Image-Based Phenotypic Screening with Human Primary T Cells Using One-Dimensional Imaging Cytometry with Self-Tuning Statistical-Gating Algorithms.

The parallel microfluidic cytometer (PMC) is an imaging flow cytometer that operates on statistical analysis of low-pixel-count, one-dimensional (1D) line scans. It is highly efficient in data collection and operates on suspension cells. In this article, we present a supervised automated pipeline for the PMC that minimizes operator intervention by incorporating multivariate logistic regression for data scoring.

Image-based cell-resolved screening assays in flow.

A parallel microfluidic cytometer (PMC) is based on a one-dimensional (1D) scanning detector, a parallel array of flow channels, and new multiparameter analysis algorithms that operate on low-pixel-count 1D images. In this article, we explore a series of image-based live- and fixed-cell screening assays, including two NF-kB nuclear translocations and T-cell capping. We then develop a new multiparametric linear weighted classifier that achieves a Z' factor sufficient for scaled pharmaceutical discovery with Jurkat cells in suspension.

Intracellular protein and nucleic acid measured in eight cell types using deep-ultraviolet mass mapping.

We present measurements by deep-ultraviolet mass mapping of nucleic acid (NA) and protein for five commonly cultured and three primary cell types. The dry mass distribution at submicron resolution was determined on a single-cell basis for 250-500 cells from each of these types. Since the method carries a direct reference to a spectrophotometric standard (molar extinction coefficient), we are able to calibrate the absolute weight distributions both on a cell-to-cell basis within each type and across types.

Using molecular mechanics to predict bulk material properties of fibronectin fibers.

The structural proteins of the extracellular matrix (ECM) form fibers with finely tuned mechanical properties matched to the time scales of cell traction forces. Several proteins such as fibronectin (Fn) and fibrin undergo molecular conformational changes that extend the proteins and are believed to be a major contributor to the extensibility of bulk fibers. The dynamics of these conformational changes have been thoroughly explored since the advent of single molecule force spectroscopy and molecular dynamics simulations but remarkably, these data have not been rigorously applied to the understanding of the time dependent mechanics of bulk ECM fibers.

Deep ultraviolet mapping of intracellular protein and nucleic acid in femtograms per pixel.

By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 100× objective with a numerical aperture of 1.2 NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (∼100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal organelles.

Parallel imaging microfluidic cytometer.

By adding an additional degree of freedom from multichannel flow, the parallel microfluidic cytometer (PMC) combines some of the best features of fluorescence-activated flow cytometry (FCM) and microscope-based high-content screening (HCS). The PMC (i) lends itself to fast processing of large numbers of samples, (ii) adds a 1D imaging capability for intracellular localization assays (HCS), (iii) has a high rare-cell sensitivity, and (iv) has an unusual capability for time-synchronized sampling. An inability to practically handle large sample numbers has restricted applications of conventional flow cytometers and microscopes in combinatorial cell assays, network biology, and drug discovery.

A parallel microfluidic flow cytometer for high-content screening.

A parallel microfluidic cytometer (PMC) uses a high-speed scanning photomultiplier-based detector to combine low-pixel-count, one-dimensional imaging with flow cytometry. The 384 parallel flow channels of the PMC decouple count rate from signal-to-noise ratio. Using six-pixel one-dimensional images, we investigated protein localization in a yeast model for human protein misfolding diseases and demonstrated the feasibility of a nuclear-translocation assay in Chinese hamster ovary (CHO) cells expressing an NFκB-EGFP reporter.

Vertical hydrodynamic focusing in glass microchannels.

Vertical hydrodynamic focusing in microfluidic devices is investigated through simulation and through direct experimental verification using a confocal microscope and a novel form of stroboscopic imaging. Optimization for microfluidic cytometry of biological cells is examined. By combining multiple crossing junctions, it is possible to confine cells to a single analytic layer of interest.

Scaling of nucleic acid assays on microelectrophoresis array devices: high-dynamic range multi-gene readout from less than ten transcripts.

In this paper we describe progress in using the prodigious data-collecting ability of multilane microelectrophoresis instruments to bear on problems in scaled nucleic acid assays. We emphasize compound stacking and solid-support loading as means to concentrate <100 pg samples for direct injection. Reaction Mapping is applied to readout quantitative polymerase chain reaction gene-expression and as a way to practically overcome difficulty in interpreting amplification curves of multiplexed quantitative polymerase chain reaction at 20-50 gene/well complexity.

384-channel parallel microfluidic cytometer for rare-cell screening.

We have constructed a 384-channel parallel microfluidic cytometer (PMC). The multichannel architecture allows 384 unique samples for a cell-based screen to be read out in approximately 6-10 min, about 30-times the speed of a conventional fluorescence-activated cytometer system (FACS). This architecture also allows the signal integration time to be varied over a larger range than is practical in single-channel FACS and is suitable for detection of rare-cells in a high background of negatives.

Reaction-mapped quantitative multiplexed polymerase chain reaction on a microfluidic device.

We have applied multiple-time-point reaction mapping to generate high-dynamic-range quantitative data from PCR multiplexes. The approach measures, then compensates, numerous PCR slope nonidealities across the multiplex without prejudice. A multilane microelectophoresis device with a novel scanning detector that reports redundantly over more than six decades in signal strength was used to collect data with multiple readings for each amplification point and with double internal calibration (lane standards and gene standards).

Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy.

We developed a deep-ultraviolet (UV) microscope capable of imaging cell mitosis and motility at 280 nm for 45 min with minimal UV-induced toxicity, and for 6 h before the onset of visible cell death in cultured human and mouse cells. Combined with computational methods that convert the intensity of each pixel into an estimate of mass, deep-UV microscopy images generate maps of nucleic acid mass, protein mass and fluorescence yield in unlabeled cells.

Numerical model for DNA loading in microdevices: stacking and autogating effects.

Many electrophoresis-based DNA sequencing and genotyping microdevices rely on field-driven effects to load and preconcentrate the sample. A quantitative model is developed for a broad class of electrophoresis-based microfabricated sample injectors. Quantitative predictions of DNA preconcentration are compared with experimental data and are shown to qualitatively reproduce the detailed time-evolving sample distribution in the injector.

Solid-support sample loading for DNA sequencing.

We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel.

Microdevice DNA forensics by the simple tandem repeat method.

We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system.

A 768-lane microfabricated system for high-throughput DNA sequencing.

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm x 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor.

Enhanced detection sensitivity using a novel solid-phase incorporated affinity fluorescent protein biosensor.

We engineered green fluorescent protein (GFP) into affinity fluorescent proteins (aFPs) biosensors. The aFPs detect protein-protein interactions by enhanced fluorescence intensity. In a proof of principle demonstration, aFPs containing haemagglutinin (HA) tag bind specifically to the anti-HA antibody.

Thirty-cycle temperature optimization of a closed-cycle capillary PCR machine.

The performance of a novel thermal cycler has been characterized in a 30-cycle PCR. The device consists of a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm i.