Recovering the dynamics of root growth and development using novel image acquisition and analysis methods

Roots are highly responsive to environmental signals encountered in the rhizosphere, such as nutrients, mechanical resistance and gravity. As a result, root growth and development is very plastic. If this complex and vital process is to be understood, methods and tools are required to capture the dynamics of root responses. Tools are needed which are high-throughput, supporting large-scale experimental work, and provide accurate, high-resolution, quantitative data. We describe and demonstrate the efficacy of the high-throughput and high-resolution root imaging systems recently developed within the Centre for Plant Integrative Biology (CPIB). This toolset includes (i) robotic imaging hardware to generate time-lapse datasets from standard cameras under infrared illumination and (ii) automated image analysis methods and software to extract quantitative information about root growth and development both from these images and via high-resolution light microscopy. These methods are demonstrated using data gathered during an experimental study of the gravitropic response of Arabidopsis thaliana.     

The use of ROI overlays and a semi-automated method for measuring cortical area in ImageJ for histological analysis

A major part of histologic studies is the use of high resolution imaging for data collection and analysis. ImageJ, a freely available software from the NIH designed for image analysis, has many features that are not well-known among bone histologists and can be incredibly beneficial in terms of stream-lining data collection and maximizing limited resources. The aims of this technical note are twofold: (a) to describe methods for image annotation and measurement using region of interest overlays in ImageJ, and (b) to present a new code for a semi-automated method of measuring cortical bone areas from high resolution cross-sectional images also using ImageJ.     

Advancing fine root research with minirhizotrons

Minirhizotrons provide a nondestructive, in situ method for directly viewing and studying fine roots. Although many insights into fine roots have been gained using minirhizotrons, a review of the literature indicates a wide variation in how minirhizotrons and minirhizotron data are used. Tube installation is critical, and steps must be taken to insure good soil/tube contact without compacting the soil. Ideally, soil adjacent to minirhizotrons will mimic bulk soil. Tube installation causes some degree of soil disturbance and has the potential to create artifacts in subsequent root data and analysis. We therefore recommend a waiting period between tube installation and image collection of 6-12 months to allow roots to recolonize the space around the tubes and to permit nutrients to return to pre-disturbance levels. To make repeated observations of individual roots for the purposes of quantifying their dynamic properties (e.g. root production, turnover or lifespan), tubes should be secured to prevent movement. The frequency of image collection depends upon the root parameters being measured or calculated and the time and resources available for collecting images and extracting data. However, long sampling intervals of 8 weeks or more can result in large underestimates of root dynamic properties because more fine roots will be born and die unobserved between sampling events. A sampling interval of 2 weeks or less reduces these underestimates to acceptable levels. While short sample intervals are desirable, they can lead to a potential trade-off between the number of minirhizotron tubes used and the number of frames analyzed per tube. Analyzing fewer frames per minirhizotron tube is one way to reduce costs with only minor effects on data variation. The quality of minirhizotron data should be assessed and reported; procedures for quantifying the quality of minirhizotron data are presented here. Root length is a more sensitive metric for dynamic root properties than the root number. To make minirhizotron data from separate experiments more easily comparable, idiosyncratic units should be avoided. Volumetric units compatible with aboveground plant measures make minirhizotron-based estimates of root standing crop, production and turnover more useful. Methods for calculating the volumetric root data are discussed and an example presented. Procedures for estimating fine root lifespan are discussed.     

Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography

In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.     

Smooth surface meshing for automated finite element model generation from 3D image data

Finite element (FE) modelling based on data from three-dimensional high-resolution computed tomography (CT) imaging systems provides a non-invasive method to assess structural mechanics. Automated mesh generation from these voxel based image data can be achieved by direct conversion to hexahedron elements, however these model representations have jagged edges. This paper proposes an automated method to generate smoothed FE meshes from voxel-based image data. Mesh fairing processes are utilized that allow constraints that control the smoothing process, and are computationally efficient. Surfaces of the mesh on the exterior, as well as interfaces between two tissues, can be smoothed by varying fairing parameters and constraint criteria. The method was tested on a variety of real and simulated three-dimensional data sets, resulting in both hexahedron and tetrahedron meshes. It was shown that the fairing process is linearly related to the number of smoothing iterations, and that peak stresses are reduced in FE simulations of the smoothed models. Although developed for micro-CT data sets, this fast and reliable mesh smoothing method could be applied to any three-dimensional image data where node and element connectivity have been defined.     

Technical advance: an aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.     

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.     

Analysis of Maize (Zea mays L.) Seedling Roots with the High-Throughput Image Analysis Tool ARIA (Automatic Root Image Analysis)

The maize root system is crucial for plant establishment as well as water and nutrient uptake. There is substantial genetic and phenotypic variation for root architecture, which gives opportunity for selection. Root traits, however, have not been used as selection criterion mainly due to the difficulty in measuring them, as well as their quantitative mode of inheritance. Seedling root traits offer an opportunity to study multiple individuals and to enable repeated measurements per year as compared to adult root phenotyping. We developed a new software framework to capture various traits from a single image of seedling roots. This framework is based on the mathematical notion of converting images of roots into an equivalent graph. This allows automated querying of multiple traits simply as graph operations. This framework is furthermore extendable to 3D tomography image data. In order to evaluate this tool, a subset of the 384 inbred lines from the Ames panel, for which extensive genotype by sequencing data are available, was investigated. A genome wide association study was applied to this panel for two traits, Total Root Length and Total Surface Area, captured from seedling root images from WinRhizo Pro 9.0 and the current framework (called ARIA) for comparison using 135,311 single nucleotide polymorphism markers. The trait Total Root Length was found to have significant SNPs in similar regions of the genome when analyzed by both programs. This high-throughput trait capture software system allows for large phenotyping experiments and can help to establish relationships between developmental stages between seedling and adult traits in the future.     

FRAP Analysis: Accounting for Bleaching during Image Capture

The analysis of Fluorescence Recovery After Photobleaching (FRAP) experiments involves mathematical modeling of the fluorescence recovery process. An important feature of FRAP experiments that tends to be ignored in the modeling is that there can be a significant loss of fluorescence due to bleaching during image capture. In this paper, we explicitly include the effects of bleaching during image capture in the model for the recovery process, instead of correcting for the effects of bleaching using reference measurements. Using experimental examples, we demonstrate the usefulness of such an approach in FRAP analysis.     

A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography

We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.     

Input-output analysis of in vivo photoassimilate translocation using Positron-Emitting Tracer Imaging System (PETIS) data

The Positron-Emitting Tracer Imaging System (PETIS) is introduced for monitoring the distribution of (11)C-labelled photoassimilates in Sorghum. The obtained two-dimensional image data were quantitatively analysed using a transfer function analysis approach. While one half of a Sorghum root in a split root system was treated with either 0, 100, or 500 mM NaCl dissolved in the nutrient solution, tracer images of the root halves and the lower stem section were recorded using PETIS. From the observed tracer levels, parameters were estimated, from which the mean speed of tracer transport and the proportion of tracer moved between specified image positions were deduced. Transport speed varied between 0.7 and 1.8 cm min(-1) with the difference depending on which part of the stem was involved. When data were collected in the lowest 0.5-1 cm of the stem, which included the point where the roots emerge, transport speed was less. Rapid changes in NaCl concentration, from 0 to 100 mM, resulted in short-term increases of assimilate import into the treated root. This response represented a transient osmotic effect, that was compensated for in the medium-term by osmotic adaptation. Higher concentrations of NaCl (500 mM) resulted in distinctly less photoassimilate transport into the treated root half. The present results agree with earlier observations, showing that transport of (11)C-labelled photoassimilates measured with the PETIS detector system can be quantified using the method of input-output analysis. It is worth noting that with the PETIS detector system, areas of interest do not need to be defined until after data collection. This means that unexpected behaviour of a plant organ will be seen, which is not necessarily the case with conventional detector systems looking at predefined areas of interest.     

Determination of the unknown age at first capture of western rock lobsters (Panulirus cygnus) by random effects model

We propose a method for fitting growth curves to multiple recapture data of lobsters when the age at first capture is unknown. The von Bertalanffy growth curve is used to model the growth. To account for individual variability, the unknown age in logarithmic scale of a lobster at first capture, the individual asymptotic size, and the individual growth coefficient of its carapace length are modeled as random effects with a trivariate normal distribution. Unlike previously suggested models, the present model permits correlation between the growth coefficient and the age at first capture and can be fitted readily using existing software. The error structures between consecutive recaptures of a lobster are assumed to be a first-order autoregressive process with unequally spaced time points. A comparison between this model and the Fabens growth equation is given. The proposed method is a flexible method and can be applied to fit different growth equations when the age at first capture is unknown.     

Cryo-Cooling Effect on DHFR Crystal Studied by Replica-Exchange Molecular Dynamics Simulations

Cryo-cooling is routinely performed before x-ray diffraction image collection to reduce the damage to crystals due to ionizing radiation. It has been suggested that although backbone structures are usually very similar between room temperature and cryo-temperature, cryo-cooling may hamper biologically relevant dynamics. In this study, the crystal of Escherichia coli dihydrofolate reductase is studied with replica-exchange molecular dynamics simulation, and the results are compared with the crystal structure determined at cryo-temperature and room temperature with the time-averaged ensemble method. Although temperature dependence of unit cell compaction and root mean-square fluctuation of Cα is found in accord with experiment, it is found that the protein structure at low temperature can be more heterogeneous than the ensemble of structures reported by using the time-averaged ensemble method, encouraging further development of the time-averaged ensemble method and indicating that data should be examined carefully to avoid overinterpretation of one average structure.     

A novel approach to spot detection for two-dimensional gel electrophoresis images using pixel value collection

Separation of complex mixtures of proteins by two-dimensional gel electrophoresis (2-DE) is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for the identification of alterations in protein expression within a given biological system. Despite the availability of several commercially available software packages designed for this purpose, image analysis is extremely resource intensive, subjective and remains a major bottleneck. In addition to reducing throughput, the requirement for manual intervention results in the introduction of operator subjectivity, which can limit the statistical significance of the numerical data generated. A key requirement of image analysis is the accurate definition of protein spot boundaries using a suitable method of image segmentation. We describe a method of spot detection applicable to 2-DE image files using a segmentation method involving pixel value collection via serial analysis of the image through its range of density levels. This algorithm is reproducible, sensitive, accurate and primarily designed to be automatic, removing operator subjectivity. Furthermore, it is believed that this method may offer the potential for improved spot detection over currently available software.     

Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes

Rapid development of next generation sequencing (NGS) technologies in recent years has made whole genome sequencing of bacterial genomes widely accessible. However, it is often unnecessary or not feasible to sequence the whole genome for most applications of genetic analyses in bacteria. Selectively capturing defined genomic regions followed by NGS analysis could be a promising approach for high-resolution molecular typing of a large set of strains. In this study, we describe a novel and straightforward PCR-based target-capturing method, hairpin-primed multiplex amplification (HPMA), which allows for simultaneous amplification of numerous target genes. To test the feasibility of NGS-based strain typing using HPMA, 20 target gene sequences were simultaneously amplified with barcode tagging in each of 41 Salmonella strains. The amplicons were then pooled and analyzed by 454 pyrosequencing. Analysis of the sequence data, as an extension of multilocus sequence typing (MLST), demonstrated the utility and potential of this novel typing method, MLST-seq, as a high-resolution strain typing method. With the rapidly increasing sequencing capacity of NGS, MLST-seq or its variations using different target enrichment methods can be expected to become a high-resolution typing method in the near future for high-throughput analysis of a large collection of bacterial strains.     

Kinematic study of root elongation in Arabidopsis thaliana with a novel image-analysis program

The measurement of the spatial profile of root elongation needs to determine matching points between time-lapse images and calculate their displacement. These data have been obtained by laborious manual methods in the past. Some computer-based programs have been developed to improve the measurement, but they require many time-series digital images or sprinkling graphite particles on the root prior to image capture. Here, we have developed GrowthTracer, a new image-analysis program for the kinematic study of root elongation. GrowthTracer employs a multiresolution image matching method with a nonlinear filter called the critical point filter (CPF), which extracts critical points from images at each resolution and can determine precise matching points by analysis of only two intact images, without pre-marking by graphite particles. This program calculates the displacement of each matching point and determines the displacement velocity profile along the medial axis of the root. In addition, a manual input of distinct matching points increases the matching accuracy. We show a successful application of this novel program for the kinematic analysis of root growth in Arabidopsis thaliana.     

Automated acquisition of electron microscopic random conical tilt sets

Single particle reconstruction using the random conical tilt data collection geometry is a robust method for the initial determination of macromolecular structures by electron microscopy. Unfortunately, the broad adoption of this powerful approach has been limited by the practical challenges inherent in manual data collection of the required pairs of matching high and low tilt images (typically 60 degrees and 0 degrees). The microscopist is obliged to keep the imaging area centered during tilting as well as to maintain accurate focus in the tilted image while minimizing the overall electron dose, a challenging and time consuming process. To help solve these problems, we have developed an automated system for the rapid acquisition of accurately aligned and focused tilt pairs. The system has been designed to minimize the dose incurred during alignment and focusing, making it useful in both negative stain and cryo-electron microscopy. The system includes a feature for montaging untilted images to ensure that all of the particles in the tilted image may be used in the reconstruction.     

Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation

In the present work, we demonstrate a method for concurrent collection of EEG/fMRI data. In our setup, EEG data are collected using a high-density 256-channel sensor net. The EEG amplifier itself is contained in a field isolation containment system (FICS), and MRI clock signals are synchronized with EEG data collection for subsequent MR artifact characterization and removal. We demonstrate this method first for resting state data collection. Thereafter, we demonstrate a protocol for EEG/fMRI data recording, while subjects listen to a tape asking them to visualize that their left hand is immersed in a cold-water bath and referred to, here, as the cold glove paradigm. Thermal differentials between each hand are measured throughout EEG/fMRI data collection using an MR compatible temperature sensor that we developed for this purpose. We collect cold glove EEG/fMRI data along with simultaneous differential hand temperature measurements both before and after hypnotic induction. Between pre and post sessions, single modality EEG data are collected during the hypnotic induction and depth assessment process. Our representative results demonstrate that significant changes in the EEG power spectrum can be measured during hypnotic induction, and that hand temperature changes during the cold glove paradigm can be detected rapidly using our MR compatible differential thermometry device.     

A method to separate plant roots from soil and analyze root surface area

Analysis of the effects of soil management practices on crop production requires knowledge of these effects on plant roots. Much time is required to wash plant roots from soil and separate the living plant roots from organic debris and previous years’ roots. We developed a root washer that can accommodate relatively large soil samples for washing. The root washer has a rotary design and will accommodate up to 24 samples (100 mm diam. by 240 mm long) at one time. We used a flat-bed scanner to digitize an image of the roots from each sample and used a grid system with commercially-available image analysis software to analyze each sample for root surface area. Sensitivity analysis and subsequent comparisons of ‘dirty’ samples containing the roots and all the organic debris contained in the sample and ‘clean’ samples where the organic debris was manually removed from each sample showed that up to 15% of the projected image could be coveredwith debris without affecting accuracy and precision of root surface area measurements. Samples containing a large amount of debris may need to be partitioned into more than one scanning tray to allow accurate measurements of the root surface area. Sample processing time was reduced from 20 h, when hand separation of roots from debris was used, to about 0.5 h, when analyzing the image from an uncleaned sample. The method minimizes the need for preprocessing steps such as dying the roots to get better image contrast for image analysis. Some information, such as root length, root diameter classes and root weights, is not obtained when using this technique. Root length measurements, if needed, could be made by hand on the digital images. Root weight measurement would require sample cleaning and the advantage of less processing time per sample with this method would be lost. The significance of the tradeoff between information not obtained using this technique and the ability to process a greater number of samples with the time and personnel resources available must be determined by the individual researcher and research objectives.