High-Speed Wide-Field Time-Correlated Single-Photon Counting Fluorescence Lifetime Imaging Microscopy

Fluorescence microscopy is a powerful imaging technique used in the biological sciences to identify labeled components of a sample with specificity. This is usually accomplished through labeling with fluorescent dyes, isolating these dyes by their spectral signatures with optical filters, and recording the intensity of the fluorescent response. Although these techniques are widely used, fluorescence intensity images can be negatively affected by a variety of factors that impact the fluorescence intensity. Fluorescence lifetime imaging microscopy (FLIM) is an imaging technique that is relatively immune to intensity fluctuations and also provides the unique ability to directly monitor the microenvironment surrounding a fluorophore. Despite the benefits associated with FLIM, the applications to which it is applied are fairly limited due to long image acquisition times and high cost of traditional hardware. Recent advances in complementary metal-oxide-semiconductor (CMOS) single-photon avalanche diodes (SPADs) have enabled the design of low-cost imaging arrays that are capable of recording lifetime images with acquisition times greater than one order of magnitude faster than existing systems. However, these SPAD arrays have yet to realize the full potential of the technology due to limitations in their ability to handle the vast amount of data generated during the commonly used time-correlated single-photon counting (TCSPC) lifetime imaging technique. This thesis presents the design, implementation, characterization, and demonstration of a high speed FLIM imaging system. The components of this design include a CMOS imager chip in a standard 0.13 μm technology containing a custom CMOS SPAD, a 64-by-64 array of these SPADs, pixel control circuitry, independent time-to-digital converters (TDCs), a FLIM specific datapath, and high bandwidth output buffers. In addition to the CMOS imaging array, a complete system was designed and implemented using a printed circuit board (PCB) for capturing data from the imager, creating histograms for the photon arrival data using field-programmable gate arrays, and transferring the data to a computer using a cabled PCIe interface. Finally, software is used to communicate between the imaging system and a computer.The dark count rate of the SPAD was measured to be only 231 Hz at room temperature while maintaining a photon detection probability of up to 30\%. TDCs included on the array have a 62.5 ps resolution and a 64 ns range, which is suitable for measuring the lifetime of most biological fluorophores. Additionally, the on-chip datapath was designed to handle continuous data transfers at rates capable of supporting TCSPC-based lifetime imaging at 100 frames per second. The system level implementation also provides sufficient data throughput for transferring up to 750 frames per second from the imaging system to a computer. The lifetime imaging system was characterized using standard techniques for evaluating SPAD performance and an electrical delay signal for measuring the TDC performance. This thesis concludes with a demonstration of TCSPC-FLIM imaging at 100 frames per second -- the fastest 64-by-64 TCSPC FLIM that has been demonstrated. This system overcomes some of the limitations of existing FLIM systems and has the potential to enable new application domains in dynamic FLIM imaging

Satisfiers and dissatisfiers for international vocational education students: A case study using narrative frames

While the satisfaction of international students is frequently surveyed, much of this research is based on a very limited range of closed-item data collection methods, producing findings that partially reflect the researchers’ assumptions in designing the survey items. Recognizing the potential value in using methods that are more open-ended and qualitative analyses, the present study employed narrative frames and follow-up interviews to explore the satisfiers and dissatisfiers for international vocational education students at one institution in New Zealand. Reporting on perceptions of the class, institution, and community, the findings identify the participants’ top-of-mind (dis)satisfiers and complement the existing literature by identifying seldom-reported themes. Discussion of issues in analyzing narrative frames is also provided

Evaluating Arctic clouds modelled with the Unified Model and Integrated Forecasting System

By synthesising remote-sensing measurements made in the central Arctic into a model-gridded Cloudnet cloud product, we evaluate how well the Met Office Unified Model (UM) and the European Centre for Medium-Range Weather Forecasting (ECMWF) Integrated Forecasting System (IFS) capture Arctic clouds and their associated interactions with the surface energy balance and the thermodynamic structure of the lower troposphere. This evaluation was conducted using a 4-week observation period from the Arctic Ocean 2018 expedition, where the transition from sea ice melting to freezing conditions was measured. Three different cloud schemes were tested within a nested limited-area model (LAM) configuration of the UM – two regionally operational single-moment schemes (UM_RA2M and UM_RA2T) and one novel double-moment scheme (UM_CASIM-100) – while one global simulation was conducted with the IFS, utilising its default cloud scheme (ECMWF_IFS). Consistent weaknesses were identified across both models, with both the UM and IFS overestimating cloud occurrence below 3 km. This overestimation was also consistent across the three cloud configurations used within the UM framework, with >90 % mean cloud occurrence simulated between 0.15 and 1 km in all the model simulations. However, the cloud microphysical structure, on average, was modelled reasonably well in each simulation, with the cloud liquid water content (LWC) and ice water content (IWC) comparing well with observations over much of the vertical profile. The key microphysical discrepancy between the models and observations was in the LWC between 1 and 3 km, where most simulations (all except UM_RA2T) overestimated the observed LWC. Despite this reasonable performance in cloud physical structure, both models failed to adequately capture cloud-free episodes: this consistency in cloud cover likely contributes to the ever-present near-surface temperature bias in every simulation. Both models also consistently exhibited temperature and moisture biases below 3 km, with particularly strong cold biases coinciding with the overabundant modelled cloud layers. These biases are likely due to too much cloud-top radiative cooling from these persistent modelled cloud layers and were consistent across the three UM configurations tested, despite differences in their parameterisations of cloud on a sub-grid scale. Alarmingly, our findings suggest that these biases in the regional model were inherited from the global model, driving a cause–effect relationship between the excessive low-altitude cloudiness and the coincident cold bias. Using representative cloud condensation nuclei concentrations in our double-moment UM configuration while improving cloud microphysical structure does little to alleviate these biases; therefore, no matter how comprehensive we make the cloud physics in the nested LAM configuration used here, its cloud and thermodynamic structure will continue to be overwhelmingly biased by the meteorological conditions of its driving model

Kernel Flow:a high channel count scalable time-domain functional near-infrared spectroscopy system

Significance: Time-domain functional near-infrared spectroscopy (TD-fNIRS) has been considered as the gold standard of noninvasive optical brain imaging devices. However, due to the high cost, complexity, and large form factor, it has not been as widely adopted as continuous wave NIRS systems. Aim: Kernel Flow is a TD-fNIRS system that has been designed to break through these limitations by maintaining the performance of a research grade TD-fNIRS system while integrating all of the components into a small modular device. Approach: The Kernel Flow modules are built around miniaturized laser drivers, custom integrated circuits, and specialized detectors. The modules can be assembled into a system with dense channel coverage over the entire head. Results: We show performance similar to benchtop systems with our miniaturized device as characterized by standardized tissue and optical phantom protocols for TD-fNIRS and human neuroscience results. Conclusions: The miniaturized design of the Kernel Flow system allows for broader applications of TD-fNIRS.</p

The listening talker: A review of human and algorithmic context-induced modifications of speech

International audienceSpeech output technology is finding widespread application, including in scenarios where intelligibility might be compromised - at least for some listeners - by adverse conditions. Unlike most current algorithms, talkers continually adapt their speech patterns as a response to the immediate context of spoken communication, where the type of interlocutor and the environment are the dominant situational factors influencing speech production. Observations of talker behaviour can motivate the design of more robust speech output algorithms. Starting with a listener-oriented categorisation of possible goals for speech modification, this review article summarises the extensive set of behavioural findings related to human speech modification, identifies which factors appear to be beneficial, and goes on to examine previous computational attempts to improve intelligibility in noise. The review concludes by tabulating 46 speech modifications, many of which have yet to be perceptually or algorithmically evaluated. Consequently, the review provides a roadmap for future work in improving the robustness of speech output

The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data

Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions

Lung Cancer Risk in Never-Smokers of European Descent is Associated With Genetic Variation in the 5(p)15.33 TERT-CLPTM1Ll Region

Introduction: Inherited susceptibility to lung cancer risk in never-smokers is poorly understood. The major reason for this gap in knowledge is that this disease is relatively uncommon (except in Asians), making it difficult to assemble an adequate study sample. In this study we conducted a genome-wide association study on the largest, to date, set of European-descent never-smokers with lung cancer. Methods: We conducted a two-phase (discovery and replication) genome-wide association study in never-smokers of European descent. We further augmented the sample by performing a meta-analysis with never-smokers from the recent OncoArray study, which resulted in a total of 3636 cases and 6295 controls. We also compare our findings with those in smokers with lung cancer. Results: We detected three genome-wide statistically significant single nucleotide polymorphisms rs31490 (odds ratio [OR]: 0.769, 95% confidence interval [CI]: 0.722-0.820; p value 5.31 x 10(-16)), rs380286 (OR: 0.770, 95% CI: 0.723-0.820; p value 4.32 x 10(-16)), and rs4975616 OR: 0.778, 95% CI: 0.730-0.829; p value 1.04 x 10(-14)). All three mapped to Chromosome 5 CLPTM1L-TERT region, previously shown to be associated with lung cancer risk in smokers and in never-smoker Asian women, and risk of other cancers including breast, ovarian, colorectal, and prostate. Conclusions: We found that genetic susceptibility to lung cancer in never-smokers is associated to genetic variants with pan-cancer risk effects. The comparison with smokers shows that top variants previously shown to be associated with lung cancer risk only confer risk in the presence of tobacco exposure, underscoring the importance of gene-environment interactions in the etiology of this disease. (C) 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Australia\u27s health 2002 : the eighth biennial report of the Australian Institute of Health and Welfare

Australia\u27s Health 2002 is the eighth biennial health report of the Australian Institute of Health and Welfare. It is the nation\u27s authoritative source of information on patterns of health and illness, determinants of health, the supply and use of health services, and health service costs and performance. Australia\u27s Health 2002 is an essential reference and information resource for all Australians with an interest in health

Satellite-based terrestrial production efficiency modeling

Production efficiency models (PEMs) are based on the theory of light use efficiency (LUE) which states that a relatively constant relationship exists between photosynthetic carbon uptake and radiation receipt at the canopy level. Challenges remain however in the application of the PEM methodology to global net primary productivity (NPP) monitoring. The objectives of this review are as follows: 1) to describe the general functioning of six PEMs (CASA; GLO-PEM; TURC; C-Fix; MOD17; and BEAMS) identified in the literature; 2) to review each model to determine potential improvements to the general PEM methodology; 3) to review the related literature on satellite-based gross primary productivity (GPP) and NPP modeling for additional possibilities for improvement; and 4) based on this review, propose items for coordinated research

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes