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.

In-Droplet Electromechanical Cell Lysis and Enhanced Enzymatic Assay Driven by Ion Concentration Polarization.

Droplets enable the encapsulation of cells for their analysis in isolated domains. The study of molecular signatures (including genes, proteins, and metabolites) from a few or single cells is critical for identifying key subpopulations. However, dealing with biological analytes at low concentrations requires long incubation times and amplification to achieve the requisite signal strength. Further, cell lysis requires additional chemical lysing agents or heat, which can interfere with assays. Here, we leverage ion concentration polarization (ICP) in droplets to rapidly lyse breast cancer cells within 2 s under a DC voltage bias of 30 V. Numerical simulations attribute cell lysis to an ICP-based electric field and shear stress. We further achieve up to 19-fold concentration enrichment of an enzymatic assay product resulting from cell lysis and a 3.8-fold increase in the reaction rate during enrichment. Our technique for sensitive in-droplet cell analysis provides scope for rapid, high-throughput detection of low-abundance intracellular analytes.

Thinner biological tissues induce leaflet flutter in aortic heart valve replacements.

Valvular heart disease has recently become an increasing public health concern due to the high prevalence of valve degeneration in aging populations. For patients with severely impacted aortic valves that require replacement, catheter-based bioprosthetic valve deployment offers a minimally invasive treatment option that eliminates many of the risks associated with surgical valve replacement. Although recent percutaneous device advancements have incorporated thinner, more flexible biological tissues to streamline safer deployment through catheters, the impact of such tissues in the complex, mechanically demanding, and highly dynamic valvular system remains poorly understood. The present work utilized a validated computational fluid-structure interaction approach to isolate the behavior of thinner, more compliant aortic valve tissues in a physiologically realistic system. This computational study identified and quantified significant leaflet flutter induced by the use of thinner tissues that initiated blood flow disturbances and oscillatory leaflet strains. The aortic flow and valvular dynamics associated with these thinner valvular tissues have not been previously identified and provide essential information that can significantly advance fundamental knowledge about the cardiac system and support future medical device innovation. Considering the risks associated with such observed flutter phenomena, including blood damage and accelerated leaflet deterioration, this study demonstrates the potentially serious impact of introducing thinner, more flexible tissues into the cardiac system.

Hydrogel-based transparent soils for root phenotyping in vivo.

Root phenotypes are increasingly explored as predictors of crop performance but are still challenging to characterize. Media that mimic field conditions (e.g., soil, sand) are opaque to most forms of radiation, while transparent media do not provide field-relevant growing conditions and phenotypes. We describe here a "transparent soil" formed by the spherification of hydrogels of biopolymers. It is specifically designed to support root growth in the presence of air, water, and nutrients, and allows the time-resolved phenotyping of roots in vivo by both photography and microscopy. The roots developed by soybean plants in this medium are significantly more similar to those developed in real soil than those developed in hydroponic conditions and do not show signs of hypoxia. Lastly, we show that the granular nature and tunable properties of these hydrogel beads can be leveraged to investigate the response of roots to gradients in water availability and soil stiffness.

Shared Genetic Control of Root System Architecture between Zea mays and Sorghum bicolor.

Determining the genetic control of root system architecture (RSA) in plants via large-scale genome-wide association study (GWAS) requires high-throughput pipelines for root phenotyping. We developed Core Root Excavation using Compressed-air (CREAMD), a high-throughput pipeline for the cleaning of field-grown roots, and Core Root Feature Extraction (COFE), a semiautomated pipeline for the extraction of RSA traits from images. CREAMD-COFE was applied to diversity panels of maize (Zea mays) and sorghum (Sorghum bicolor), which consisted of 369 and 294 genotypes, respectively. Six RSA-traits were extracted from images collected from >3,300 maize roots and >1,470 sorghum roots. Single nucleotide polymorphism (SNP)-based GWAS identified 87 TAS (trait-associated SNPs) in maize, representing 77 genes and 115 TAS in sorghum. An additional 62 RSA-associated maize genes were identified via expression read depth GWAS. Among the 139 maize RSA-associated genes (or their homologs), 22 (16%) are known to affect RSA in maize or other species. In addition, 26 RSA-associated genes are coregulated with genes previously shown to affect RSA and 51 (37% of RSA-associated genes) are themselves transe-quantitative trait locus for another RSA-associated gene. Finally, the finding that RSA-associated genes from maize and sorghum included seven pairs of syntenic genes demonstrates the conservation of regulation of morphology across taxa.

An explainable deep machine vision framework for plant stress phenotyping.

Current approaches for accurate identification, classification, and quantification of biotic and abiotic stresses in crop research and production are predominantly visual and require specialized training. However, such techniques are hindered by subjectivity resulting from inter- and intrarater cognitive variability. This translates to erroneous decisions and a significant waste of resources. Here, we demonstrate a machine learning framework's ability to identify and classify a diverse set of foliar stresses in soybean [Glycine max (L.) Merr.] with remarkable accuracy. We also present an explanation mechanism, using the top-K high-resolution feature maps that isolate the visual symptoms used to make predictions. This unsupervised identification of visual symptoms provides a quantitative measure of stress severity, allowing for identification (type of foliar stress), classification (low, medium, or high stress), and quantification (stress severity) in a single framework without detailed symptom annotation by experts. We reliably identified and classified several biotic (bacterial and fungal diseases) and abiotic (chemical injury and nutrient deficiency) stresses by learning from over 25,000 images. The learned model is robust to input image perturbations, demonstrating viability for high-throughput deployment. We also noticed that the learned model appears to be agnostic to species, seemingly demonstrating an ability of transfer learning. The availability of an explainable model that can consistently, rapidly, and accurately identify and quantify foliar stresses would have significant implications in scientific research, plant breeding, and crop production. The trained model could be deployed in mobile platforms (e.g., unmanned air vehicles and automated ground scouts) for rapid, large-scale scouting or as a mobile application for real-time detection of stress by farmers and researchers.

Concentration Enrichment, Separation, and Cation Exchange in Nanoliter-Scale Water-in-Oil Droplets.

Droplet-based techniques have had a profound impact in chemistry, owing to their ability to perform rapid and massively parallel reactions in minute fluid volumes. In many applications, concentration enrichment is required to increase the speed of reactions or the sensitivity of assays; but in-droplet concentration enrichment remains challenging. Here, we interface electrokinetic concentration polarization with droplet microfluidics to accomplish in-droplet demixing. This result is significant because the concentration of any charged species in the droplet can be enriched and the approach can be readily integrated into existing droplet workflows. Further, we show that such electrokinetic enrichment is rapid, on the order of seconds, and is robust, occurring over a wide parametric space. We further demonstrate electrokinetic separation of two anionic fluorophores within the droplet. Such a capability potentiates the droplet-templated synthesis of particles with gradient composition and the development of mobility-shift assays, which rely on discrimination of multiple species tagged with a single color fluorophore. Finally, by using a calcium-binding dye as an indicator, we demonstrate in-droplet cation exchange. This demonstration of cation exchange in droplets is significant because of its broad applicability to strategies for synthesis and bioassays. These results lay the foundation for new advanced droplet techniques with transformative applications.

Semiautomated Feature Extraction from RGB Images for Sorghum Panicle Architecture GWAS.

Because structural variation in the inflorescence architecture of cereal crops can influence yield, it is of interest to identify the genes responsible for this variation. However, the manual collection of inflorescence phenotypes can be time consuming for the large populations needed to conduct genome-wide association studies (GWAS) and is difficult for multidimensional traits such as volume. A semiautomated phenotyping pipeline, TIM (Toolkit for Inflorescence Measurement), was developed and used to extract unidimensional and multidimensional features from images of 1,064 sorghum (Sorghum bicolor) panicles from 272 genotypes comprising a subset of the Sorghum Association Panel. GWAS detected 35 unique single-nucleotide polymorphisms associated with variation in inflorescence architecture. The accuracy of the TIM pipeline is supported by the fact that several of these trait-associated single-nucleotide polymorphisms (TASs) are located within chromosomal regions associated with similar traits in previously published quantitative trait locus and GWAS analyses of sorghum. Additionally, sorghum homologs of maize (Zea mays) and rice (Oryza sativa) genes known to affect inflorescence architecture are enriched in the vicinities of TASs. Finally, our TASs are enriched within genomic regions that exhibit high levels of divergence between converted tropical lines and cultivars, consistent with the hypothesis that these chromosomal intervals were targets of selection during modern breeding.

Determination of the Free Energies of Mixing of Organic Solutions through a Combined Molecular Dynamics and Bayesian Statistics Approach.

As new generations of thin-film semiconductors are moving toward solution-based processing, the development of printing formulations will require information pertaining to the free energies of mixing of complex mixtures. From the standpoint of in silico material design, this move necessitates the development of methods that can accurately and quickly evaluate these formulations in order to maximize processing speed and reproducibility. Here, we make use of molecular dynamics (MD) simulations, in combination with the two-phase thermodynamic (2PT) model, to explore the free energy of mixing surfaces for a series of halogenated solvents and high-boiling point solvent additives used in the development of thin-film organic semiconductors. Although the combined methods generally show good agreement with available experimental data, the computational cost to traverse the free-energy landscape is considerable. Hence, we demonstrate how a Bayesian optimization scheme, coupled with the MD and 2PT approaches, can drastically reduce the number of simulations required, in turn shrinking both the computational cost and time.

Crowdsourcing image analysis for plant phenomics to generate ground truth data for machine learning.

The accuracy of machine learning tasks critically depends on high quality ground truth data. Therefore, in many cases, producing good ground truth data typically involves trained professionals; however, this can be costly in time, effort, and money. Here we explore the use of crowdsourcing to generate a large number of training data of good quality. We explore an image analysis task involving the segmentation of corn tassels from images taken in a field setting. We investigate the accuracy, speed and other quality metrics when this task is performed by students for academic credit, Amazon MTurk workers, and Master Amazon MTurk workers. We conclude that the Amazon MTurk and Master Mturk workers perform significantly better than the for-credit students, but with no significant difference between the two MTurk worker types. Furthermore, the quality of the segmentation produced by Amazon MTurk workers rivals that of an expert worker. We provide best practices to assess the quality of ground truth data, and to compare data quality produced by different sources. We conclude that properly managed crowdsourcing can be used to establish large volumes of viable ground truth data at a low cost and high quality, especially in the context of high throughput plant phenotyping. We also provide several metrics for assessing the quality of the generated datasets.

A methodology for optimal placement of sensors in enclosed environments: A dynamical systems approach.

Air quality has been an important issue in public health for many years. Sensing the level and distributions of impurities help in the control of building systems and mitigate long term health risks. Rapid detection of infectious diseases in large public areas like airports and train stations may help limit exposure and aid in reducing the spread of the disease. Complete coverage by sensors to account for any release scenario of chemical or biological warfare agents may provide the opportunity to develop isolation and evacuation plans that mitigate the impact of the attack. All these scenarios involve strategic placement of sensors to promptly detect and rapidly respond. This paper presents a data driven sensor placement algorithm based on a dynamical systems approach. The approach utilizes the finite dimensional Perron-Frobenius (PF) concept. The PF operator (or the Markov matrix) is used to construct an observability gramian that naturally incorporates sensor accuracy, location constraints, and sensing constraints. The algorithm determines the response times, sensor coverage maps, and the number of sensors needed. The utility of the procedure is illustrated using four examples: a literature example of the flow field inside an aircraft cabin and three air flow fields in different geometries. The effect of the constraints on the response times for different sensor placement scenarios is investigated. Knowledge of the response time and coverage of the multiple sensors aides in the design of mechanical systems and response mechanisms. The methodology provides a simple process for place sensors in a building, analyze the sensor coverage maps and response time necessary during extreme events, as well as evaluate indoor air quality. The theory established in this paper also allows for future work in topics related to construction of classical estimator problems for the sensors, real-time contaminant transport, and development of agent dispersion, contaminant isolation/removal, and evacuation strategies.

Genome-wide association analysis of seedling root development in maize (Zea mays L.).

BACKGROUND: Plants rely on the root system for anchorage to the ground and the acquisition and absorption of nutrients critical to sustaining productivity. A genome wide association analysis enables one to analyze allelic diversity of complex traits and identify superior alleles. 384 inbred lines from the Ames panel were genotyped with 681,257 single nucleotide polymorphism markers using Genotyping-by-Sequencing technology and 22 seedling root architecture traits were phenotyped. RESULTS: Utilizing both a general linear model and mixed linear model, a GWAS study was conducted identifying 268 marker trait associations (p ≤ 5.3×10(-7)). Analysis of significant SNP markers for multiple traits showed that several were located within gene models with some SNP markers localized within regions of previously identified root quantitative trait loci. Gene model GRMZM2G153722 located on chromosome 4 contained nine significant markers. This predicted gene is expressed in roots and shoots. CONCLUSION: This study identifies putatively associated SNP markers associated with root traits at the seedling stage. Some SNPs were located within or near (<1 kb) gene models. These gene models identify possible candidate genes involved in root development at the seedling stage. These and respective linked or functional markers could be targets for breeders for marker assisted selection of seedling root traits.

Constructing Markov matrices for real-time transient contaminant transport analysis for indoor environments.

Predicting the movement of contaminants in the indoor environment has applications in tracking airborne infectious disease, ventilation of gaseous contaminants, and the isolation of spaces during biological attacks. Markov matrices provide a convenient way to perform contaminant transport analysis. However, no standardized method exists for calculating these matrices. A methodology based on set theory is developed for calculating contaminant transport in real-time utilizing Markov matrices from CFD flow data (or discrete flow field data). The methodology provides a rigorous yet simple strategy for determining the number and size of the Markov states, the time step associated with the Markov matrix, and calculation of individual entries of the Markov matrix. The procedure is benchmarked against scalar transport of validated airflow fields in enclosed and ventilated spaces. The approach can be applied to any general airflow field, and is shown to calculate contaminant transport over 3000 times faster than solving the corresponding scalar transport partial differential equation. This near real-time methodology allows for the development of more robust sensing and control procedures of critical care environments (clean rooms and hospital wards), small enclosed spaces (like airplane cabins) and high traffic public areas (train stations and airports).

Rapid Estimation of the Intermolecular Electronic Couplings and Charge-Carrier Mobilities of Crystalline Molecular Organic Semiconductors through a Machine Learning Pipeline.

Organic semiconductors (OSC) offer tremendous potential across a wide range of (opto)electronic applications. OSC development, however, is often limited by trial-and-error design, with computational modeling approaches deployed to evaluate and screen candidates through a suite of molecular and materials descriptors that generally require hours to days of computational time to accumulate. Such bottlenecks slow the pace and limit the exploration of the vast chemical space comprising OSC. When considering charge-carrier transport in OSC, a key parameter of interest is the intermolecular electronic coupling. Here, we introduce a machine learning (ML) model to predict intermolecular electronic couplings in organic crystalline materials from their three-dimensional (3D) molecular geometries. The ML predictions take only a few seconds of computing time compared to hours by density functional theory (DFT) methods. To demonstrate the utility of the ML predictions, we deploy the ML model in conjunction with mathematical formulations to rapidly screen the charge-carrier mobility anisotropy for more than 60,000 molecular crystal structures and compare the ML predictions to DFT benchmarks.

Out-of-Distribution Detection Algorithms for Robust Insect Classification.

Plants encounter a variety of beneficial and harmful insects during their growth cycle. Accurate identification (i.e., detecting insects' presence) and classification (i.e., determining the type or class) of these insect species is critical for implementing prompt and suitable mitigation strategies. Such timely actions carry substantial economic and environmental implications. Deep learning-based approaches have produced models with good insect classification accuracy. Researchers aim to implement identification and classification models in agriculture, facing challenges when input images markedly deviate from the training distribution (e.g., images like vehicles, humans, or a blurred image or insect class that is not yet trained on). Out-of-distribution (OOD) detection algorithms provide an exciting avenue to overcome these challenges as they ensure that a model abstains from making incorrect classification predictions on images that belong to non-insect and/or untrained insect classes. As far as we know, no prior in-depth exploration has been conducted on the role of the OOD detection algorithms in addressing agricultural issues. Here, we generate and evaluate the performance of state-of-the-art OOD algorithms on insect detection classifiers. These algorithms represent a diversity of methods for addressing an OOD problem. Specifically, we focus on extrusive algorithms, i.e., algorithms that wrap around a well-trained classifier without the need for additional co-training. We compared three OOD detection algorithms: (a) maximum softmax probability, which uses the softmax value as a confidence score; (b) Mahalanobis distance (MAH)-based algorithm, which uses a generative classification approach; and (c) energy-based algorithm, which maps the input data to a scalar value, called energy. We performed an extensive series of evaluations of these OOD algorithms across three performance axes: (a) Base model accuracy: How does the accuracy of the classifier impact OOD performance? (b) How does the level of dissimilarity to the domain impact OOD performance? (c) Data imbalance: How sensitive is OOD performance to the imbalance in per-class sample size? Evaluating OOD algorithms across these performance axes provides practical guidelines to ensure the robust performance of well-trained models in the wild, which is a key consideration for agricultural applications. Based on this analysis, we proposed the most effective OOD algorithm as wrapper for the insect classifier with highest accuracy. We presented the results of its OOD detection performance in the paper. Our results indicate that OOD detection algorithms can significantly enhance user trust in insect pest classification by abstaining classification under uncertain conditions.

Cyber-agricultural systems for crop breeding and sustainable production.

The cyber-agricultural system (CAS) represents an overarching framework of agriculture that leverages recent advances in ubiquitous sensing, artificial intelligence, smart actuators, and scalable cyberinfrastructure (CI) in both breeding and production agriculture. We discuss the recent progress and perspective of the three fundamental components of CAS - sensing, modeling, and actuation - and the emerging concept of agricultural digital twins (DTs). We also discuss how scalable CI is becoming a key enabler of smart agriculture. In this review we shed light on the significance of CAS in revolutionizing crop breeding and production by enhancing efficiency, productivity, sustainability, and resilience to changing climate. Finally, we identify underexplored and promising future directions for CAS research and development.

Optimization framework for patient-specific modeling under uncertainty.

Estimating a patient-specific computational model's parameters relies on data that is often unreliable and ill-suited for a deterministic approach. We develop an optimization-based uncertainty quantification framework for probabilistic model tuning that discovers model inputs distributions that generate target output distributions. Probabilistic sampling is performed using a surrogate model for computational efficiency, and a general distribution parameterization is used to describe each input. The approach is tested on seven patient-specific modeling examples using CircAdapt, a cardiovascular circulatory model. Six examples are synthetic, aiming to match the output distributions generated using known reference input data distributions, while the seventh example uses real-world patient data for the output distributions. Our results demonstrate the accurate reproduction of the target output distributions, with a correct recreation of the reference inputs for the six synthetic examples. Our proposed approach is suitable for determining the parameter distributions of patient-specific models with uncertain data and can be used to gain insights into the sensitivity of the model parameters to the measured data.

Self-supervised maize kernel classification and segmentation for embryo identification.

Introduction: Computer vision and deep learning (DL) techniques have succeeded in a wide range of diverse fields. Recently, these techniques have been successfully deployed in plant science applications to address food security, productivity, and environmental sustainability problems for a growing global population. However, training these DL models often necessitates the large-scale manual annotation of data which frequently becomes a tedious and time-and-resource- intensive process. Recent advances in self-supervised learning (SSL) methods have proven instrumental in overcoming these obstacles, using purely unlabeled datasets to pre-train DL models. Methods: Here, we implement the popular self-supervised contrastive learning methods of NNCLR Nearest neighbor Contrastive Learning of visual Representations) and SimCLR (Simple framework for Contrastive Learning of visual Representations) for the classification of spatial orientation and segmentation of embryos of maize kernels. Maize kernels are imaged using a commercial high-throughput imaging system. This image data is often used in multiple downstream applications across both production and breeding applications, for instance, sorting for oil content based on segmenting and quantifying the scutellum's size and for classifying haploid and diploid kernels. Results and discussion: We show that in both classification and segmentation problems, SSL techniques outperform their purely supervised transfer learning-based counterparts and are significantly more annotation efficient. Additionally, we show that a single SSL pre-trained model can be efficiently finetuned for both classification and segmentation, indicating good transferability across multiple downstream applications. Segmentation models with SSL-pretrained backbones produce DICE similarity coefficients of 0.81, higher than the 0.78 and 0.73 of those with ImageNet-pretrained and randomly initialized backbones, respectively. We observe that finetuning classification and segmentation models on as little as 1% annotation produces competitive results. These results show SSL provides a meaningful step forward in data efficiency with agricultural deep learning and computer vision.

Electrokinetic Enrichment and Label-Free Electrochemical Detection of Nucleic Acids by Conduction of Ions along the Surface of Bioconjugated Beads.

In this paper, we report a method to integrate the electrokinetic pre-enrichment of nucleic acids within a bed of probe-modified microbeads with their label-free electrochemical detection. In this detection scheme, hybridization of locally enriched target nucleic acids to the beads modulates the conduction of ions along the bead surfaces. This is a fundamental advancement in that this mechanism is similar to that observed in nanopore sensors, yet occurs in a bed of microbeads with microscale interstices. In application, this approach has several distinct advantages. First, electrokinetic enrichment requires only a simple DC power supply, and in combination with nonoptical detection, it makes this method amenable to point-of-care applications. Second, the sensor is easy to fabricate and comprises a packed bed of commercially available microbeads, which can be readily modified with a wide range of probe types, thereby making this a versatile platform. Finally, the sensor is highly sensitive (picomolar) despite the modest 100-fold pre-enrichment we employ here by faradaic ion concentration polarization (fICP). Further gains are anticipated under conditions for fICP focusing that are known to yield higher enrichment factors (up to 100,000-fold enrichment). Here, we demonstrate the detection of 3.7 pM single-stranded DNA complementary to the bead-bound oligoprobe, following a 30 min single step of enrichment and hybridization. Our results indicate that a shift in the slope of a current-voltage curve occurs upon hybridization and that this shift is proportional to the logarithm of the concentration of target DNA. Finally, we investigate the proposed mechanism of sensing by developing a numerical simulation that shows an increase in ion flux through the bed of insulating beads, given the changes in surface charge and zeta potential, consistent with our experimental conditions.

A computational framework for transmission risk assessment of aerosolized particles in classrooms.

Infectious airborne diseases like the recent COVID-19 pandemic render confined spaces high-risk areas. However, in-person activities like teaching in classroom settings and government services are often expected to continue or restart quickly. It becomes important to evaluate the risk of airborne disease transmission while accounting for the physical presence of humans, furniture, and electronic equipment, as well as ventilation. Here, we present a computational framework and study based on detailed flow physics simulations that allow straightforward evaluation of various seating and operating scenarios to identify risk factors and assess the effectiveness of various mitigation strategies. These scenarios include seating arrangement changes, presence/absence of computer screens, ventilation rate changes, and presence/absence of mask-wearing. This approach democratizes risk assessment by automating a key bottleneck in simulation-based analysis-creating an adequately refined mesh around multiple complex geometries. Not surprisingly, we find that wearing masks (with at least 74% inward protection efficiency) significantly reduced transmission risk against unmasked and infected individuals. While the use of face masks is known to reduce the risk of transmission, we perform a systematic computational study of the transmission risk due to variations in room occupancy, seating layout and air change rates. In addition, our findings on the efficacy of face masks further support use of face masks. The availability of such an analysis approach will allow education administrators, government officials (courthouses, police stations), and hospital administrators to make informed decisions on seating arrangements and operating procedures. Supplementary Information: The online version contains supplementary material available at 10.1007/s00366-022-01773-9.