Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid ß1-42 pathologies.

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-ß 1-42 (Aß42) 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.

Concepts for structured illumination microscopy with extended axial resolution through mirrored illumination.

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.

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.

Novel cortex lytic enzymes in Bacillus megaterium QM B1551 spores.

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.

Improved methodology for performing the inverse Abel transform of flame images for color ratio pyrometry.

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.

ELM: super-resolution analysis of wide-field images of fluorescent shell structures.

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.

Proteins Encoded by the gerP Operon Are Localized to the Inner Coat in Bacillus cereus Spores and Are Dependent on GerPA and SafA for Assembly.

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.

Homographically generated light sheets for the microscopy of large specimens.

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.

Generation of strength in a drying film: How fracture toughness depends on dispersion properties.

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.

Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.

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.

triSPIM: light sheet microscopy with isotropic super-resolution.

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.

Terahertz correlation spectroscopy infers particle velocity and rheological properties.

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.

Analysis of the chronic wound microbiota of 2,963 patients by 16S rDNA pyrosequencing.

The extent to which microorganisms impair wound healing is an ongoing controversy in the management of chronic wounds. Because the high diversity and extreme variability of the microbiota between individual chronic wounds lead to inconsistent findings in small cohort studies, evaluation of a large number of chronic wounds using identical sequencing and bioinformatics methods is necessary for clinicians to be able to select appropriate empiric therapies. In this study, we utilized 16S rDNA pyrosequencing to analyze the composition of the bacterial communities present in samples obtained from patients with chronic diabetic foot ulcers (N = 910), venous leg ulcers (N = 916), decubitus ulcers (N = 767), and nonhealing surgical wounds (N = 370). The wound samples contained a high proportion of Staphylococcus and Pseudomonas species in 63 and 25% of all wounds, respectively; however, a high prevalence of anaerobic bacteria and bacteria traditionally considered commensalistic was also observed. Our results suggest that neither patient demographics nor wound type influenced the bacterial composition of the chronic wound microbiome. Collectively, these findings indicate that empiric antibiotic selection need not be based on nor altered for wound type. Furthermore, the results provide a much clearer understanding of chronic wound microbiota in general; clinical application of this new knowledge over time may help in its translation to improved wound healing outcomes.

Ellipsoid Localization Microscopy Infers the Size and Order of Protein Layers in Bacillus Spore Coats.

Multilayered protein coats are crucial to the dormancy, robustness, and germination of bacterial spores. In Bacillus subtilis spores, the coat contains over 70 distinct proteins. Identifying which proteins reside in each layer may provide insight into their distinct functions. We present image analysis methods that determine the order and geometry of concentric protein layers by fitting a model description for a spheroidal fluorescent shell image to optical micrographs of spores incorporating fluorescent fusion proteins. The radius of a spherical protein shell can be determined with <10 nm error by fitting an equation to widefield fluorescence micrographs. Ellipsoidal shell axes can be fitted with comparable precision. The layer orders inferred for B. subtilis and B. megaterium are consistent with measurements in the literature. The aspect ratio of elongated spores and the tendency of some proteins to localize near their poles can be quantified, enabling measurement of structural anisotropy.

Structural analysis of herpes simplex virus by optical super-resolution imaging.

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

A quantitative protocol for intensity-based live cell FRET imaging.

Förster resonance energy transfer (FRET) has become one of the most ubiquitous and powerful methods to quantify protein interactions in molecular biology. FRET refers to the sensitization of an acceptor molecule through transfer of energy from a nearby donor, and it can occur if the emission band of the donor exhibits spectral overlap with the absorption band of the acceptor molecule. Numerous methods exist to quantify FRET levels from interacting protein labels including fluorescence lifetime, acceptor photobleaching, and polarization-resolved imaging (Lakowicz, Principles of fluorescence spectroscopy, 2006; Jares-Erijman and Jovin, Curr Opin Chem Biol 10(5):409-416, 2006; van Munster and Gadella, Microscopy techniques, 2005). For live cell imaging, however, sensitized emission FRET (seFRET) is the most powerful and robust method of FRET signal quantification (Chakrabortee et al., Proc Natl Acad Sci USA 107(37):16084-16089, 2010). It is fast, can be applied using straight forward microscopy equipment, and offers information not only on strength of interaction but, uniquely, also on the relative changes between interacting and noninteracting moieties in the reaction, referred to as FRET stoichiometry. A rigorous and quantitative application of seFRET is far from trivial, however, and requires appropriate calibration experiments and constructs, control over hardware settings, and appropriate image processing steps.This protocol presents a rigorous method to perform quantitative seFRET measurements in live cells, providing the maximum possible information content from the measurement. The theoretical development and validation of the method is described in detail in Elder et al. (J R Soc Interface 6:S59-S81, 2009) where it is also demonstrated in the kinetic ("time-lapse") analysis of protein interactions governing mitosis. The present protocol gives a detailed recipe for application of seFRET. It is written specifically for use with CFP (cyan fluorescence protein) as donor fluorophore and YFP (yellow fluorescent protein) as acceptor fluorophore, a popular choice for many experiments. The protocol is however valid for any other FRET fluorophore pair, and we indicate how to adapt the protocol in such situations. We also provide a software program that automates the calibration tasks outlined in this protocol and which is available for free to download ( http://wiki.laser.ceb.cam.ac.uk/wiki/index.php/Resources ).

Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing.

BACKGROUND: Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing (culture-free 16S ribosomal DNA sequencing), and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture. METHODS: Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria. RESULTS: One hundred forty-five (145) unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14) unique genera were identified using aerobic culture methods. One-third (31/92) of the cultures were determined to be < 1% of the relative abundance of the wound microbiota using molecular testing. At the genus level, molecular testing identified 85% (78/92) of the bacteria that were identified by culture. Conversely, culturing detected 15.7% (78/497) of the aerotolerant bacteria and detected 54.9% of the collective aerotolerant relative abundance of the samples. Aerotolerant bacterial genera (and individual species including Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling. CONCLUSION: Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture.

Periodic interactions between solitons and dispersive waves during the generation of non-coherent supercontinuum radiation.

We present a numerical study of interactions between dispersive waves (DWs) and solitons during supercontinuum generation in photonic crystal fibers pumped with picosecond laser pulses. We show how the soliton-induced trapping potential evolves along the fiber and affects the dynamics of a DW-soliton pair. Individual frequency components of the DW periodically interact with the soliton resulting in stepwise frequency blue shifts. In contrast, the ensemble blue shifts of all frequency components in the DW appear to be quasi-continuous. The step size of frequency up-conversion and the temporal separation between subsequent soliton-DW interactions are governed by the potential well which confines the soliton-DW pair and which changes in time.

An adaptive filter for studying the life cycle of optical rogue waves.

We present an adaptive numerical filter for analyzing fiber-length dependent properties of optical rogue waves, which are highly intense and extremely red-shifted solitons that arise during supercontinuum generation in photonic crystal fiber. We use this filter to study a data set of 1000 simulated supercontinuum pulses, produced from 5 ps pump pulses containing random noise. Optical rogue waves arise in different supercontinuum pulses at various positions along the fiber, and exhibit a lifecycle: their intensity peaks over a finite range of fiber length before declining slowly.

Predicting supercontinuum pulse collisions with simulations exhibiting temporal aliasing.

Interactions between supercontinuum (SC) light pulses, produced by the propagation of rapidly sequenced picosecond pump laser pulses along a photonic crystal fiber, result in spectral broadening, which we attribute to interpulse soliton collisions. This phenomenon was measured experimentally, following our observation of spectral broadening in numerical simulations that exhibit so-called "pulse wraparound" or "temporal aliasing." This occurs in simulations with narrow time grids: as early parts of the SC pulse leave the computational time domain, they "reenter" at the beginning and so interact with later parts of the evolving SC pulse. We show that this provides an effective model to predict the experimentally observed spectral changes.