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Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we theoretically and numerically investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

Funder: Boehringer Ingelheim Fonds; FundRef: http://dx.doi.org/10.13039/501100001645, Funder: Herchel Smith Fund, Funder: Fitzwilliam College, Univeristy of Cambridge, To analyse neuron data at scale, neuroscientists expend substantial effort reading documentation, installing dependencies and moving between analysis and visualisation environments. To facilitate this, we have developed a suite of interoperable open-source R packages called the natverse. The natverse allows users to read local and remote data, perform popular analyses including visualisation and clustering and graph-theoretic analysis of neuronal branching. Unlike most tools, the natverse enables comparison across many neurons of morphology and connectivity after imaging or co-registration within a common template space. The natverse also enables transformations between different template spaces and imaging modalities. We demonstrate tools that integrate the vast majority of Drosophila neuroanatomical light microscopy and electron microscopy connectomic datasets. The natverse is an easy-to-use environment for neuroscientists to solve complex, large-scale analysis challenges as well as an open platform to create new code and packages to share with the community.

Many countries have a rapidly ageing population, placing strain on health services and creating a growing market for assistive technology for older people. We have, through a student-led, 12-week project for 10 students from a variety of science and engineering backgrounds, developed an integrated sensor system to enable older people, or those at risk, to live independently in their own homes for longer, while providing reassurance for their family and carers. We provide details on the design procedure and performance of our sensor system and the management and execution of a short-term, student-led research project. Detailed information on the design and use of our devices, including a door sensor, power monitor, fall detector, general in-house sensor unit and easy-to-use location-aware communications device, is given, with our open designs being contrasted with closed proprietary systems. A case study is presented for the use of our devices in a real-world context, along with a comparison with commercially available systems. We discuss how the system could lead to improvements in the quality of life of older users and increase the effectiveness of their associated care network. We reflect on how recent developments in open source technology and rapid prototyping increase the scope and potential for the development of powerful sensor systems and, finally, conclude with a student perspective on this team effort and highlight learning outcomes, arguing that open technologies will revolutionize the way in which technology will be deployed in academic research in the future.

To analyse neuron data at scale, neuroscientists expend substantial effort reading documentation, installing dependencies and moving between analysis and visualisation environments. To facilitate this, we have developed a suite of interoperable open-source R packages called the natverse. The natverse allows users to read local and remote data, perform popular analyses including visualisation and clustering and graph-theoretic analysis of neuronal branching. Unlike most tools, the natverse enables comparison across many neurons of morphology and connectivity after imaging or co-registration within a common template space. The natverse also enables transformations between different template spaces and imaging modalities. We demonstrate tools that integrate the vast majority of Drosophila neuroanatomical light microscopy and electron microscopy connectomic datasets. The natverse is an easy-to-use environment for neuroscientists to solve complex, large-scale analysis challenges as well as an open platform to create new code and packages to share with the community.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

To analyse neuron data at scale, neuroscientists expend substantial effort reading documentation, installing dependencies and moving between analysis and visualisation environments. To facilitate this, we have developed a suite of interoperable open-source R packages called the natverse. The natverse allows users to read local and remote data, perform popular analyses including visualisation and clustering and graph-theoretic analysis of neuronal branching. Unlike most tools, the natverse enables comparison across many neurons of morphology and connectivity after imaging or co-registration within a common template space. The natverse also enables transformations between different template spaces and imaging modalities. We demonstrate tools that integrate the vast majority of Drosophila neuroanatomical light microscopy and electron microscopy connectomic datasets. The natverse is an easy-to-use environment for neuroscientists to solve complex, large-scale analysis challenges as well as an open platform to create new code and packages to share with the community.

Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.