High‐throughput two‐dimensional root system phenotyping platform facilitates genetic analysis of root growth and development

High‐throughput phenotyping of root systems requires a combination of specialized techniques and adaptable plant growth, root imaging and software tools. A custom phenotyping platform was designed to capture images of whole root systems, and novel software tools were developed to process and analyse these images. The platform and its components are adaptable to a wide range root phenotyping studies using diverse growth systems (hydroponics, paper pouches, gel and soil) involving several plant species, including, but not limited to, rice, maize, sorghum, tomato and Arabidopsis. The RootReader2D software tool is free and publicly available and was designed with both user‐guided and automated features that increase flexibility and enhance efficiency when measuring root growth traits from specific roots or entire root systems during large‐scale phenotyping studies. To demonstrate the unique capabilities and high‐throughput capacity of this phenotyping platform for studying root systems, genome‐wide association studies on rice (Oryza sativa) and maize (Zea mays) root growth were performed and root traits related to aluminium (Al) tolerance were analysed on the parents of the maize nested association mapping (NAM) population.     

RootAnalyzer: A Cross-Section Image Analysis Tool for Automated Characterization of Root Cells and Tissues

The morphology of plant root anatomical features is a key factor in effective water and nutrient uptake. Existing techniques for phenotyping root anatomical traits are often based on manual or semi-automatic segmentation and annotation of microscopic images of root cross sections. In this article, we propose a fully automated tool, hereinafter referred to as RootAnalyzer, for efficiently extracting and analyzing anatomical traits from root-cross section images. Using a range of image processing techniques such as local thresholding and nearest neighbor identification, RootAnalyzer segments the plant root from the image’s background, classifies and characterizes the cortex, stele, endodermis and epidermis, and subsequently produces statistics about the morphological properties of the root cells and tissues. We use RootAnalyzer to analyze 15 images of wheat plants and one maize plant image and evaluate its performance against manually-obtained ground truth data. The comparison shows that RootAnalyzer can fully characterize most root tissue regions with over 90% accuracy.     

The Athena semi-automated karyotyping system

In this article we describe Athena, a system that provides for semi-automated karyotyping of metaphase spreads. The system is based upon the Macintosh II computer. It uses software that is written entirely in C and consists of approximately 200 Kbytes of executable code. Athena provides automated segmentation of metaphase images into individual chromosomes, automated measurements on each banded chromosome, and automated classification into the standard Paris-convention karyotype. Furthermore, the system provides the ability to construct one or more chromosome data bases to represent the types of metaphase spreads and staining techniques that may be used in a given laboratory. Because we believe that it is impossible to construct a system that can achieve perfect segmentation, perfect separation of touching and overlapping chromosomes, perfect localization of the centromeres, and perfect classification, the system offers the possibility for interaction at each of the above stages using the well-accepted Macintosh user interface.     

ARADEEPOPSIS,an Automated Workflow for Top-View Plant Phenomics using Semantic Segmentation of Leaf States

Linking plant phenotype to genotype is a common goal to both plant breeders and geneticists. However, collecting phenotypic data for large numbers of plants remain a bottleneck. Plant phenotyping is mostly image based and therefore requires rapid and robust extraction of phenotypic measurements from image data. However, because segmentation tools usually rely on color information, they are sensitive to background or plant color deviations. We have developed a versatile, fully open-source pipeline to extract phenotypic measurements from plant images in an unsupervised manner. ARADEEPOPSIS (https://github.com/Gregor-Mendel-Institute/aradeepopsis) uses semantic segmentation of top-view images to classify leaf tissue into three categories: healthy, anthocyanin rich, and senescent. This makes it particularly powerful at quantitative phenotyping of different developmental stages, mutants with aberrant leaf color and/or phenotype, and plants growing in stressful conditions. On a panel of 210 natural Arabidopsis (Arabidopsis thaliana) accessions, we were able to not only accurately segment images of phenotypically diverse genotypes but also to identify known loci related to anthocyanin production and early necrosis in genome-wide association analyses. Our pipeline accurately processed images of diverse origin, quality, and background composition, and of a distantly related Brassicaceae. ARADEEPOPSIS is deployable on most operating systems and high-performance computing environments and can be used independently of bioinformatics expertise and resources.     

Phenotiki: an open software and hardware platform for affordable and easy image‐based phenotyping of rosette‐shaped plants

Phenotyping is important to understand plant biology, but current solutions are costly, not versatile or are difficult to deploy. To solve this problem, we present Phenotiki, an affordable system for plant phenotyping that, relying on off‐the‐shelf parts, provides an easy to install and maintain platform, offering an out‐of‐box experience for a well‐established phenotyping need: imaging rosette‐shaped plants. The accompanying software (with available source code) processes data originating from our device seamlessly and automatically. Our software relies on machine learning to devise robust algorithms, and includes an automated leaf count obtained from 2D images without the need of depth (3D). Our affordable device (~€200) can be deployed in growth chambers or greenhouse to acquire optical 2D images of approximately up to 60 adult Arabidopsis rosettes concurrently. Data from the device are processed remotely on a workstation or via a cloud application (based on CyVerse). In this paper, we present a proof‐of‐concept validation experiment on top‐view images of 24 Arabidopsis plants in a combination of genotypes that has not been compared previously. Phenotypic analysis with respect to morphology, growth, color and leaf count has not been performed comprehensively before now. We confirm the findings of others on some of the extracted traits, showing that we can phenotype at reduced cost. We also perform extensive validations with external measurements and with higher fidelity equipment, and find no loss in statistical accuracy when we use the affordable setting that we propose. Device set‐up instructions and analysis software are publicly available ( http://phenotiki.com ).     

A growth phenotyping pipeline for Arabidopsis thaliana integrating image analysis and rosette area modeling for robust quantification of genotype effects

? To gain a deeper understanding of the mechanisms behind biomass accumulation, it is important to study plant growth behavior. Manually phenotyping large sets of plants requires important human resources and expertise and is typically not feasible for detection of weak growth phenotypes. Here, we established an automated growth phenotyping pipeline for Arabidopsis thaliana to aid researchers in comparing growth behaviors of different genotypes. ? The analysis pipeline includes automated image analysis of two-dimensional digital plant images and evaluation of manually annotated information of growth stages. It employs linear mixed-effects models to quantify genotype effects on total rosette area and relative leaf growth rate (RLGR) and ANOVAs to quantify effects on developmental times. ? Using the system, a single researcher can phenotype up to 7000 plants d?1. Technical variance is very low (typically < 2%). We show quantitative results for the growth-impaired starch-excess mutant sex4-3 and the growth-enhanced mutant grf9. ? We show that recordings of environmental and developmental variables reduce noise levels in the phenotyping datasets significantly and that careful examination of predictor variables (such as d after sowing or germination) is crucial to avoid exaggerations of recorded phenotypes and thus biased conclusions.     

Hyperspectral imaging combined with machine learning as a tool to obtain high‐throughput plant salt‐stress phenotyping

The rapid selection of salinity‐tolerant crops to increase food production in salinized lands is important for sustainable agriculture. Recently, high‐throughput plant phenotyping technologies have been adopted that use plant morphological and physiological measurements in a non‐destructive manner to accelerate plant breeding processes. Here, a hyperspectral imaging (HSI) technique was implemented to monitor the plant phenotypes of 13 okra (Abelmoschus esculentus L.) genotypes after 2 and 7 days of salt treatment. Physiological and biochemical traits, such as fresh weight, SPAD, elemental contents and photosynthesis‐related parameters, which require laborious, time‐consuming measurements, were also investigated. Traditional laboratory‐based methods indicated the diverse performance levels of different okra genotypes in response to salinity stress. We introduced improved plant and leaf segmentation approaches to RGB images extracted from HSI imaging based on deep learning. The state‐of‐the‐art performance of the deep‐learning approach for segmentation resulted in an intersection over union score of 0.94 for plant segmentation and a symmetric best dice score of 85.4 for leaf segmentation. Moreover, deleterious effects of salinity affected the physiological and biochemical processes of okra, which resulted in substantial changes in the spectral information. Four sample predictions were constructed based on the spectral data, with correlation coefficients of 0.835, 0.704, 0.609 and 0.588 for SPAD, sodium concentration, photosynthetic rate and transpiration rate, respectively. The results confirmed the usefulness of high‐throughput phenotyping for studying plant salinity stress using a combination of HSI and deep‐learning approaches.     

A direction‐selective local‐thresholding method,DSLT, in combination with a dye‐based method for automated three‐dimensional segmentation of cells and airspaces in developing leaves

Quantifying the anatomical data acquired from three‐dimensional (3D) images has become increasingly important in recent years. Visualization and image segmentation are essential for acquiring accurate and detailed anatomical data from images; however, plant tissues such as leaves are difficult to image by confocal or multi‐photon laser scanning microscopy because their airspaces generate optical aberrations. To overcome this problem, we established a staining method based on Nile Red in silicone‐oil solution. Our staining method enables color differentiation between lipid bilayer membranes and airspaces, while minimizing any damage to leaf development. By repeated applications of our staining method we performed time‐lapse imaging of a leaf over 5 days. To counteract the drastic decline in signal‐to‐noise ratio at greater tissue depths, we also developed a local thresholding method (direction‐selective local thresholding, DSLT) and an automated iterative segmentation algorithm. The segmentation algorithm uses the DSLT to extract the anatomical structures. Using the proposed methods, we accurately segmented 3D images of intact leaves to single‐cell resolution, and measured the airspace volumes in intact leaves.     

RooTrak: automated recovery of three-dimensional plant root architecture in soil from x-ray microcomputed tomography images using visual tracking

X-ray microcomputed tomography (μCT) is an invaluable tool for visualizing plant root systems within their natural soil environment noninvasively. However, variations in the x-ray attenuation values of root material and the overlap in attenuation values between roots and soil caused by water and organic materials represent major challenges to data recovery. We report the development of automatic root segmentation methods and software that view μCT data as a sequence of images through which root objects appear to move as the x-y cross sections are traversed along the z axis of the image stack. Previous approaches have employed significant levels of user interaction and/or fixed criteria to distinguish root and nonroot material. RooTrak exploits multiple, local models of root appearance, each built while tracking a specific segment, to identify new root material. It requires minimal user interaction and is able to adapt to changing root density estimates. The model-guided search for root material arising from the adoption of a visual-tracking framework makes RooTrak less sensitive to the natural ambiguity of x-ray attenuation data. We demonstrate the utility of RooTrak using μCT scans of maize (Zea mays), wheat (Triticum aestivum), and tomato (Solanum lycopersicum) grown in a range of contrasting soil textures. Our results demonstrate that RooTrak can successfully extract a range of root architectures from the surrounding soil and promises to facilitate future root phenotyping efforts.     

Black Spot: a platform for automated and rapid estimation of leaf area from scanned images

Leaf area and its derivatives (e.g. specific leaf area) are widely used in ecological assessments, especially in the fields of plant–animal interactions, plant community assembly, ecosystem functioning and global change. Estimating leaf area is highly time-consuming, even when using specialized software to process scanned leaf images, because manual inputs are invariably required for scale detection and leaf surface digitisation. We introduce Black Spot Leaf Area Calculator (hereafter, Black Spot), a technique and stand-alone software package for rapid and automated leaf area assessment from images of leaves taken with standard flatbed scanners. Black Spot operates on comprehensive rule-sets for colour band ratios to carry out pixel-based classification which isolates leaf surfaces from the image background. Importantly, the software extracts information from associated image meta-data to detect image scale, thereby eliminating the need for time-consuming manual scale calibration. Black Spot’s output provides the user with estimates of leaf area as well as classified images for error checking. We tested this method and software combination on a set of 100 leaves of 51 different plant species collected from the field. Leaf area estimates generated using Black Spot and by manual processing of the images using an image editing software generated statistically identical results. Mean error rate in leaf area estimates from Black Spot relative to manual processing was ?0.4 % (SD = 0.76). The key advantage of Black Spot is the ability to rapidly batch process multi-species datasets with minimal user effort and at low cost, thus making it a valuable tool for field ecologists.     

Enabling user-guided segmentation and tracking of surface-labeled cells in time-lapse image sets of living tissues

To study the process of morphogenesis, one often needs to collect and segment time-lapse images of living tissues to accurately track changing cellular morphology. This task typically involves segmenting and tracking tens to hundreds of individual cells over hundreds of image frames, a scale that would certainly benefit from automated routines; however, any automated routine would need to reliably handle a large number of sporadic, and yet typical problems (e.g., illumination inconsistency, photobleaching, rapid cell motions, and drift of focus or of cells moving through the imaging plane). Here, we present a segmentation and cell tracking approach based on the premise that users know their data best-interpreting and using image features that are not accounted for in any a priori algorithm design. We have developed a program, SeedWater Segmenter, that combines a parameter-less and fast automated watershed algorithm with a suite of manual intervention tools that enables users with little to no specialized knowledge of image processing to efficiently segment images with near-perfect accuracy based on simple user interactions.     

Minimizing Manual Image Segmentation Turn-Around Time for Neuronal Reconstruction by Embracing Uncertainty

The ability to automatically segment an image into distinct regions is a critical aspect in many visual processing applications. Because inaccuracies often exist in automatic segmentation, manual segmentation is necessary in some application domains to correct mistakes, such as required in the reconstruction of neuronal processes from microscopic images. The goal of the automated segmentation tool is traditionally to produce the highest-quality segmentation, where quality is measured by the similarity to actual ground truth, so as to minimize the volume of manual correction necessary. Manual correction is generally orders-of-magnitude more time consuming than automated segmentation, often making handling large images intractable. Therefore, we propose a more relevant goal: minimizing the turn-around time of automated/manual segmentation while attaining a level of similarity with ground truth. It is not always necessary to inspect every aspect of an image to generate a useful segmentation. As such, we propose a strategy to guide manual segmentation to the most uncertain parts of segmentation. Our contributions include 1) a probabilistic measure that evaluates segmentation without ground truth and 2) a methodology that leverages these probabilistic measures to significantly reduce manual correction while maintaining segmentation quality.     

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).     

基于图像识别技术的金针菇表型高通量采集与分析

金针菇Flammulina filiformis作为国内产销量大、工厂化生产程度高的食用菌种类之一,其育种工作因表型信息采集效率低下而受到限制.本研究以金针菇子实体可见光图像为信息来源,基于育种工作对表型信息的需求,利用图像识别技术和深度学习模型,提出了金针菇高通量表型信息采集分析方法,并开发了相应分析系统软件.利用该...     

Oufti: an integrated software package for high‐accuracy,high‐throughput quantitative microscopy analysis

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills.     

Identification and utilization of genetic determinants of trait measurement errors in image-based,high-throughput phenotyping

The accuracy of trait measurements greatly affects the quality of genetic analyses. During automated phenotyping, trait measurement errors, i.e. differences between automatically extracted trait values and ground truth, are often treated as random effects that can be controlled by increasing population sizes and/or replication number. In contrast, there is some evidence that trait measurement errors may be partially under genetic control. Consistent with this hypothesis, we observed substantial nonrandom, genetic contributions to trait measurement errors for five maize (Zea mays) tassel traits collected using an image-based phenotyping platform. The phenotyping accuracy varied according to whether a tassel exhibited “open” versus. “closed” branching architecture, which is itself under genetic control. Trait-associated SNPs (TASs) identified via genome-wide association studies (GWASs) conducted on five tassel traits that had been phenotyped both manually (i.e. ground truth) and via feature extraction from images exhibit little overlap. Furthermore, identification of TASs from GWASs conducted on the differences between the two values indicated that a fraction of measurement error is under genetic control. Similar results were obtained in a sorghum (Sorghum bicolor) plant height dataset, demonstrating that trait measurement error is genetically determined in multiple species and traits. Trait measurement bias cannot be controlled by increasing population size and/or replication number.

The accuracy of high-throughput phenotyping can be affected by genetically determined measurement biases, which can alter the results of genetic analyses.     

Mathematical morphologic segmentation dedicated to quantitative immunohistochemistry

OBJECTIVE: To develop automatic segmentation sequences for fully automated quantitative immunohistochemistry of cancer cell nuclei by image analysis. STUDY DESIGN: The study focused on the automated delineation of cancer cell lobules and nuclei, taking breast carcinoma as an example. A hierarchic segmentation was developed, employing mainly the chaining of mathematical morphology operators. The proposed sequence was tested on 22 images of various situations, collected from 18 different cases of breast carcinoma. A quality control procedure was applied, comparing the automated method with manual outlining of cancer cell foci and with manual pricking of cancer cell nuclei. RESULTS: Good concordance was found between automated and manual segmentation procedures (90% for cancer cell clumps, 97% for cancer cell nuclei on average), but the rate of false positive nuclei (small regions labeled as nuclei by the segmentation procedure) could be relatively high (11% on average, with a maximum of 35%) and can result in underestimation of the immunostaining ratio. CONCLUSION: This study examined a preliminary approach to automated immunoquantification, limited to automated segmentation without any color characterization. The automated hierarchic segmentation presented here leads to good discrimination of cancer cell nuclei at the chosen magnification.