Widefield, Spatiotemporal Mapping of Spontaneous Activity of Mouse Cultured Neuronal Networks Using Quantum Diamond Sensors

Quantum diamond sensors containing Nitrogen Vacancy (NV) centers were integrated into the culture of spontaneously electrically active cultures of live mouse primary cortical neurons. Two diamond formats were used enabling extracellular studies of cells cultured directly on a planar diamond plate and intracellular studies via endocytosis mediated integration of nanoscale diamond particles within cells. Over a 14 days culture period functional neuronal networks formed as confirmed by endogenous calcium transients detected via fluorescent imaging of a calcium probe (Fluo-5 AM). Spatially correlated images of photoluminescence from NV centers were also recorded demonstrating successful acquisition of optically detected magnetic resonance from regions of endogenously firing neuronal cultures. Further, a fast and simple measurement protocol based on acquisition of NV photoluminescence without and with the application of near resonant microwaves is presented. This protocol was applied to live signaling cells and dead cells. The relative photoluminescence from NVs with and without microwaves varied for the case of live as compared to dead cells warranting future investigation. Overall this work presents a protocol for integration of NV diamond sensors in a spontaneously active network of neuronal cells of relevance to the field of neuroscience. Photoluminescence from NV centers was detected with high spatiotemporal resolution using measurement protocols potentially capable of single shot detection of neuronal activity. It is anticipated that the methodologies introduced in this work will underpin the establishment of NV diamond sensors as a radically new measurement platform capable of rapid, non-destructive functional studies of cells

Thawing out the Cold Record: Some Thoughts on How Videotaped Records May Affect Traditional Standards of Deference on Direct and Collateral Review

Appellate courts are unable to “smell the smoke of battle” from a trial. For this reason, a trial court’s decision is owed deference when examining an appeal. Video technology makes this reason for deference less relevant

Thawing out the Cold Record: Some Thoughts on How Videotaped Records May Affect Traditional Standards of Deference on Direct and Collateral Review

Appellate courts are unable to “smell the smoke of battle” from a trial. For this reason, a trial court’s decision is owed deference when examining an appeal. Video technology makes this reason for deference less relevant

Cytoplasmic RNA in undifferentiated neural stem cells: a potential label-free Raman spectral marker for assessing the undifferentiated status

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA

Barriers and facilitators to clinical behaviour change by primary care practitioners: a theory-informed systematic review of reviews using the Theoretical Domains Framework and Behaviour Change Wheel.

BACKGROUND: Understanding the barriers and facilitators to behaviour change by primary care practitioners (PCPs) is vital to inform the design and implementation of successful Behaviour Change Interventions (BCIs), embed evidence-based medicine into routine clinical practice, and improve quality of care and population health outcomes. METHODS: A theory-led systematic review of reviews examining barriers and facilitators to clinical behaviour change by PCPs in high-income primary care contexts using PRISMA. Embase, MEDLINE, PsychInfo, HMIC and Cochrane Library were searched. Content and framework analysis was used to map reported barriers and facilitators to the Theoretical Domains Framework (TDF) and describe emergent themes. Intervention functions and policy categories to change behaviour associated with these domains were identified using the COM-B Model and Behaviour Change Wheel (BCW). RESULTS: Four thousand three hundred eighty-eight reviews were identified. Nineteen were included. The average quality score was 7.5/11. Reviews infrequently used theory to structure their methods or interpret their findings. Barriers and facilitators most frequently identified as important were principally related to 'Knowledge', 'Environmental context and resources' and 'Social influences' TDF domains. These fall under the 'Capability' and 'Opportunity' domains of COM-B, and are linked with interventions related to education, training, restriction, environmental restructuring and enablement. From this, three key areas for policy change include guidelines, regulation and legislation. Factors least frequently identified as important were related to 'Motivation' and other psychological aspects of 'Capability' of COM-B. Based on this, BCW intervention functions of persuasion, incentivisation, coercion and modelling may be perceived as less relevant by PCPs to change behaviour. CONCLUSIONS: PCPs commonly perceive barriers and facilitators to behaviour change related to the 'Capability' and 'Opportunity' domains of COM-B. PCPs may lack insight into the role that 'Motivation' and aspects of psychological 'Capability' have in behaviour change and/or that research methods have been inadequate to capture their function. Future research should apply theory-based frameworks and appropriate design methods to explore these factors. With no 'one size fits all' intervention, these findings provide general, transferable insights into how to approach changing clinical behaviour by PCPs, based on their own views on the barriers and facilitators to behaviour change. SYSTEMATIC REVIEW REGISTRATION: A protocol was submitted to the London School of Hygiene and Tropical Medicine via the Ethics and CARE form submission on 16.4.2020, ref number 21478 (available on request). The project was not registered on PROSPERO

Ultrasound-mediation of self-illuminating reporters improves imaging resolution in optically scattering media

In vivo imaging of self-illuminating bio-and chemiluminescent reporters is used to observe the physiology of small animals. However, strong light scattering by biological tissues results in poor spatial resolution of the optical imaging, which also degrades the quantitative accuracy. To overcome this challenging problem, focused ultrasound is used to modulate the light from the reporter at the ultrasound frequency. This produces an ultrasound switchable light ‘beacon’ that reduces the influence of light scattering in order to improve spatial resolution. The experimental results demonstrate that apart from light modulation at the ultrasound frequency (AC signal at 3.5 MHz), ultrasound also increases the DC intensity of the reporters. This is shown to be due to a temperature rise caused by insonification that was minimized to be within acceptable mammalian tissue safety thresholds by adjusting the duty cycle of the ultrasound. Line scans of bio-and chemiluminescent objects embedded within a scattering medium were obtained using ultrasound modulated (AC) and ultrasound enhanced (DC) signals. Lateral resolution is improved by a factor of 12 and 7 respectively, as compared to conventional CCD imaging. Two chemiluminescent sources separated by ~10mm at ~20 mm deep inside a 50 mm thick chicken breast have been successfully resolved with an average signal-to-noise ratio of approximately 8-10 dB

Anomalous absorption of bulk shear sagittal acoustic waves in a layered structure with viscous fluid

It is demonstrated theoretically that the absorptivity of bulk shear sagittal waves by an ultra-thin layer of viscous fluid between two different elastic media has a strong maximum (in some cases as good as 100%) at an optimal layer thickness. This thickness is usually much smaller than the penetration depths and lengths of transverse and longitudinal waves in the fluid. The angular dependencies of the absorptivity are demonstrated to have significant and unusual structure near critical angles of incidence. The effect of non-Newtonian properties and non-uniformities of the fluid layer on the absorptivity is also investigated. In particular, it is shown that the absorption in a thin layer of viscous fluid is much more sensitive to non-zero relaxation time(s) in the fluid layer than the absorption at an isolated solid-fluid interface.Comment: 14 pages, 8 figure

Progress in low-field benchtop NMR spectroscopy in chemical and biochemical analysis

The employment of spectroscopically-resolved NMR techniques as analytical probes have previously been both prohibitively expensive and logistically challenging in view of the large sizes of high-field facilities. However, with recent advances in the miniaturisation of magnetic resonance technology, low-field, cryogen-free “benchtop” NMR instruments are seeing wider use. Indeed, these miniaturised spectrometers are utilised in areas ranging from food and agricultural analyses, through to human biofluid assays and disease monitoring. Therefore, it is both intrinsically timely and important to highlight current applications of this analytical strategy, and also provide an outlook for the future, where this approach may be applied to a wider range of analytical problems, both qualitatively and quantitatively