An automated, parallel processing approach to biomolecular sample preparation

Sample preparation for DNA and RNA assays is a prime candidate for laboratory automation. A novel, parallel processing device that performs the three separate liquid-handling functions necessary for such sample preparation-dispensing, pipetting, and pressurizing-is presented. The device comprises an array of fine nozzles connected by fluidic channels to automatically and precisely distribute flow between one source and an array of points. The design principles, as well as the experimental and computational methods used to develop the device, are described. Test results, including accuracy, uniformity, volume range, and timing, are presented. The functionality of the device is demonstrated by performing a solid-phase extraction of DNA with two types of microcolumns.     

Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens

The potential of the developing zebrafish model for toxicology and drug discovery is limited by inefficient approaches to manipulating and chemically exposing zebrafish embryos-namely, manual placement of embryos into 96- or 384-well plates and exposure of embryos while still in the chorion, a barrier of poorly characterized permeability enclosing the developing embryo. We report the automated dechorionation of 1600 embryos at once at 4 h postfertilization (hpf) and placement of the dechorionated embryos into 96-well plates for exposure by 6 hpf. The process removed ≥95% of the embryos from their chorions with 2% embryo mortality by 24 hpf, and 2% of the embryos malformed at 120 hpf. The robotic embryo placement allocated 6-hpf embryos to 94.7% ± 4.2% of the wells in multiple 96-well trials. The rate of embryo mortality was 2.8% (43 of 1536) from robotic handling, the rate of missed wells was 1.2% (18 of 1536), and the frequency of multipicks was <0.1%. Embryo malformations observed at 24 hpf occurred nearly twice as frequently from robotic handling (16 of 864; 1.9%) as from manual pipetting (9 of 864; 1%). There was no statistical difference between the success of performing the embryo placement robotically or manually.     

Dynex: multiplex ELISA technology

Conventional enzyme-linked immunosorbent assay (ELISA) is a gold standard for screening antibodies and testing for protein or antigen presence. A significant limitation of this assay resides in the fact that only one analyte can be assessed per microplate well. Here, we describe and investigate a new technology consisting of an automated ELISA system in which up to 10 analytes can be measured within one single well, thus improving productivity, accuracy, and repeatability by reducing the amount of human labor required. Another strength of the platform is that a user can load any necessary sets/subsets of beads to perform required assays, with improved flexibility compared to manufactured-loaded arrays for multiplex analysis. We also demonstrate that this system can be used to determine the pathogenicity (i.e., presence of Shiga toxins) and serotype (i.e., Escherichia coli O157) of E. coli isolates.     

Computed tomographic imaging of the coronary arterial tree-use of local tomography

It has been demonstrated previously (see E.L. Ritman and A.A. Bove, in State of the Art in Quantitative Coronary Arteriography, p.67-78, 1986) that coronary artery anatomy can be visualized using high-speed, volume-scanning X-ray CT (computed tomography). In the current study it is demonstrated that local image reconstruction (i.e. the reconstruction kernel is ~2(+) mm long), as distinct from more conventional global image reconstruction (i.e. 200(+) mm kernel length), has the advantage of reducing the need for operator interactive image processing. In addition, the local reconstruction algorithm eliminates the need for recording the X-ray projection data over the full transaxial extent of the thorax because it requires only the X-ray attenuation data over the region of the heart. This latter aspect reduces the dynamic range requirements for the sensors and could reduce total X-ray exposure.     

Color quantitation through image processing in dermatology

Classical color models and their applications to computer vision are reviewed. The performances of color quantitation from digitized images are compared with those derived from a chromameter. The color quantitation obtained from either digitized color slides or directly digitized images is proved to be more efficient than the conventional visual assessment of observers. A methodology is proposed for determining the specific color indices which are needed in dermatology. An application of this methodology is developed for designing a blanching quantitation index in order to replace the visual assessment during McKenzie tests.     

Real-time evaluation and visualization of learner performance in a mixed-reality environment for clinical breast examination

We investigate the efficacy of incorporating real-time feedback of user performance within mixed-reality environments (MREs) for training real-world tasks with tightly coupled cognitive and psychomotor components. This paper presents an approach to providing real-time evaluation and visual feedback of learner performance in an MRE for training clinical breast examination (CBE). In a user study of experienced and novice CBE practitioners (n = 69), novices receiving real-time feedback performed equivalently or better than more experienced practitioners in the completeness and correctness of the exam. A second user study (n = 8) followed novices through repeated practice of CBE in the MRE. Results indicate that skills improvement in the MRE transfers to the real-world task of CBE of human patients. This initial case study demonstrates the efficacy of MREs incorporating real-time feedback for training real-world cognitive-psychomotor tasks.     

Morse set classification and hierarchical refinement using Conley index

Morse decomposition provides a numerically stable topological representation of vector fields that is crucial for their rigorous interpretation. However, Morse decomposition is not unique, and its granularity directly impacts its computational cost. In this paper, we propose an automatic refinement scheme to construct the Morse Connection Graph (MCG) of a given vector field in a hierarchical fashion. Our framework allows a Morse set to be refined through a local update of the flow combinatorialization graph, as well as the connection regions between Morse sets. The computation is fast because the most expensive computation is concentrated on a small portion of the domain. Furthermore, the present work allows the generation of a topologically consistent hierarchy of MCGs, which cannot be obtained using a global method. The classification of the extracted Morse sets is a crucial step for the construction of the MCG, for which the Poincare′ index is inadequate. We make use of an upper bound for the Conley index, provided by the Betti numbers of an index pair for a translation along the flow, to classify the Morse sets. This upper bound is sufficiently accurate for Morse set classification and provides supportive information for the automatic refinement process. An improved visualization technique for MCG is developed to incorporate the Conley indices. Finally, we apply the proposed techniques to a number of synthetic and realworld simulation data to demonstrate their utility.