A fundamental question in cell biology is how cells assemble their outer layers. The bacterial endospore is a well-established model for cell layer assembly. However, the assembly of the exosporium, a complex protein shell comprising the outermost layer in the pathogen Bacillus anthracis, remains poorly understood. Exosporium assembly begins with the deposition of proteins at one side of the spore surface, followed by the progressive encirclement of the spore. We seek to resolve a major open question: the mechanism directing exosporium assembly to the spore, and then into a closed shell. We hypothesized that material directly underneath the exosporium (the interspace) directs exosporium assembly to the spore and drives encirclement. In support of this, we show that the interspace possesses at least two distinct layers of polysaccharide. Secondly, we show that putative polysaccharide biosynthetic genes are required for exosporium encirclement, suggesting a direct role for the interspace. These results not only significantly clarify the mechanism of assembly of the exosporium, an especially widespread bacterial outer layer, but also suggest a novel mechanism in which polysaccharide layers drive the assembly of a protein shell.

SignificanceDespite advances in directed evolution and computational design, engineering of functional proteins remains challenging. Here we demonstrate that rearrangement of modular repeats from BRCA2 can yield chimeras with improved interaction properties, while deconstructing the relative energetic contributions of different modules across a large interaction interface and resolving the effects of shape complementary or physicochemical properties by empirical observation. This de- and reconstruction of binding interfaces supports a mix-and-match model for proteins in which repeat units can be manipulated and used to construct functional proteins that interfere with dsDNA repair. Our approach may aid in creating biochemical and therapeutic tools from natural modules, with minimal screening effort.

We present a tomographic reconstruction algorithm (flOPT), which is applied to Optical Projection Tomography (OPT) images, that is robust to mechanical jitter and systematic angular and spatial drift. OPT relies on precise mechanical rotation and is less mechanically stable than large-scale computer tomography (CT) scanning systems, leading to reconstruction artefacts. The algorithm uses multiple (5+) tracked fiducial beads to recover the sample pose and the image rays are then back-projected at each orientation. The quality of the image reconstruction using the proposed algorithm shows an improvement when compared to the Radon transform. Moreover, when adding a systematic spatial and angular mechanical drift, the reconstruction shows a significant improvement over the Radon transform.

Mitochondria have long been implicated in Parkinson’s disease (PD), however, it is not clear how mitochondrial impairment and alpha-synuclein pathology are coupled. We report here that intra-mitochondrial protein homeostasis plays a major role in alpha-synuclein fibril elongation, as interference with intra-mitochondrial proteases and mitochondrial protein import significantly aggravate alpha-synuclein aggregation. In contrast, direct inhibition of mitochondrial complex I, increase in intracellular calcium concentration or formation of reactive oxygen species (ROS), all of which have been associated with mitochondrial stress, did not affect alpha-synuclein pathology. We further demonstrate that similar mechanisms are involved in Amyloid β 1-42 (Aβ42) aggregation, suggesting that mitochondria are directly capable of influencing cytosolic protein homeostasis of aggregation-prone proteins.

Implementations of light-sheet microscopes are often incompatible with standard methods of sample mounting. Light-sheet microscopy uses orthogonal illumination and detection to create a thin sheet of light which does not illuminate the sample outside of the depth of field of the detection axis. Typically, this configuration involves a pair of orthogonal objectives which constrains the positioning of a length of coverslips or microscopes in range of the detection objective. We present an open-hardware (1, 2) sample mounting system for light-sheet microscopes using large detection objectives.

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.

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.

A new method is presented for performing the Abel inversion by fitting the line-of-sight projection of a predefined intensity distribution (FLiPPID) to the recorded 2D projections. The aim is to develop a methodology that is less prone to experimental noise when analyzing the projection of axisymmetric objects-in this case, co-flow diffusion flame images for color ratio pyrometry. A regression model is chosen for the light emission intensity distribution of the flame cross section as a function of radial distance from the flame center line. The forward Abel transform of this model function is fitted to the projected light intensity recorded by a color camera. For each of the three color channels, the model function requires three fitting parameters to match the radial intensity profile at each height above the burner. This results in a very smooth Abel inversion with no artifacts such as oscillations or negative values of the light source intensity, as is commonly observed for alternative Abel inversion techniques, such as the basis-set expansion or onion peeling. The advantages of the new FLiPPID method are illustrated by calculating the soot temperature and volume fraction profiles inside a co-flow diffusion flame, both being significantly smoother than those produced by the alternative inversion methods. The developed FLiPPID methodology can be applied to numerous other optical techniques for which smooth inverse Abel transforms are required.

Present models for spore germination in Bacillus species include a requirement for either the SleB or CwlJ cortex lytic enzymes to efficiently depolymerise the spore cortex. Previous work has demonstrated that B. megaterium spores may differ to other species in this regard, since sleB cwlJ null mutant spores complemented with the gene in trans for the non-peptidoglycan lysin YpeB can efficiently degrade the cortex. Here, we identify two novel cortex lytic enzymes, encoded at the BMQ 2391 and BMQ 3234 loci, which are essential for cortex hydrolysis in the absence of SleB and CwlJ. Ellipsoid localisation microscopy places the BMQ 3234 protein within the inner-spore coat, a region of the spore that is populated by other cortex lytic enzymes. The findings reinforce the idea that there is a degree of variation in mechanisms of cortex hydrolysis across the Bacillales, raising potential implications for environmental decontamination strategies based upon targeted inactivation of components of the spore germination apparatus.

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.

We compare the performance of linear and nonlinear methods for aligning the excitation and detection planes throughout volumes of large specimens in digitally scanned light sheet microscopy. An effective nonlinear method involves the registration of four corner extrema of the imaging volume via a projective transform. We show that this improves the light collection efficiency of the commonly used three-point affine registration by an average of 42% over a typical specimen volume. Accurate illumination/detection registration methods are now pertinent to biological research in view of current trends towards imaging large or expanded samples, at depth, with diffraction limited resolution.

The germination of Bacillus spores is triggered by certain amino acids and sugar molecules which permeate the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here, we use the ellipsoid localization microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex-lytic enzymes within the inner coat. We also reveal that the GerPA protein alone can localize in the absence of all other GerP proteins and that it has an essential role for the localization of all other GerP proteins within the spore. Its essential role is also demonstrated to be dependent on SafA, but not CotE, for localization, which is consistent with an inner coat location. GerP-null spores are shown also to have reduced permeability to fluorescently labeled dextran molecules compared to wild-type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability.IMPORTANCE The bacterial spore coat comprises a multilayered proteinaceous structure that influences the distribution, survival, and germination properties of spores in the environment. The results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ellipsoid localization microscopy (ELM) image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, health care, and environmental sectors.

It is often necessary to precisely quantify the size of specimens in biological studies. When measuring feature size in fluorescence microscopy, significant biases can arise due to blurring of its edges if the feature is smaller than the diffraction limit of resolution. This problem is avoided if an equation describing the feature's entire image is fitted to its image data. In this paper we present open-source software, ELM, which uses this approach to measure the size of spheroidal or cylindrical fluorescent shells with a precision of around 10 nm. This has been used to measure coat protein locations in bacterial spores and cell wall diameter in vegetative bacilli, and may also be valuable in microbiological studies of algae, fungi and viruses. ELM is available for download at https://github.com/quantitativeimaging/ELM.

Refer to the readme. txt file for a description of folder contents. The ELM software provided in this archive, written in MATLAB, has its own instructions and sample data in the readme. md file in the folder\ELM-master

The fracture toughness of colloidal films is measured by characterizing cracks which form during directional drying. Images from a confocal microscope are processed to measure the crack width as a function of distance from the crack tip. Applying theory for thin elastic films the fracture toughness is extracted. It is found that the fracture toughness scales with the particle size to the -0.8 power and that the critical energy release rate scales with the particle size to the -1.3 power. In addition, the fracture toughness is found to increase at lower evaporation rates, but the film thickness does not have a significant effect.

Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein-peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.

We propose a three-objective light sheet microscopy geometry which, through a combination of skewed lattice light sheet excitation through two objectives and the computational fusion of images taken from two separate lens pairings, would allow for isotropic super-resolution in mesoscopic samples. We also show that simultaneous coherent excitation through two excitation objectives could further substantially increase resolution. Simulations demonstrate that our design could achieve a resolution of 120 nm for EGFP imaging while minimizing photodamage.

Correlation spectroscopy is an analytical technique that can identify the residence time of reflective or fluorescent particles in a measurement spot, allowing particle velocity or diffusion to be inferred. We show that the technique can be applied to data measured with a time-domain terahertz sensor. The speed of reflectors such as silica ballotini or bubbles can thus be measured in fluid samples. Time-domain terahertz sensors can therefore be used, for the first time, to measure rheological properties of optically opaque fluids that contain entrained reflectors, such as polyethylene beads.

In experiments we generate images of cracks in drying suspensions The code determines the crack tip and then calculates the crack width as a function of distance from the crack tip.