Jennifer Sturgis

Microfluidic Biochip for Impedance Spectroscopy of Biological Species

This paper describes the fabrication and characterization of a microelectronic device for the electrical interrogation and impedance spectroscopy of biological species. Key features of the device include an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the non-planar topography of the chip using spin-on-glass as an intermediate bonding layer. The total fluidic path volume in the device is on the order of 30 nl. Flow fields in the closed chip were mapped by particle image velocimetry. Electrical impedance measurements of suspensions of the live microorganism Listeria innocua injected into the chip demonstrate an easy method for detecting the viability of a few bacterial cells. By-products of the bacterial metabolism modify the ionic strength of a low conductivity suspension medium, significantly altering its electrical characteristics.

Intracellularly grown gold nanoparticles as potential surface-enhanced Raman scattering probes

Gold nanoparticles grown within the intracellular confines of living cells are introduced as potential surface-enhanced Raman scattering (SERS) substrates for confocal Raman spectrometry. Electron microscopy and a silver-enhanced reflectance laser scanning confocal microscopic approach were used to visualize the size, shape, and distribution of intracellularly grown gold nanoparticles (IGAuN) as small as 1 nm. Passive uptake as the conventional approach for delivering nanoparticles inside cells faces the insurmountable challenge of escaping the endosomal/lysosomal pathway. In contrast, IGAuN provides an unprecedented advantage of providing access to cytoplasm and nucleus.

Automated quantification and reconstruction of collagen matrix from 3D confocal datasets

The geometrical structure of fibrous extracellular matrix (ECM) impacts on its biological function. In this report, we demonstrate a new algorithm designed to extract quantitative structural information about individual collagen fibres (orientation, length and diameter) from 3D backscattered‐light confocal images of collagen gels. The computed quantitative data allowed us to create surface‐rendered 3D images of the investigated sample.

Factors necessary to produce basoapical polarity in human glandular epithelium formed in conventional and high-throughput three-dimensional culture: example of the breast epithelium

BackgroundBasoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated.ResultsWe demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity.ConclusionThese results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.

Differential Mitochondrial Toxicity Screening and Multi- Parametric Data Analysis

Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.

Phenotypic Profiling of Raf Inhibitors and Mitochondrial Toxicity in 3D Tissue Using Biodynamic Imaging

The existence of phenotypic differences in the drug responses of 3D tissue relative to 2D cell culture is a concern in high-content drug screening. Biodynamic imaging is an emerging technology that probes 3D tissue using short-coherence dynamic light scattering to measure the intracellular motions inside tissues in their natural microenvironments. The information content of biodynamic imaging is displayed through tissue dynamics spectroscopy (TDS) but has not previously been correlated against morphological image analysis of 2D cell culture. In this article, a set of mitochondria-affecting compounds (FCCP, valinomycin, nicardipine, ionomycin) and Raf kinase inhibitors (PLX4032, PLX4720, GDC, and sorafenib) are applied to multicellular tumor spheroids from two colon adenocarcinoma cell lines (HT-29 and DLD-1). These were screened by TDS and then compared against conventional image-based high-content analysis (HCA). The responses to the Raf inhibitors PLX4032 and PLX4720 are grouped separately by cell line, reflecting the Braf/Kras difference in these cell lines. There is a correlation between TDS and HCA phenotypic clustering for most cases, which demonstrates the ability of dynamic measurements to capture phenotypic responses to drugs. However, there are significant 2D versus 3D phenotypic differences exhibited by several of the drugs/cell lines.

High-Throughput Secondary Screening at the Single-Cell Level

We have developed an automated system for drug screening using a single-cell–multiple functional response technology. The approach uses a semiautomated preparatory system, high-speed sample collection, and a unique analytical tool that provides instantaneous results for compound dilutions using 384-well plates. The combination of automation and rapid robotic sampling increases quality control and robustness. High-speed flow cytometry is used to collect single-cell results together with a newly defined analytical tool for extraction of IC50 curves for multiple assays per cell. The principal advantage is the extreme speed of sample collection, with results from a 384-well plate being completed for both collection and data processing in less than 10 min. Using this approach, it is possible to extract detailed drug response information in a highly controlled fashion. The data are based on single-cell results, not populations. With simultaneous assays for different functions, it is possible to gain a more detailed understanding of each drug/compound interaction. Combined with integrated advanced data processing directly from raw data files, the process from sampling to analytical results is highly intuitive. Direct PubMed links allow review of drug structure and comparisons with similar compounds.

Portable bacterial identification system based on elastic light scatter patterns

BackgroundConventional diagnosis and identification of bacteria requires shipment of samples to a laboratory for genetic and biochemical analysis. This process can take days and imposes significant delay to action in situations where timely intervention can save lives and reduce associated costs. To enable faster response to an outbreak, a low-cost, small-footprint, portable microbial-identification instrument using forward scatterometry has been developed.ResultsThis device, weighing 9 lb and measuring 12 × 6 × 10.5 in., utilizes elastic light scatter (ELS) patterns to accurately capture bacterial colony characteristics and delivers the classification results via wireless access. The overall system consists of two CCD cameras, one rotational and one translational stage, and a 635-nm laser diode. Various software algorithms such as Hough transform, 2-D geometric moments, and the traveling salesman problem (TSP) have been implemented to provide colony count and circularity, centering process, and minimized travel time among colonies.ConclusionsExperiments were conducted with four bacteria genera using pure and mixed plate and as proof of principle a field test was conducted in four different locations where the average classification rate ranged between 95 and 100%.

Alternatives to current flow cytometry data analysis for clinical and research studies

Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment.

Reflected scatterometry for noninvasive interrogation of bacterial colonies

Abstract. A phenotyping of bacterial colonies on agar plates using forward-scattering diffraction-pattern analysis provided promising classification of several different bacteria such as Salmonella, Vibrio, Listeria, and E. coli. Since the technique is based on forward-scattering phenomena, light transmittance of both the colony and the medium is critical to ensure quality data. However, numerous microorganisms and their growth media allow only limited light penetration and render the forward-scattering measurement a challenging task. For example, yeast, Lactobacillus, mold, and several soil bacteria form colorful and dense colonies that obstruct most of the incoming light passing through them. Moreover, blood agar, which is widely utilized in the clinical field, completely blocks the incident coherent light source used in forward scatterometry. We present a newly designed reflection scatterometer and validation of the resolving power of the instrument. The reflectance-type instrument can acquire backward elastic scatter patterns for both highly opaque media and colonies and has been tested with three different bacterial genera grown on blood agar plates. Cross-validation results show a classification rate above 90% for four genera.