Visualisation of gene expression within the context of tissues using an X-ray computed tomography-based multimodal approach.

The development of an organism is orchestrated by the spatial and temporal expression of genes. Accurate visualisation of gene expression patterns in the context of the surrounding tissues offers a glimpse into the mechanisms that drive morphogenesis. We developed correlative light-sheet fluorescence microscopy and X-ray computed tomography approach to map gene expression patterns to the whole organism`s 3D anatomy. We show that this multimodal approach is applicable to gene expression visualized by protein-specific antibodies and fluorescence RNA in situ hybridisation offering a detailed understanding of individual phenotypic variations in model organisms. Furthermore, the approach offers a unique possibility to identify tissues together with their 3D cellular and molecular composition in anatomically less-defined in vitro models, such as organoids. We anticipate that the visual and quantitative insights into the 3D distribution of gene expression within tissue architecture, by multimodal approach developed here, will be equally valuable for reference atlases of model organisms development, as well as for comprehensive screens, and morphogenesis studies of in vitro models.

Environmental and health impacts assessment of long-term naturally-weathered municipal solid waste incineration ashes deposited in soil-old burden in Bratislava city, Slovakia.

Municipal solid waste incineration (MSWI) is an effective method for reducing the volume/mass of waste. However, MSWI ashes contain high concentrations of many substances, including trace metal (loid)s, that could be released into the environment and contaminate soils and groundwater. In this study, attention was focused on the site near the municipal solid waste incinerator where MSWI ashes are deposited on the surface without any control. Here, combined results (chemical and mineralogical analyses, leaching tests, speciation modelling, groundwater chemistry and human health risk assessment) are presented to assess the impact of MSWI ash on the surrounding environment. The mineralogy of ∼forty years old MSWI ash was diverse, and quartz, calcite, mullite, apatite, hematite, goethite, amorphous glasses and several Cu-bearing minerals (e.g. malachite, brochantite) were commonly detected. In general, the total concentrations of metal (loid)s in MSWI ashes were high, following the order: Zn (6731 mg/kg) > Ba (1969 mg/kg) ≈ Mn (1824 mg/kg) > Cu (1697 mg/kg) > Pb (1453 mg/kg) > Cr (247 mg/kg) > Ni (132 mg/kg) > Sb (59.4 mg/kg) > As (22.9 mg/kg) ≈ Cd (20.6 mg/kg). Cadmium, Cr, Cu, Pb, Sb and Zn exceeded the indication or even intervention criteria for industrial soils defined by the Slovak legislation. Batch leaching experiments with diluted citric and oxalic acids that simulate the leaching of chemical elements under rhizosphere conditions documented low dissolved fractions of metals (0.00-2.48%) in MSWI ash samples, showing their high geochemical stability. Non-carcinogenic and carcinogenic risks were below the threshold values of 1.0 and 1 × 10-6, respectively, with soil ingestion being the most important exposure route for workers. The groundwater chemistry was unaffected by deposited MSWI ashes. This study may be useful in determining the environmental risks of trace metal (loid)s in weathered MSWI ashes that are loosely deposited on the soil surface.

Tofu: a fast, versatile and user-friendly image processing toolkit for computed tomography.

Tofu is a toolkit for processing large amounts of images and for tomographic reconstruction. Complex image processing tasks are organized as workflows of individual processing steps. The toolkit is able to reconstruct parallel and cone beam as well as tomographic and laminographic geometries. Many pre- and post-processing algorithms needed for high-quality 3D reconstruction are available, e.g. phase retrieval, ring removal and de-noising. Tofu is optimized for stand-alone GPU workstations on which it achieves reconstruction speed comparable with costly CPU clusters. It automatically utilizes all GPUs in the system and generates 3D reconstruction code with minimal number of instructions given the input geometry (parallel/cone beam, tomography/laminography), hence yielding optimal run-time performance. In order to improve accessibility for researchers with no previous knowledge of programming, tofu contains graphical user interfaces for both optimization of 3D reconstruction parameters and batch processing of data with pre-configured workflows for typical computed tomography reconstruction. The toolkit is open source and extensive documentation is available for both end-users and developers. Thanks to the mentioned features, tofu is suitable for both expert users with specialized image processing needs (e.g. when dealing with data from custom-built computed tomography scanners) and for application-specific end-users who just need to reconstruct their data on off-the-shelf hardware.

Evolution of flexible biting in hyperdiverse parasitoid wasps.

One key event in insect evolution was the development of mandibles with two joints, which allowed powerful biting but restricted their movement to a single degree of freedom. These mandibles define the Dicondylia, which constitute over 99% of all extant insect species. It was common doctrine that the dicondylic articulation of chewing mandibles remained unaltered for more than 400 million years. We report highly modified mandibles overcoming the restrictions of a single degree of freedom and hypothesize their major role in insect diversification. These mandibles are defining features of parasitoid chalcid wasps, one of the most species-rich lineages of insects. The shift from powerful chewing to precise cutting likely facilitated adaptations to parasitize hosts hidden in hard substrates, which pose challenges to the emerging wasps. We reveal a crucial step in insect evolution and highlight the importance of comprehensive studies even of putatively well-known systems.

Juvenile ecology drives adult morphology in two insect orders.

Most animals undergo ecological niche shifts between distinct life phases, but such shifts can result in adaptive conflicts of phenotypic traits. Metamorphosis can reduce these conflicts by breaking up trait correlations, allowing each life phase to independently adapt to its ecological niche. This process is called adaptive decoupling. It is, however, yet unknown to what extent adaptive decoupling is realized on a macroevolutionary scale in hemimetabolous insects and if the degree of adaptive decoupling is correlated with the strength of ontogenetic niche shifts. It is also unclear whether the degree of adaptive decoupling is correlated with phenotypic disparity. Here, we quantify nymphal and adult trait correlations in 219 species across the whole phylogeny of earwigs and stoneflies to test whether juvenile and adult traits are decoupled from each other. We demonstrate that adult head morphology is largely driven by nymphal ecology, and that adult head shape disparity has increased with stronger ontogenetic niche shifts in some stonefly lineages. Our findings implicate that the hemimetabolan metamorphosis in earwigs and stoneflies does not allow for high degrees of adaptive decoupling, and that high phenotypic disparity can even be realized when the evolution of distinct life phases is coupled.

Contaminated soils of different natural pH and industrial origin: The role of (nano) iron- and manganese-based amendments in As, Sb, Pb, and Zn leachability.

Soils containing a large proportion of industrial waste can pose a health risk due to high environmentally available concentrations of toxic metal(loid)s. Nano zero-valent iron (nZVI) and amorphous manganese oxide (AMO) were applied as immobilising amendments (1 wt%) to soils with different industrial origin of As and Sb, and leaching of As, Sb, Pb, and Zn was investigated using a single extraction with deionised water. The different industrial impact was reflected in the mineralogy, chemical composition and pH of these soils. Water-soluble As ratios positively correlated with pH in all experimental treatments. A significant decrease of water-soluble As ratios was observed in all nZVI-amended soils (~65-93% of the control) except for one sample with the lowest solution pH. Nano zero-valent iron was also successful in Sb immobilisation (~76-90% of the control). Highly variable results were obtained for AMO, which only led to a decrease of water-soluble As in soils with solution pH of ≥7 (~70-80% of the control), probably due to lower stability of AMO in acidic conditions. In each case, nZVI was more efficient at decreasing water-soluble As ratios than AMO. Dissolved Pb concentrations remained unchanged after the application of nZVI and AMO, and the decrease of Zn leaching using AMO was controlled mainly by soil pH increase induced by its application. According to the calculated saturation indices, tripuhyite (FeSbO4) was predicted to be the key mineral controlling Sb solubility in mine soils. Secondary Fe (hydr)oxides either originally present or newly formed due to nZVI oxidation were instrumentally identified at different stages of their transformation and metal(loid) retention. To conclude, nZVI is suitable for application to contaminated soils at a wide pH range, while the use of AMO for decreasing As leaching is limited to soils with pH ≥ 7.

Introducing Biomedisa as an open-source online platform for biomedical image segmentation.

We present Biomedisa, a free and easy-to-use open-source online platform developed for semi-automatic segmentation of large volumetric images. The segmentation is based on a smart interpolation of sparsely pre-segmented slices taking into account the complete underlying image data. Biomedisa is particularly valuable when little a priori knowledge is available, e.g. for the dense annotation of the training data for a deep neural network. The platform is accessible through a web browser and requires no complex and tedious configuration of software and model parameters, thus addressing the needs of scientists without substantial computational expertise. We demonstrate that Biomedisa can drastically reduce both the time and human effort required to segment large images. It achieves a significant improvement over the conventional approach of densely pre-segmented slices with subsequent morphological interpolation as well as compared to segmentation tools that also consider the underlying image data. Biomedisa can be used for different 3D imaging modalities and various biomedical applications.

Drought-Induced Xylem Embolism Limits the Recovery of Leaf Gas Exchange in Scots Pine.

Climate change increases the occurrence of prolonged drought periods with large implications for forest functioning. Scots pine (Pinus sylvestris) is one of the most abundant conifers worldwide, and evidence is rising that its resilience to severe drought is limited. However, we know little about its ability to recover from drought-induced embolism. To analyze postdrought hydraulic recovery, we investigated stress and recovery dynamics of leaf gas exchange, nonstructural carbohydrates, and hydraulic properties in 2.5-year-old Scots pine seedlings. We quantified the degree of xylem embolism by combining in vivo x-ray microtomography with intrusive techniques including measurements of hydraulic conductivity and dye staining during drought progression and short-term (2 d) and long-term (4 weeks) recovery. Seedlings were grown under controlled conditions, and irrigation was withheld until stomata closed and xylem water potential declined to -3.2 MPa on average, causing a 46% loss of stem hydraulic conductivity. Following drought release, we found a gradual recovery of leaf gas exchange to 50% to 60% of control values. This partial recovery indicates hydraulic limitations due to drought-induced damage. Whereas xylem water potential recovered close to control values within 2 d, both x-ray microtomography and intrusive measurements revealed no recovery of stem hydraulic conductivity. Moreover, we did not find indications for nonstructural carbohydrate reserves limiting hydraulic recovery. Our findings demonstrate that Scots pine is able to survive severe drought and to partially recover, although we assume that xylem development during the next growing season might compensate for some of the hydraulic impairment. Such drought-induced legacy effects are important when considering vegetation responses to extreme events.

Parasitoid biology preserved in mineralized fossils.

About 50% of all animal species are considered parasites. The linkage of species diversity to a parasitic lifestyle is especially evident in the insect order Hymenoptera. However, fossil evidence for host-parasitoid interactions is extremely rare, rendering hypotheses on the evolution of parasitism assumptive. Here, using high-throughput synchrotron X-ray microtomography, we examine 1510 phosphatized fly pupae from the Paleogene of France and identify 55 parasitation events by four wasp species, providing morphological and ecological data. All species developed as solitary endoparasitoids inside their hosts and exhibit different morphological adaptations for exploiting the same hosts in one habitat. Our results allow systematic and ecological placement of four distinct endoparasitoids in the Paleogene and highlight the need to investigate ecological data preserved in the fossil record.

syris: a flexible and efficient framework for X-ray imaging experiments simulation.

An open-source framework for conducting a broad range of virtual X-ray imaging experiments, syris, is presented. The simulated wavefield created by a source propagates through an arbitrary number of objects until it reaches a detector. The objects in the light path and the source are time-dependent, which enables simulations of dynamic experiments, e.g. four-dimensional time-resolved tomography and laminography. The high-level interface of syris is written in Python and its modularity makes the framework very flexible. The computationally demanding parts behind this interface are implemented in OpenCL, which enables fast calculations on modern graphics processing units. The combination of flexibility and speed opens new possibilities for studying novel imaging methods and systematic search of optimal combinations of measurement conditions and data processing parameters. This can help to increase the success rates and efficiency of valuable synchrotron beam time. To demonstrate the capabilities of the framework, various experiments have been simulated and compared with real data. To show the use case of measurement and data processing parameter optimization based on simulation, a virtual counterpart of a high-speed radiography experiment was created and the simulated data were used to select a suitable motion estimation algorithm; one of its parameters was optimized in order to achieve the best motion estimation accuracy when applied on the real data. syris was also used to simulate tomographic data sets under various imaging conditions which impact the tomographic reconstruction accuracy, and it is shown how the accuracy may guide the selection of imaging conditions for particular use cases.

Generalized pupil function of a compound X-ray refractive lens.

Quality of a refractive compound X-ray lens can be limited by imperfections in surfaces of unit lenses and stacking precision. In general case both the lens transmission and optical aberrations define properties of a beam in the lens exit plane; together they can be expressed in terms of the generalized pupil function. In this work we measure this function for a diamond single crystal compound refractive lens. Consequently, we apply the pupil function to evaluate the performance of the examined compound refractive X-ray lens. A number of practically important conclusions can be drawn from such analysis.

Real-time image-content-based beamline control for smart 4D X-ray imaging.

Real-time processing of X-ray image data acquired at synchrotron radiation facilities allows for smart high-speed experiments. This includes workflows covering parameterized and image-based feedback-driven control up to the final storage of raw and processed data. Nevertheless, there is presently no system that supports an efficient construction of such experiment workflows in a scalable way. Thus, here an architecture based on a high-level control system that manages low-level data acquisition, data processing and device changes is described. This system is suitable for routine as well as prototypical experiments, and provides specialized building blocks to conduct four-dimensional in situ, in vivo and operando tomography and laminography.

Three-dimensional-printed gas dynamic virtual nozzles for x-ray laser sample delivery.

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

On the origin and nature of the grating interferometric dark-field contrast obtained with low-brilliance x-ray sources.

The x-ray dark-field contrast accessible via grating interferometry is sensitive to features at length scales well below what is resolvable by a detector system. It is commonly explained as arising from small-angle x-ray scattering (SAXS), and can be implemented both at synchrotron beamlines and with low-brilliance sources such as x-ray tubes. Here, we demonstrate that for tube based setups the underlying process of image formation can be fundamentally different. For focal spots or detector pixels that comprise multiple grating periods, we show that dark-field images contain a strong artificial and system-specific component not arising from SAXS. Based on experiments carried out with a nanofocus x-ray tube and the example of an excised rat lung, we demonstrate that the dark-field contrast observed for porous media transforms into a differential phase contrast for large geometric magnifications. Using a photon counting detector with an adjustable point spread function, we confirm that a dark-field image can indeed be formed by an intra-pixel differential phase contrast that cannot be resolved as such due to a dephasing between the periodicities of the absorption grating and the Talbot carpet. Our findings are further corroborated by a link between the strength of this pseudo-dark-field contrast and our x-ray tube's focal spot size in a three-grating setup. These results must not be ignored when measurements are intended to be reproducible across systems.