Imaging techniques for elements and element species in plant science

Revealing the uptake, transport, localization and speciation of both essential and toxic elements in plants is important for understanding plant homeostasis and metabolism, subsequently, providing information for food and nutrient studies, agriculture activities, as well as environmental research. In the last decade, emerging techniques for elemental imaging and speciation analysis allowed us to obtain increasing knowledge of elemental distribution and availabilities in plants. Chemical imaging techniques include mass spectrometric methods such as secondary ionization mass spectrometry (SIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron-based techniques such as X-ray fluorescence spectroscopy (SRXRF), and so forth. On the other hand, X-ray absorption spectroscopy (XAS) based on synchrotron radiation is capable of in situ investigation of local atomic structure around the central element of interest. This technique can also be operated in tandem with SRXRF to image each element species of interest within plant tissue. In this review, the principles and state-of-the-art of these techniques regarding sample preparation, advantages and limitations, and improvement of sensitivity and spatial resolution are discussed. New results with respect to elemental distribution and speciation in plants revealed by these techniques are presented.     

Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

Background

Angiogenesis is widely investigated in conjunction with cancer development, in particular because of the possibility of early stage detection and of new therapeutic strategies. However, such studies are negatively affected by the limitations of imaging techniques in the detection of microscopic blood vessels (diameter 3-5 ??m) grown under angiogenic stress. We report that synchrotron-based X-ray imaging techniques with very high spatial resolution can overcome this obstacle, provided that suitable contrast agents are used.

Results

We tested different contrast agents based on gold nanoparticles (AuNPs) for the detection of cancer-related angiogenesis by synchrotron microradiology, microtomography and high resolution X-ray microscopy. Among them only bare-AuNPs in conjunction with heparin injection provided sufficient contrast to allow in vivo detection of small capillary species (the smallest measured lumen diameters were 3-5 ??m). The detected vessel density was 3-7 times higher than with other nanoparticles. We also found that bare-AuNPs with heparin allows detecting symptoms of local extravascular nanoparticle diffusion in tumor areas where capillary leakage appeared.

Conclusions

Although high-Z AuNPs are natural candidates as radiology contrast agents, their success is not guaranteed, in particular when targeting very small blood vessels in tumor-related angiography. We found that AuNPs injected with heparin produced the contrast level needed to reveal--for the first time by X-ray imaging--tumor microvessels with 3-5 ??m diameter as well as extravascular diffusion due to basal membrane defenestration. These results open the interesting possibility of functional imaging of the tumor microvasculature, of its development and organization, as well as of the effects of anti-angiogenic drugs.     

Chemical elements in the mineralization of plant residues under soil pollution with heavy metals

Mineralization processes of plant residues in background and anthropogenically affected areas are analyzed. The multielement composition of plant residues before and after their exposure in soil is provided with the help of synchrotron radiation (XPA SR). It is demonstrated that the destruction process is accompanied by changes in the chemical composition of plant residues. Chemical elements with concentrations increasing as they are filtrated from soil into the exposed samples are determined. Agrobotanical groups of plants with the highest infiltration rate are revealed.     

Biological applications of synchrotron radiation infrared spectromicroscopy

Extremely brilliant infrared (IR) beams provided by synchrotron radiation sources are now routinely used in many facilities with available commercial spectrometers coupled to IR microscopes. Using these intense non-thermal sources, a brilliance two or three order of magnitude higher than a conventional source is achievable through small pinholes (< 10 μm) with a high signal to-noise ratio. IR spectroscopy is a powerful technique to investigate biological systems and offers many new imaging opportunities. The field of infrared biological imaging covers a wide range of fundamental issues and applied researches such as cell imaging or tissue imaging. Molecular maps with a spatial resolution down to the diffraction limit may be now obtained with a synchrotron radiation IR source also on thick samples. Moreover, changes of the protein structure are detectable in an IR spectrum and cellular molecular markers can be identified and used to recognize a pathological status of a tissue. Molecular structure and functions are strongly correlated and this aspect is particularly relevant for imaging. We will show that the brilliance of synchrotron radiation IR sources may enhance the sensitivity of a molecular signal obtained from small biosamples, e.g., a single cell, containing extremely small amounts of organic matter. We will also show that SR IR sources allow to study chemical composition and to identify the distribution of organic molecules in cells at submicron resolution is possible with a high signal-to-noise ratio. Moreover, the recent availability of two-dimensional IR detectors promises to push forward imaging capabilities in the time domain. Indeed, with a high current synchrotron radiation facility and a Focal Plane Array the chemical imaging of individual cells can be obtained in a few minutes. Within this framework important results are expected in the next years using synchrotron radiation and Free Electron Laser (FEL) sources for spectro-microscopy and spectral-imaging, alone or in combination with Scanning Near-field Optical Microscopy methods to study the molecular composition and dynamic changes in samples of biomedical interest at micrometric and submicrometric scales, respectively.     

Perspectives in three-dimensional analysis of bone samples using synchrotron radiation microtomography.

In this paper we present a methodology based on 3D synchrotron radiation microtomography to analyze non-destructively 3D bone samples. After a technical presentation of the imaging system and the image analysis techniques, we report results on three-dimensional analysis of vertebral samples from women of different ages. The new capabilities of this technique for the investigation of bone are discussed. They include a high spatial resolution down to the micron level, a high density resolution allowing a local quantification of bone mineralization, phase contrast imaging and advances in 3D image analysis.     

Trace metal imaging with high spatial resolution: applications in biomedicine

New generations of analytical techniques for imaging of metals are pushing hitherto boundaries of spatial resolution and quantitative analysis in biology. Because of this, the application of these imaging techniques described herein to the study of the organization and dynamics of metal cations and metal-containing biomolecules in biological cell and tissue is becoming an important issue in biomedical research. In the current review, three common metal imaging techniques in biomedical research are introduced, including synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). These are exemplified by a demonstration of the dopamine-Fe complexes, by assessment of boron distribution in a boron neutron capture therapy cell model, by mapping Cu and Zn in human brain cancer and a rat brain tumor model, and by the analysis of metal topography within neuromelanin. These studies have provided solid evidence that demonstrates that the sensitivity, spatial resolution, specificity, and quantification ability of metal imaging techniques is suitable and highly desirable for biomedical research. Moreover, these novel studies on the nanometre scale (e.g., of individual single cells or cell organelles) will lead to a better understanding of metal processes in cells and tissues.     

Contact microscopy with synchrotron radiation

Soft X-ray contact microscopy with synchrotron radiation offers the biologist, and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM, or SEM methods (i.e., hydrated samples, samples easily damaged by an electron beam, electron-dense samples, thick specimens, unstained, low-contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash X-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of X-ray wavelengths or specific individual wavelengths that optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of X-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples.     

Functional lung imaging with synchrotron radiation: Methods and preclinical applications

Many lung disease processes are characterized by structural and functional heterogeneity that is not directly appreciable with traditional physiological measurements. Experimental methods and lung function modeling to study regional lung function are crucial for better understanding of disease mechanisms and for targeting treatment. Synchrotron radiation offers useful properties to this end: coherence, utilized in phase-contrast imaging, and high flux and a wide energy spectrum which allow the selection of very narrow energy bands of radiation, thus allowing imaging at very specific energies. K-edge subtraction imaging (KES) has thus been developed at synchrotrons for both human and small animal imaging. The unique properties of synchrotron radiation extend X-ray computed tomography (CT) capabilities to quantitatively assess lung morphology, and also to map regional lung ventilation, perfusion, inflammation and biomechanical properties, with microscopic spatial resolution. Four-dimensional imaging, allows the investigation of the dynamics of regional lung functional parameters simultaneously with structural deformation of the lung as a function of time. This review summarizes synchrotron radiation imaging methods and overviews examples of its application in the study of disease mechanisms in preclinical animal models, as well as the potential for clinical translation both through the knowledge gained using these techniques and transfer of imaging technology to laboratory X-ray sources.     

In situ analysis of metal(loid)s in plants: State of the art and artefacts

Metals and metalloids play important roles in plant function and metabolism. Likewise, plants subsequently introduce vital dietary nutrition to people and animals. Understanding the transport, localisation and speciation of these elements is critical for understanding availability and metabolic pathways. Subsequently this knowledge can be applied to plant physiology and agricultural research, food science and genetic engineering.This review focuses on the most recent status of in situ techniques to visualise spatial distributions and assess the speciation of metals and metalloids. The techniques addressed include: histochemical analysis, autoradiography, LA-ICP-MS, SIMS, SEM including EDX, PIXE; and synchrotron methods: XRF, differential and fluorescence tomography, and X-ray absorption techniques.This review has been written with the intent of plant researchers to gain familiarity with techniques to which they are not accustom but wish to extend their research with alternative, but complementary, capabilities. Importantly, the disadvantages as well as advantages, have been highlighted for each technique and potential artefacts induced by the analysis or sample preparation are reviewed. These often overlooked aspects are the points critical for novice use of unfamiliar techniques and are offered for advancing research approaches commensurate with the accelerating interest regarding metal(loid)s in botanical specimens.     

Phospholipid phase transitions: kinetics and structural mechanisms

A brief review is given on the principles and methods used to investigate structural phase transitions in phospholipid supramolecular structures. The conceptual differences of approaches close to and far from equilibrium are addressed, and the consequences in terms of the limits of interpretation for different types of methods, in particular referring to jump-relaxation and steady-state techniques, are surveyed. With the emphasis on connecting dynamic and structural information, the results obtained so far from different techniques are reviewed, and the open questions addressed. The more recent advances by millisecond time-resolved X-ray diffraction with synchrotron radiation and their main results obtained for transitions triggered by IR-laser temperature jumps are summarized. As a major novel aspect in the field, the necessity of considering martensitic, diffusionless transformation mechanisms and the occurrence of intermediate structures is highlighted.     

无人机遥感技术在景观生态学中的应用

野外数据的获取是生态学研究的挑战之一,而通过遥感技术能够实现对地球表面的多面立体观测,获取丰富多样的空间信息数据,开展从微观到宏观不同尺度上的景观单元(包括物种、种群、群落、生态系统等)的空间关系研究。传统卫星遥感影像受空间和时间分辨率的限制,难以满足局域尺度或者时间序列上的景观空间生态学研究需求。无人机遥感技术为生态学研究的野外数据获取提供了一种新方法,以其灵活、高效、简便等特点弥补了传统卫星遥感的空间分辨率低、重访周期长、云雾影响等方面的不足,在景观空间生态学研究中受到越来越多的关注。简要介绍无人机类型及其搭载常见的传感器类型,分别从不同尺度的景观单元,即物种、种群、群落以及生态系统水平上探讨其应用进展,并指出当前无人机技术在景观生态学研究中存在的挑战与困难,同时展望了未来可能的研究热点,以期对今后无人机遥感技术在景观生态学领域的应用研究有所启发。     

The tertiary structure of azurin from Pseudomonas denitrificans as determined by Cu resonant diffraction using synchrotron radiation

The structure of Pseudomonas denitrificans azurin has been solved at 3.0 A resolution by film data collection methods using synchrotron radiation. Bijvoet pairs of reflections were collected using 8.98 keV radiation where both delta f' and delta f", the real and imaginary corrections to azurin's Cu prosthetic group scattering, respectively, are large, and using 8.00 keV radiation where these corrections are smaller. The Cu atom was located by difference Patterson syntheses and used to phase the observed protein structure-factor moduli of selected reflections. An atomic model of the protein was built using a restricted data set and phases for all observed reflections to 3.0 A resolution were subsequently calculated from the initial model. The atomic model was subsequently rebuilt using all observed data. The results of this work are presented here and illustrate the utility of synchrotron radiation phasing techniques in solving the structures of metalloproteins without recourse to multiple isomorphous replacement.     

无人机遥感在生态学中的应用进展

无人机与遥感技术的结合,即无人机遥感。与传统的以卫星和有人机遥感相比,无人机遥感具有高时效、高时空分辨率、云下低空飞行、高机动性等优势,是传统卫星和有人机遥感手段所无法比拟的。这些优点使得无人机在生态学和保护生物学等领域获得迅速发展。首先对无人机遥感技术的发展历程、系统组成、分类与选型、应用优势等进行了介绍。在此基础上,对无人机在生态学中的应用案例进行了总结,内容涉及生境监测、植物物候调查、动物监测等方面。最后通过比较国内外相关领域的研究进展对无人机生态学存在的问题(技术门槛较高和法律法规不完善等)和潜在应用前景进行了探讨。     

Comparing different approaches to calculate the effects of heterogeneous root distribution on nutrient uptake: a case study on subsoil nitrate uptake by a barley root system

Simulation models of nutrient uptake of root systems starting with one-dimensional single root approaches up to complex three-dimensional models are increasingly used for examining the interacting of root distribution and nutrient uptake. However, their accuracy was seldom systematically tested. The objective of the study is to compare one-dimensional and two-dimensional modelling approaches and to test their applicability for simulation of nutrient uptake of heterogeneously distributed root systems giving particular attention to the impact of spatial resolution. Therefore, a field experiment was carried out with spring barley (Hordeum vulgare L. cv. Barke) in order to obtain data of in situ root distribution patterns as model input. Results indicate that a comparable coarse spatial resolution can be used with sufficient modelling results when a steady state approximation is applied to the sink cells of the two-dimensional model. Furthermore, the accuracy of the model was clearly improved compared to a simple zero sink approach assuming both near zero concentrations within the sink cell and a linear gradient between the sink cell and its adjacent neighbours. However, for modelling nitrate uptake of a heterogeneous root system a minimum number of grid cells is still necessary. The tested single root approach provided a computational efficient opportunity to simulate nitrate uptake of an irregular distributed root system. Nevertheless, two-dimensional models are better suited for a number of applications (e.g. surveys made on the impact of soil heterogeneity on plant nutrient uptake). Different settings for the suggested modelling techniques are discussed.     

Chemical imaging of biological tissue with synchrotron infrared light

Fourier transform infrared micro-spectroscopy (FTIRM) and imaging (FTIRI) have become valuable techniques for examining the chemical makeup of biological materials by probing their vibrational motions on a microscopic scale. Synchrotron infrared (S-IR) light is an ideal source for FTIRM and FTIRI due to the combination of its high brightness (i.e., flux density), also called brilliance, and broadband nature. Through a 10-μm pinhole, the brightness of a synchrotron source is 100-1000 times higher than a conventional thermal (globar) source. Accordingly, the improvement in spatial resolution and in spectral quality to the diffraction limit has led to a plethora of applications that is just being realized. In this review, we describe the development of synchrotron-based FTIRM, illustrate its advantages in many applications to biological systems, and propose some potential future directions for the technique.     

Chemical imaging of biological tissue with synchrotron infrared light

Fourier transform infrared micro-spectroscopy (FTIRM) and imaging (FTIRI) have become valuable techniques for examining the chemical makeup of biological materials by probing their vibrational motions on a microscopic scale. Synchrotron infrared (S-IR) light is an ideal source for FTIRM and FTIRI due to the combination of its high brightness (i.e., flux density), also called brilliance, and broadband nature. Through a 10-microm pinhole, the brightness of a synchrotron source is 100-1000 times higher than a conventional thermal (globar) source. Accordingly, the improvement in spatial resolution and in spectral quality to the diffraction limit has led to a plethora of applications that is just being realized. In this review, we describe the development of synchrotron-based FTIRM, illustrate its advantages in many applications to biological systems, and propose some potential future directions for the technique.     

Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions.     

Monitoring and validating active site redox states in protein crystals

High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Ecology and evolution of bacterial microdiversity

Using high resolution molecular fingerprinting techniques like random amplification of polymorphic DNA, repetitive extragenic palindromic PCR and multilocus enzyme electrophoresis, a high bacterial diversity below the species and subspecies level (microdiversity) is revealed. It became apparent that bacteria of a certain species living in close association with different plants either as associated rhizosphere bacteria or as plant pathogens or symbiotic organisms, typically reflect this relationship in their genetic relatedness. The strain composition within a population of soil bacterial species at a given field site, which can be identified by these high resolution fingerprinting techniques, was markedly influenced by soil management and soil features. The observed bacterial microdiversity reflected the conditions of the habitat, which select for better adapted forms. In addition, influences of spatial separation on specific groupings of bacteria were found, which argue for the occurrence of isolated microevolution. In this review, examples are presented of bacterial microdiversity as influenced by different ecological factors, with the main emphasis on bacteria from the natural environment. In addition, information available from some of the first complete genome sequences of bacteria (Helicobacter pylori and Escherichia coli) was used to highlight possible mechanisms of molecular evolution through which mutations are created; these include mutator enzymes. Definitions of bacterial species and subspecies ranks are discussed in the light of detailed information from whole genome typing approaches.