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Monomeric alpha-synuclein (aSyn) is a well characterised as a lipid binding protein. aSyn is known to form amyloid fibrils which are also localised with lipids and organelles in so called Lewy bodies, insoluble structures found in Parkinson’s disease patient’s brains. It is still unclear under which conditions the aSyn-lipid interaction can start to become pathological. Previous work to address pathological interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes which not only have a more complex lipid composition, but also contain lipid interacting proteins. Here, we investigate how either monomeric or fibrillar aSyn interact with physiological synaptic vesicles (SV) isolated from rodent brain. Using small angle neutron scattering and high-resolution imaging we observe that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV. Furthermore, SV enhance the aggregation rate of aSyn, however increasing the SV:aSyn ratio causes a reduction in aggregation propensity. SV lipids appear as an integrated part of aSyn fibrils and while the fibril morphology differs to aSyn fibrils alone, the core fibril structure remains the same. We finally demonstrate that lipid-associated aSyn fibrils are more easily taken up into cortical i3Neurons derived from induced pluripotent stem cells. Our study sheds light on differences between interactions of aSyn with synthetic lipid vesicles and physiological SV. We show how aSyn fibrils may enhance pathology by disintegrating SV, which in turn may have fatal consequences for neurons. Furthermore, disease burden may additionally be impacted by an increased uptake of lipid-associated aSyn by neurons, leading to more SV damage and enhancing aSyn aggregation.

General Materials Science

Materials science, Nano, Particle, Nanoparticle, Nanotechnology, Nanofluidics, Photoresist, Nanoscopic scale, Lithography, and Soft lithography

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

lcsh:Applied optics. Photonics, Microscope, Materials science, 123, 639/624/1107/328/2237, 01 natural sciences, law.invention, 010309 optics, 03 medical and health sciences, Optics, Optical microscope, law, 0103 physical sciences, Microscopy, lcsh:QC350-467, 030304 developmental biology, 0303 health sciences, 132, business.industry, Light-sheet microscopy, article, lcsh:TA1501-1820, Image plane, Atomic and Molecular Physics, and Optics, Electronic, Optical and Magnetic Materials, Lens (optics), Cardinal point, Light sheet fluorescence microscopy, 639/624/1107/328, Axial symmetry, business, and lcsh:Optics. Light

Funder: MedImmune, and Infinitus (China) Ltd.
In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10–100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.

Materials science, Microscope, Cell, General Physics and Astronomy, 02 engineering and technology, 01 natural sciences, Retinal ganglion, General Biochemistry, Genetics and Molecular Biology, law.invention, 010309 optics, 03 medical and health sciences, Optics, law, 0103 physical sciences, Microscopy, medicine, Animals, General Materials Science, 030304 developmental biology, VDP::Mathematics and natural science: 400, Neurons, 0303 health sciences, High contrast, Photons, Total internal reflection fluorescence microscope, business.industry, 010401 analytical chemistry, General Engineering, General Chemistry, VDP::Matematikk og Naturvitenskap: 400, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Rats, medicine.anatomical_structure, Microscopy, Fluorescence, Photonics, 0210 nano-technology, business, and Waveguide

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1, 2] and have also been demonstrated for short-term live-imaging of robust cell types [3–5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGC). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.

0301 basic medicine, Computer science, ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, lcsh:Medicine, Bioengineering, Imaging techniques, 01 natural sciences, Optical projection tomography, 5105 Medical and Biological Physics, Article, 010309 optics, Set (abstract data type), 03 medical and health sciences, 0302 clinical medicine, 46 Information and Computing Sciences, Computer graphics (images), 0103 physical sciences, Microscopy, lcsh:Science, Lung, 030304 developmental biology, 0303 health sciences, Multidisciplinary, 639/766/930/2735, lcsh:R, Sample (graphics), 030104 developmental biology, Networking and Information Technology R&D (NITRD), 14/63, Biomedical Imaging, lcsh:Q, 51 Physical Sciences, Biological fluorescence, 030217 neurology & neurosurgery, and 631/57/2267

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 µm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples.

Fabrication, Materials science, Materials Science (miscellaneous), Interface (computing), Microfluidics, Nanotechnology, Nanofluidics, 02 engineering and technology, Photoresist, Multiphoton lithography, lcsh:Technology, Industrial and Manufacturing Engineering, 03 medical and health sciences, Wafer, Electrical and Electronic Engineering, 030304 developmental biology, 0303 health sciences, lcsh:T, Nanofabrication and nanopatterning, article, 639/925/927/351, 021001 nanoscience & nanotechnology, Condensed Matter Physics, 639/925/350/2251, Atomic and Molecular Physics, and Optics, Nanolithography, lcsh:TA1-2040, lcsh:Engineering (General). Civil engineering (General), and 0210 nano-technology

Nanofluidic devices have great potential for applications in areas ranging from renewable energy to human health. A crucial requirement for the successful operation of nanofluidic devices is the ability to interface them in a scalable manner with the outside world. Here, we demonstrate a hybrid two photon nanolithography approach interfaced with conventional mask whole-wafer UV-photolithography to generate master wafers for the fabrication of integrated micro and nanofluidic devices. Using this approach we demonstrate the fabrication of molds from SU-8 photoresist with nanofluidic features down to 230 nm lateral width and channel heights from micron to sub-100 nm. Scanning electron microscopy and atomic force microscopy were used to characterize the printing capabilities of the system and show the integration of nanofluidic channels into an existing microfluidic chip design. The functionality of the devices was demonstrated through super-resolution microscopy, allowing the observation of features below the diffraction limit of light produced using our approach. Single molecule localization of diffusing dye molecules verified the successful imprint of nanochannels and the spatial confinement of molecules to 200 nm across the nanochannel molded from the master wafer. This approach integrates readily with current microfluidic fabrication methods and allows the combination of microfluidic devices with locally two-photon-written nano-sized functionalities, enabling rapid nanofluidic device fabrication and enhancement of existing microfluidic device architectures with nanofluidic features.
Nanoengineering: Flexible production of micro- and nanofluidic devices A laser-based manufacturing process can produce combined nanofluidic and microfluidic devices in a rapid and scalable manner. Nanofluidic devices allow for the observation of biological processes at the single-molecule level, but current fabrication methods are slow and costly. In this article, Tuomas Knowles and his team from the UK’s University of Cambridge used conventional light-blocking masks and laser technology to craft a light-sensitive material into devices with micron to nano-sized channels. The researchers used microscopy and single-molecule tracking of dyes to verify the functionality of the final imprinted device, which also demonstrates the integration of nanofluidic channels into existing microfluidics designs. The team says that further research may optimize their new platform, which they hope will offer a reliable and flexible pathway for nanofluidic device fabrication.

Wavelength modulation spectroscopy, Optics, Absorption spectroscopy, Wavelength modulation, business.industry, Chemistry, ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, Harmonic, Modulation spectroscopy, business, and Absorption (electromagnetic radiation)

This work aims to develop a cost-effective absorption tomographic system based on wavelength modulation spectroscopy and multi-harmonic detections. This paper presents the mathematical formulation and numerical demonstration.

Photon, Materials science, business.industry, law, Biophysics, Optoelectronics, Lab-on-a-chip, business, Lithography, and law.invention