Alex David Corwin

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

High-performance surface-micromachined inchworm actuator

This work demonstrates a polycrystalline silicon surface-micromachined inchworm actuator that exhibits high-performance characteristics such as large force (/spl plusmn/0.5 millinewtons), large velocity range (0 to /spl plusmn/4.4 mm/sec), large displacement range (/spl plusmn/100 microns), small step size (/spl plusmn/10, /spl plusmn/40 or /spl plusmn/100 nanometers), low power consumption (nanojoules per cycle), continuous bidirectional operation and relatively small area (600 /spl times/ 200/spl mu/m/sup 2/). An in situ load spring calibrated on a logarithmic scale from micronewtons to millinewtons, optical microscopy and Michelson interferometry are used to characterize its performance. The actuator consists of a force-amplifying plate that spans two voltage-controlled clamps, and walking is achieved by appropriately sequencing signals to these three components. In the clamps, normal force is borne by equipotential rubbing counterfaces, enabling friction to be measured against load. Using different monolayer coatings, we show that the static coefficient of friction can be changed from 0.14 to 1.04, and that it is load-independent over a broad range. We further find that the static coefficient of friction does not accurately predict the force generated by the actuator and attribute this to nanometer-scale presliding tangential deflections.

Effect of adhesion on dynamic and static friction in surface micromachining

We measure friction of monolayer-lubricated microelectromechanical systems surfaces under both static and dynamic conditions while continuously controlling the applied normal load at positive or negative values (i.e., compression or tension). The dynamic friction experiment methodology we have devised enables fitting to the complete one-dimensional equation of motion. We observe friction at zero applied load, and quantitatively attribute this to interfacial adhesion. Within error, the adhesive force is the same under static and dynamic conditions.

Simple and robust image-based autofocusing for digital microscopy

A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.

Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation

Summary Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells); cytokeratin 7 (ductal cells); and smooth muscle &agr;-actin (myoepithelial cells) and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle &agr;-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis.

The effect of nanoparticles on rough surface adhesion

Particulates can strongly influence interfacial adhesion between rough surfaces by changing their average separation. In a cantilever beam adhesion test structure, a compressive zone exists just beyond the crack tip, which may act to deform such particles. To explore this phenomenon quantitatively, we compared finite element method calculations of the interface to load-displacement experiments of individual particles. Below a certain threshold density, we show that the stress distribution at the interface is sufficient to deform individual particles. In this regime, the adhesion is controlled by the intrinsic surface roughness and under dry conditions is mainly due to van der Waals forces across extensive noncontacting areas. Above this threshold density, however, the particles introduce a topography that is more significant than the intrinsic surface roughness. As a result, the interfacial separation is governed by the particle size and the adhesion is lower but stochastic in nature. We demonstrate that ...

Micromachined piconewton force sensor for biophysics investigations

The authors describe a micromachined force sensor that is able to measure forces as small as 1pN in both air and water. First, they measured the force field produced by an electromagnet on individual 2.8μm magnetic beads glued to the sensor. By repeating with 11 different beads, they measured a 9% standard deviation in saturation magnetization. They next demonstrated that the sensor was fully functional when immersed in physiological buffer. These results show that the force sensors can be useful for magnetic force calibration and also for measurement of biophysical forces on chip.

A novel, automated technology for multiplex biomarker imaging and application to breast cancer

Multiplexed immunofluorescence is a powerful tool for validating multigene assays and understanding the complex interplay of proteins implicated in breast cancer within a morphological context. We describe a novel technology for imaging an extended panel of biomarkers on a single, formalin‐fixed paraffin‐embedded breast sample and evaluating biomarker interaction at a single‐cell level, and demonstrate proof‐of‐concept on a small set of breast tumours, including those which co‐express hormone receptors with Her2/neu and Ki‐67.

Operational Wear and Friction in MEMS Devices

Friction and wear are extremely important issues in micromachined surfaces in design applications that allow rubbing. A polysilicon surface-micromachined inchworm device has been developed to obtain detailed in-situ information on these properties under well-controlled loading conditions. Here, we investigate the inchworm operational wear and the evolution of friction coefficient as a function of the number of imposed wear cycles. A test procedure was developed to monitor various functional parameters such as the travel distance of the inchworm under an imposed drag force for a fixed number of steps and the friction coefficient. While subject to this drag force, the travel distance decreased gradually until the foot of the device became permanently lodged in the grooves created by the wear-track. Meanwhile, it was found that the friction coefficient increased from 0.2 on a virgin surface to 3 when the accumulated number of wear cycles reached around 200,000. The friction test itself was found to interact with the wear processes. By minimizing the number of friction tests performed during the wear test, the operational life of the device was extended well beyond 700,000 cycles. Microscopic observation of the wear surfaces revealed that the early wear is characterized by the blunting of the sharp peaks on the poly silicon grains and then flattening of this fine wear debris on the surface. Evidence of plastic deformation was inferred by the spread of the wear debris over several grains. With increased number of wear cycles, material removal through scratches induced by the wear debris was observed. The device failure occurred due to a large volume of material removal (severe wear) in localized regions.Copyright

Accelerating aging failures in MEMS devices

The feasibility of using temperature and humidity to age vapor-deposited SAM-coated electrostatic-actuated MEMS devices with contacting surfaces was determined. Failures were dependent on both temperature and humidity. The trend indicated longer life at both lower temperatures and lower humidity levels. Using cantilever beams, measurements reveal degradation of the VSAM (vapor-deposited self assembled monolayer) surface coating when stressed at 300/spl deg/C with controlled humidity environments of 500 and 2000 ppmv. In particular, we have seen the surface adhesion change for these beams stressed at 300/spl deg/C for time intervals of 10, 24, 50, 100, and 200 hours. However, there is no measurable change after 2 hours. The higher humidity case promotes the same surface adhesion change in a factor of ten less time. The complex MEMS devices tested followed the same trends as the beam test structures. We definitely observe a failure of the MEMS devices due to the environment with most failures occurring at 300/spl deg/C and some failures at 200/spl deg/C. These failures are due to an adhesion site in the hub of the load gear where the typical gap is 0.3 /spl mu/m.