Bruce C. Wheeler

EMG feature evaluation for movement control of upper extremity prostheses

A variety of EMG features have been evaluated for control of myoelectric upper extremity prostheses. Movement class discrimination, robustness, and computational complexity of these features have been investigated for different time window sizes and noise levels. The measurements include novel application of the Davies-Bouldin index, a measure of cluster separability, and the K-nearest neighbor nonparametric classifier. The features evaluated are the integral of average value, the variance, the number of zero crossings, the Willison amplitude, the v-order and log detectors, and autoregressive model parameters. A new feature, the EMG Histogram, is introduced and shown to be the most effective of the group. The experiments were done on the data acquired from the residual biceps and triceps muscle of an above-elbow amputee.

Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture

Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 M sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

Microstamp patterns of biomolecules for high-resolution neuronal networks

A microstamping technique has been developed for high-resolution patterning of proteins on glass substrates for the localisation of neurons and their axons and dendrites. The patterning process uses a microfabricated polydimethylsiloxane stamp with micrometer length features to transfer multiple types of biomolecules to silanederivatised substrates, using glutaraldehyde as a homobifunctional linker. To test the efficacy of the procedure, substrates are compared in which poly-d-lysine (PDL) was physisorbed and patterned by photoresist with those stamped with PDL. Fluorescein isothiocyanate labelled poly-I-lysine was used to verify the presence and uniformity of the patterns on the glass substrates. As a biological assay, B104 neuroblastoma cells were plated on stamped and physisorbed glass coverslips. Pattern compliance was determined as the percentage of cells on the pattern 8h after plating. Results indicate that the stamping and photoresist patterning procedure are equivalent. Substrates stamped with PDL had an average pattern compliance of 52.6±4.4%, compared to 54.6±8.1% for physisorbed substrates. Measures of background avoidance were also equivalent. As the procedure permits successive stamping of multiple proteins, each with its own micropattern, it should be very useful for defining complex substrates to assist in cell patterning and other cell guidance studies.

Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine

For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C/sub 3/H/sub 7/NO/sub 3/) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-/spl mu/m lines of poly-D-lysine (PDL), which promotes neural attachment and growth. After 29 days in culture the foreground compliance, or the fraction of all live somata which rested on the desired PDL surface, averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. The authors conclude that these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons. They believe that this is the first report of high-quality long-term patterning of cultured neurons.

Modulation of neural network activity by patterning

Using neuronal cultures on microelectrode arrays, researchers have shown that recordable electrical activity can be influenced by chemicals in the culture environment, thus demonstrating potential applicability to biosensors or drug screening. Since practical success requires the design of robust networks with repeatable, reliable responses understanding the sources of variation is important. In this report, we used lithographic technologies to confine neurons to highly defined patterns (40 microm wide stripes); in turn these patterns gave us a measure of control over the local density of neurons (100-500 cells/mm(2)). We found that the apparent electrical activity of the network, as measured by the fraction of electrodes from which signals were recordable, increases 8-10-fold with greater local density. Also, average-firing rates of the active neurons increased 3-5-fold. We conclude that patterned networks offer one means of controlling and enhancing the responsiveness of cultured neural networks.

Blind estimation of reverberation time

The reverberation time (RT) is an important parameter for characterizing the quality of an auditory space. Sounds in reverberant environments are subject to coloration. This affects speech intelligibility and sound localization. Many state-of-the-art audio signal processing algorithms, for example in hearing-aids and telephony, are expected to have the ability to characterize the listening environment, and turn on an appropriate processing strategy accordingly. Thus, a method for characterization of room RT based on passively received microphone signals represents an important enabling technology. Current RT estimators, such as Schroeders method, depend on a controlled sound source, and thus cannot produce an online, blind RT estimate. Here, a method for estimating RT without prior knowledge of sound sources or room geometry is presented. The diffusive tail of reverberation was modeled as an exponentially damped Gaussian white noise process. The time-constant of the decay, which provided a measure of the RT, was estimated using a maximum-likelihood procedure. The estimates were obtained continuously, and an order-statistics filter was used to extract the most likely RT from the accumulated estimates. The procedure was illustrated for connected speech. Results obtained for simulated and real room data are in good agreement with the real RT values.

A modified microstamping technique enhances polylysine transfer and neuronal cell patterning

Macromolecular microstamping with polydimethylsiloxane (PDMS) stamps has been demonstrated to transfer proteins onto glassy substrates for antigen or antibody detection and for cell patterning. For many applications, including neuronal cell patterning, it is important to assure reliable transfer of sufficient quantity of protein. Research has shown that protein transfer is enhanced with the selection of the proper protein-stamp-substrate combination. In addition, detergent studies have shown that detergent-protein complexes detach from surfaces to a greater extent than proteins alone. Therefore, we hypothesized that stamp surface modification (termed here a release layer) can enhance polylysine transfer and benefit cell growth on microstamped substrates. We found unmodified stamps to transfer insufficient polylysine to support good cell survival of hippocampal neurons in a widely used serum-free, reduced-glia cell culture system. However, with modified stamps neuronal growth was reliably good. This enhanced cell growth can be attributed to the increased polylysine transfer due to the release layer rather than increased loading onto the stamp. This enhancement was found to be even greater for two-month old stamps that were stored in water. Furthermore, the physicochemical properties of the release layer can modulate the loading process. Thus, our data supports the conclusions that the release layer: (1) modulates polylysine loading, (2) enhances polylysine transfer, (3) enhances cellular growth on microstamped substrates, and (4) extends the durability (defined as the number of times a stamp can be reused) of PDMS microstamps.

A flexible perforated microelectrode array for extended neural recordings

A flexible and perforated 32-element planar microelectrode array has been fabricated and used to measure evoked potentials in brain slices. Electrodes are spaced 200 mu m apart in a 4*8 array and are sandwiched between layers of insulating polyimide. The polyimide sandwich is lifted off its substrate, making it flexible so that it could be shaped to contoured tissues. Prior to liftoff, holes are etched to expose recording sites 15 mu m in diameter and to create perforations which allow increased circulation of artificial cerebrospinal fluid to the recording surface of the tissue and, hence, increased viability. Comparisons of evoked potentials measured over tie showed an average increase of 10 h to the viability of the slice while using the perforated versus nonperforated arrays.<<ETX>>

Micrometer resolution silane-based patterning of hippocampal neurons: critical variables in photoresist and laser ablation processes for substrate fabrication

Toward the goal of creating patterns of primary hippocampal neurons in low density culture, the authors investigated techniques to fabricate microminiature grids of organofunctional silanes on glassy surfaces. A new photoresist (PR) process, Selective Silane Removal (SSR), was developed and compared to two previously developed techniques which use PR and laser patterning. The grid patterns consisted of 27 combinations of path width, length, and intersection (node diameter). The background consisted of squares bounded for the paths. The best neuron patterning was observed on substrates produced by the SSR process where cytophilic aminosilane is uniformly deposited and selectively removed from the background. Controlling water during aminosilane deposition was critical to good neuronal growth and patterning. Oxygen plasma etching of background regions prior to cytophobic phenylsilane binding significantly reduced off-pattern cell growth. Up to 90% of somata grown on these substrates complied to the pattern, and an average of 77% of background regions were free of neurites or cells connected to the pattern. The highest laser energy density, 120 mJ/cm/sup 2/, produced the best compliance on lased substrates, with an average of 35% of background regions free of connected cells and neurites, but considerable variation across the surface. On substrates with excellent patterning, compliance to nodes was found to be dependent on pattern dimensions, with 20-/spl mu/m node diameters and 80-/spl mu/m internodal path lengths increasing compliance.

Multisite hippocampal slice recording and stimulation using a 32 element microelectrode array

A technique has been developed in which a planar array of 32 microelectrodes, arranged in a 4 by 8 pattern with 200 micron separation, is used to record from and stimulate the hippocampal slice preparation at multiple sites. Control of media flow past the tissue is critical to observe signals and preserve viability. Active suppression circuitry is used to prevent device saturation due to large stimulation artifacts. The field potentials recorded are spatially unique and provide a 2-dimensional description of the underlying population activity in the various pyramidal strata and subpopulations. Multisite stimulation is also possible with the array, permitting the experimenter to quickly stimulate and record from brain slices in many spatial patterns.