision making. We propose combining wide-field phase imaging with existing white light endoscopy in order to provide enhanced contrast for dysplasia and early-stage cancer in an image format that is familiar to endoscopists. Wide-field phase imaging in endoscopy can be achieved using coherent illumination combined with phase retrieval algorithms. Here, we present the design and simulation of a benchtop phase imaging system that is compatible with capsule endoscopy. We have undertaken preliminary optical modelling of the phase imaging setup, including aberration correction simulations and an investigation into distinguishing between different tissue phantom scattering coefficients. As our approach is based on phase retrieval rather than interferometry, it is feasible to realize a device with low-cost components for future clinical implementation

Hierarchical statistical techniques are necessary to draw reliable conclusions from analysis of isolated cardiomyocyte studies

Aims It is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests). Methods and results Ca2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF. Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF. Conclusion Ca2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for reliable conclusions. We present cost-free simple tools for performing these analyses

An in vivo platform to select and evolve aggregation-resistant proteins

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve ‘manufacturable’ biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease

Undergraduate medical textbooks do not provide adequate information on intravenous fluid therapy: a systematic survey and suggestions for improvement

<b>Background</b><p></p> Inappropriate prescribing of intravenous (IV) fluid, particularly 0.9% sodium chloride, causes post-operative complications. Fluid prescription is often left to junior medical staff and is frequently poorly managed. One reason for poor intravenous fluid prescribing practices could be inadequate coverage of this topic in the textbooks that are used.<p></p> <b>Methods</b><p></p> We formulated a comprehensive set of topics, related to important common clinical situations involving IV fluid therapy, (routine fluid replacement, fluid loss, fluids overload) to assess the adequacy of textbooks in common use. We assessed 29 medical textbooks widely available to students in the UK, scoring the presence of information provided by each book on each of the topics. The scores indicated how fully the topics were considered: not at all, partly, and adequately. No attempt was made to judge the quality of the information, because there is no consensus on these topics.<p></p> <b>Results</b><p></p> The maximum score that a book could achieve was 52. Three of the topics we chose were not considered by any of the books. Discounting these topics as “too esoteric”, the maximum possible score became 46. One textbook gained a score of 45, but the general score was poor (median 11, quartiles 4, 21). In particular, coverage of routine postoperative management was inadequate.<p></p> <b>Conclusions</b><p></p> Textbooks for undergraduates cover the topic of intravenous therapy badly, which may partly explain the poor knowledge and performance of junior doctors in this important field. Systematic revision of current textbooks might improve knowledge and practice by junior doctors. Careful definition of the remit and content of textbooks should be applied more widely to ensure quality and “fitness for purpose”, and avoid omission of vital knowledge

Leisure-Time Sedentary Behavior, Alcohol Consumption, and Sexual Intercourse Among Adolescents Aged 12-15 Years in 19 Countries From Africa, the Americas, and Asia

BACKGROUND: The association between sedentary behavior and sexual behavior has not been investigated among adolescents. // AIM: The aim of this study was to: (i) investigate the association between leisure-time sedentary behavior and sexual intercourse, and (ii) test for mediation by alcohol consumption, drug use, physical activity, bullying victimization, parental support/ monitoring, loneliness, and depressive symptoms in a large global sample of young adolescents. // METHODS: Data were analyzed from 34,674 adolescents aged 12-15 years participating in the Global School-based Student Health Survey. Participants reported the number of hours spent in leisure-time sedentary behavior on a typical day (8 hours). Data on alcohol consumption, drug use, physical activity, bullying victimization, parental support/monitoring, loneliness, and depressive symptoms were considered as potential mediators. // OUTCOME: Participants reported whether or not they had sexual intercourse in the past 12 months (yes/no). // RESULTS: The prevalence of past 12-month sexual intercourse was 11.9%, whereas the prevalence of 8 hours per day of leisure-time sedentary behavior were 26.7%, 35.6%, 21.4%, 11.5%, and 4.9%, respectively. There was a dose-dependent relationship between sedentary behavior and odds of reporting sexual intercourse: compared with 8 hours/day of sedentary behavior were 1.12 (0.94-1.33), 1.22 (1.01-1.48), 1.34 (1.08-1.66), and 1.76 (1.37-2.27), respectively. There was no significant interaction by sex. The largest proportion of the association between sedentary behavior and sexual intercourse was explained by alcohol use (% mediated 21.2%), with other factors explaining an additional 11.2%. // CLINICAL TRANSLATION: Interventions to reduce leisure-time sedentary and/or alcohol consumption may contribute to a reduction in the proportion of adolescents engaging in sexual intercourse at a young age. The strengths and limitations of this study are the large, representative sample of adolescents from 19 countries. However, the cross-sectional design means causality or temporal associations could not be established. // CONCLUSIONS: In young adolescents, leisure-time sedentary behavior is positively associated with odds of having sexual intercourse in both boys and girls, in a dose-dependent manner. Alcohol consumption seems to be a key mediator of this relationship

Improving detection of familial hypercholesterolaemia in primary care using electronic audit and nurse-led clinics

RATIONALE, AIMS AND OBJECTIVES: In the UK fewer than 15% of familial hypercholesterolemia (FH) cases are diagnosed, representing a major gap in coronary heart disease prevention. We wished to support primary care doctors within the Medway Clinical Commissioning Group (CCG) to implement NICE guidance (CG71) and consider the possibility of FH in adults who have raised total cholesterol concentrations, thereby improving the detection of people with FH. METHODS: Utilizing clinical decision support software (Audit+) we developed an FH Audit Tool and implemented a systematic audit of electronic medical records within GP practices, first identifying all patients diagnosed with FH or possible FH and next electronically flagging patients with a recorded total cholesterol of >7.5 mmol L(-1) or LDL-C > 4.9 mmol L(-1) (in adults), for further assessment. After a 2-year period, a nurse-led clinic was introduced to screen more intensely for new FH index cases. We evaluated if these interventions increased the prevalence of FH closer to the expected prevalence from epidemiological studies. RESULTS: The baseline prevalence of FH within Medway CCG was 0.13% (1 in 750 persons). After 2 years, the recorded prevalence of diagnosed FH increased by 0.09% to 0.22% (1 in 450 persons). The nurse advisor programme ran for 9 months (October 2013-July 2014) and during this time, the recorded prevalence of patients diagnosed with FH increased to 0.28% (1 in 357 persons) and the prevalence of patients 'at risk and unscreened' reduced from 0.58% to 0.14%. CONCLUSIONS: Our study shows that two simple interventions increased the detection of FH. This systematic yet simple electronic case-finding programme with nurse-led review allowed the identification of new index cases, more than doubling the recorded prevalence of detected disease to 1 in 357 (0.28%). This study shows that primary care has an important role in identifying patients with this condition