Superresolving the kidney-a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier.

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney's glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world. Graphical abstract

Scalable integration of nano-, and microfluidics with hybrid two-photon lithography

Abstract: 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

α-Synuclein fibril and synaptic vesicle interactions lead to vesicle destruction and increased lipid-associated fibril uptake into iPSC-derived neurons

Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson’s disease patient’s brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons

OptiJ: Open-source optical projection tomography of large organ samples

Abstract: 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

OptiJ: Open-source optical projection tomography of large organ samples

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

Converting lateral scanning into axial focusing to speed up three-dimensional microscopy.

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

Nanolithography and nanoscopy methods for the study of biological samples in confined spaces

Alzheimer’s, Parkinson’s and Huntington’s disease all belong to the group of amyloid pathologies also called protein misfolding diseases. Since the first discovery of amyloid fibrils of the aggregated protein tau in inclusion of Alzheimer brains samples, research has focussed on how amyloids form and their biological relevance in neurodegenerative diseases. Microfluidics are a well-established tool for the study of protein aggregation in a controllable environment, whereas super-resolution microscopy techniques have been developed and allow imaging protein misfolding in biological models in-vitro e.g. primary neural cell cultures. It was found that during the process of aggregation, misfolded proteins develop variations in toxicity which can be related to their size. Since their aggregation kinetics for different subspecies are again partially unstable depending on buffer conditions, we seek for methods for better and faster analysis of misfolding proteins on a single molecule level. Nanofluidics are capable of sampling single molecules from a concentrated solution but are not accessible to a broad community which would greatly benefit from their potential. To allow a broader community access to nanofluidic fabrication, first, a custom-built open-source two-photon lithography was implemented to demonstrate the usage of 2-photon direct laser writing for the fabrication of master molds of nanofiltration chips. The system was characterized and its nanolithography capabilities, using electron microscopy and atomic force microscopy, evaluated. The fabrication process is outlined and important details about the integration of nanofluidic functionalities into microfluidic masters elucidated. The produced chips were then used for a variety of applications ranging from single molecule diffusional measurements of Rhodamine 6G, GFP and human tau protein in solution, for entropic trapping of biological specimen and electrophoretic concentration of fluorescently labelled DNA. The flexible fabrication scheme allowed the enhancement of nanofluidic channels with adjascent nanotraps. The nanotraps allow to observe a variety of biologically relevant species such as 100 nm colloids, 40 nm colloids, exosomes, alpha-synuclein oligomers and 40 bp fluorescently labelled DNA with up to five times increased residence times in a confocal detection volume. Fluorescence burst microscopy is used to detect the residence times of the specimen with high precision. The characteristic Kramer escape time is extracted from the particles residence time probability distribution and allows an estimate of the particle size in the confined volume. The increased residence times are beneficial for photon expensive techniques to be conducted, which are hard to achieve without immobilizing them as with conventional approaches. To study biological samples related to nanofiltration and protein misfolding diseases in vitro, a custom-built pulsed-STED system was equipped with a second excitation beam path to allow for two-colour STED imaging using the same IR depletion wavelength. The system is then used on three different biological imaging applications. Single-color STED imaging is used to study the nanofiltration barrier of the kidney in minimal change disease (MCD) and compared to other super-resolution techniques such as STORM, SIM and expansion microscopy. Results show an effective alternative imaging pathway for the study of MCD without electron microscopy techniques. Secondly, STED imaging is combined with microfluidics which allows the observation and study of misfolding proteins in neural cell cultures in microfluidic devices. A microfluidic chip design consisting of two reservoirs which are connected via microchannels, allows the selective study of axonal synaptic transport processes by fluidically isolating two neural cell cultures from each other but keeping their axonal projections intact. Live neurons were exposed to human tau monomer and results indicate the inclusion of tau aggregates in lysosomes, which are then transported along the cellular microtubule network. Thirdly, STED is used to image the morphology of the endoplasmic reticulum (ER) in primary neurons. Calnexin is a chaperone-like membrane protein on the ER surface which is due to its close connection to glucoglycans of great interest to protein misfolding tauopathies. STED and expansion microcopy are demonstrated as methods to study Calnexin on the ER membrane and microtubular network with high resolution. Lastly, correlative 2-colour STED/AFM is presented on α-synuclein fibrils and synaptic vesicles, which combines fluorescence microscopy with sub-diffraction resolution and label-free mechanical mapping at the nanoscale.EPSRC CDT in Sensor Technologies for a Healthy and Sustainable Future, EPSRC CDT in Nanoscience and Nanotechnolog

Rapid prototyping of 1xN multifocus gratings via additive direct laser writing

Multifocus gratings (MFGs) enable microscopes and other imaging systems to record entire Z-stacks of images in a single camera exposure. The exact grating shape depends on microscope parameters like wavelength and magnification and defines the multiplexing onto a grid of MxN Z-slices. To facilitate the swift production and alteration of MFGs for a system and application at hand, we have developed a fabrication protocol that allows manufacturing of 1xN MFGs within hours and without the requirement of clean room facilities or hazardous etching steps. Our approach uses photolithography with a custom-built stage-scanning direct laser writing (DLW) system. By writing MFG grating lines into spin-coated negative tone SU-8 photoresist, polymerized parts are crafted onto the substrate and thus directly become a part of the grating structure. We provide software to generate the required MFG grating line paths, details of the DLW system and fully characterize a manufactured MFG. Our produced MFG is 5.4 mm in diameter and manages to record an image volume with a Z-span of over 600 μm without spherical aberrations or noticeable loss of resolution

Scalable integration of nano-, and microfluidics with hybrid two-photon lithography

Abstract: 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

Research data supporting "Scalable integration of nano-, and microfluidics with hybrid two-photon lithography"

Raw data for the publication "Scalable integration of nano-, and microfluidics with hybrid two-photon lithography." consisting of: - a video of single molecules in a nano-channel - SEM images of polymerised SU8 illustrating the technique used for making master to create nano-channels