Tilak Jain

An array of microactuated microelectrodes for monitoring single-neuronal activity in rodents

Arrays of microelectrodes used for monitoring single- and multi-neuronal action potentials often fail to record from the same population of neurons over a period of time for several technical and biological reasons. We report here a novel Neural Probe chip with a 3-channel microactuated microelectrode array that will enable precise repositioning of the individual microelectrodes within the brain tissue after implantation. Thermal microactuators and associated microelectrodes in the Neural Probe chip are microfabricated using the Sandias Ultraplanar Multi-level MEMS Technology (SUMMiTV) process, a 5-layer polysilicon micromachining technology of the Sandia National labs, Albuquerque, NM. The Neural Probe chip enables precise bi-directional positioning of the microelectrodes in the brain with a step resolution in the order of 8.8 /spl mu/m. The thermal microactuators allow for a linear translation of the microelectrodes of up to 5 mm in either direction making it suitable for positioning microelectrodes in deep structures of a rodent brain. The overall translation in either direction was reduced to approximately 2 mm after insulation of the microelectrodes with epoxy for monitoring multi-unit activity. Single unit recordings were obtained from the somatosensory cortex of adult rats over a period of three days demonstrating the feasibility of this technology. Further optimization of the microelectrode insulation and chip packaging will be necessary before this technology can be validated in chronic experiments.

Electrostatic microactuators for precise positioning of neural microelectrodes

Microelectrode arrays used for monitoring single and multineuronal action potentials often fail to record from the same population of neurons over a period of time likely due to micromotion of neurons away from the microelectrode, gliosis around the recording site and also brain movement due to behavior. We report here novel electrostatic microactuated microelectrodes that will enable precise repositioning of the microelectrodes within the brain tissue. Electrostatic comb-drive microactuators and associated microelectrodes are fabricated using the SUMMiT V/spl trade/ (Sandias Ultraplanar Multilevel MEMS Technology) process, a five-layer polysilicon micromachining technology of the Sandia National labs, NM. The microfabricated microactuators enable precise bidirectional positioning of the microelectrodes in the brain with accuracy in the order of 1 /spl mu/m. The microactuators allow for a linear translation of the microelectrodes of up to 5 mm in either direction making it suitable for positioning microelectrodes in deep structures of a rodent brain. The overall translation was reduced to approximately 2 mm after insulation of the microelectrodes with epoxy for monitoring multiunit activity. The microactuators are capable of driving the microelectrodes in the brain tissue with forces in the order of several micro-Newtons. Single unit recordings were obtained from the somatosensory cortex of adult rats in acute experiments demonstrating the feasibility of this technology. Further optimization of the insulation, packaging and interconnect issues will be necessary before this technology can be validated in long-term experiments.

Spotiton: A prototype for an integrated inkjet dispense and vitrification system for cryo-TEM

Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the method for vitrifying specimens onto EM grids is essentially unchanged - application of ∼3 μL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.

Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays

High-throughput cell-based screens of genome-size collections of cDNAs and siRNAs have become a powerful tool to annotate the mammalian genome, enabling the discovery of novel genes associated with normal cellular processes and pathogenic states, and the unravelling of genetic networks and signaling pathways in a systems biology approach. However, the capital expenses and the cost of reagents necessary to perform such large screens have limited application of this technology. Efforts to miniaturize the screening process have centered on the development of cellular microarrays created on microscope slides that use chemical means to introduce exogenous genetic material into mammalian cells. While this work has demonstrated the feasibility of screening in very small formats, the use of chemical transfection reagents (effective only in a subset of cell lines and not on primary cells) and the lack of defined borders between cells grown in adjacent microspots containing different genetic material (to prevent cell migration and to aid spot location recognition during imaging and phenotype deconvolution) have hampered the spread of this screening technology. Here, we describe proof-of-principles experiments to circumvent these drawbacks. We have created microwell arrays on an electroporation-ready transparent substrate and established procedures to achieve highly efficient parallel introduction of exogenous molecules into human cell lines and primary mouse macrophages. The microwells confine cells and offer multiple advantages during imaging and phenotype analysis. We have also developed a simple method to load this 484-microwell array with libraries of nucleic acids using a standard microarrayer. These advances can be elaborated upon to form the basis of a miniaturized high-throughput functional genomics screening platform to carry out genome-size screens in a variety of mammalian cells that may eventually become a mainstream tool for life science research.

In situ electroporation of surface-bound siRNAs in microwell arrays

Gene silencing using RNA interference (RNAi) has become a prominent biological tool for gene annotation, pathway analysis, and target discovery in mammalian cells. High-throughput screens conducted using whole-genome siRNA libraries have uncovered rich sets of new genes involved in a variety of biological processes and cellular models of disease. However, high-throughput RNAi screening is not yet a mainstream tool in life science research because current screening platforms are expensive and onerous. Miniaturizing the RNAi screening platform to reduce cost and increase throughput will enable its widespread use and harness its potential for rapid genome annotation. With this aim, we have combined semi-conductor microfabrication and nanolitre dispensing techniques to develop miniaturized electroporation-ready microwell arrays loaded with siRNA molecules in which multiplexed gene knockdown can be achieved. Arrays of microwells are created using high-aspect ratio biocompatible photoresists on optically transparent and conductive Indium-Tin Oxide (ITO) substrates with integrated micro-electrodes to enable in situ electroporation. Non-contact inkjet microarraying allows precise dispensing of nanolitre volumes into the microwell structures. We have achieved parallel electroporation of multiple mammalian cells cultured in these microwell arrays and observed efficient knockdown of genes with surface-bound, printed siRNAs. Further integration of microfabrication and non-contact nanolitre dispensing techniques described here may enable single-substrate whole-genome siRNA screening in mammalian cells.

Microelectrode Array (MEA) Platform for Targeted Neuronal Transfection and Recording

Techniques used for nonviral gene transfection often have poor spatial resolution. In this letter, we present a microelectrode array (MEA) system that can precisely transfect exogenous molecules into targeted primary neurons using microelectroporation. An optimal cathodic pulse 4 V in amplitude and 1 ms in duration resulted in a transfection efficiency of 56% and a viability of 82%. Finally, siRNA molecules were transfected into targeted neurons in culture using the aforementioned system.

Microactuated neural probes to compensate for brain micromotion

One of the dominant failure modes of chronic neural implants is micromotion of the surrounding brain tissue relative to the implant leading to neuronal drift and shear injury. In this study, we have (a). Assessed the micromotion in the somatosensory cortex and (b). Designed, developed and tested a microactuated neural probe that can compensate for brain micromotion. We used a differential variable reluctance (DVRT) transducer in adult rats (n=8) to monitor micromotion in the somatosensory cortex. Electrostatic microactuators were fabricated using the SUMMiT (Sandias Ultraplanar Multilevel MEMS Technology) process, a 5-layer polysilicon micromachining technology of the Sandia National labs, NM. In anesthetized rats, surface micromotion was observed to be in the order of 2-25 /spl mu/m due to pressure changes during respiration and 1-3 /spl mu/m due to vascular pulsatilily. In addition there were long-term drifts in the order of 80 /spl mu/m due to changes in the anesthetic level. The microactuated neural probe was capable of moving in steps of 1/spl mu/m with an aggregate translational capability in the order of several millimeters. In conclusion, there is significant micromotion in the surface of the somatosensory cortex that could lead to failure of chronic neural implants. Microactuated neural probes are capable of compensating for this micromotion.

Multiplexed TEM Specimen Preparation and Analysis of Plasmonic Nanoparticles

We describe a system for rapidly screening hundreds of nanoparticle samples using transmission electron microscopy (TEM). The system uses a liquid handling robot to place up to 96 individual samples onto a single standard TEM grid at separate locations. The grid is then transferred into the TEM and automated software is used to acquire multiscale images of each sample. The images are then analyzed to extract metrics on the size, shape, and morphology of the nanoparticles. The system has been used to characterize plasmonically active nanomaterials.

Typhon: Multiplexed TEM Sample Preparation

Over the last decade there has been significant progress in the synthesis of inorganic and hybrid inorganic-organic nanoparticles in solution. Optimizing the synthesis conditions is often dependent on structural characterization of the nanoparticles. Transmission Electron Microscopy (TEM) provides a method for structural characterization that provides both high-resolution details of individual particles as well as particle size distribution and morphology. The draw back to this method has been that specimen preparation is typically very low-throughput; single specimens are prepared on individual TEM grids and then imaged one at a time. Imaging several tens to hundreds of nanoparticle synthesis conditions thus requires an equal number of TEM grids, which is cumbersome, time consuming, and not feasible as a routine characterization method.

Spatio-temporally controlled transfection of nucleic acid payloads in cell-culture

There is a critical need in emerging areas of high-throughput screening of large molecule libraries during drug development, to transfect multiple molecules precisely into cells in a specific location within a culture. In this study, we report the novel use of a microelectrode array technology to achieve spatio-temporally controlled transfection of siRNA molecules into fibroblasts using the mechanism of electroporation. Optimal electroporation was achieved using cathodic voltage pulses of approximately 4 V and 10 ms duration, with transfection efficiency of approximately 57% and cell viability of 60%. Further development of this technology for gene and RNAi transfection is envisioned to be useful to make significant advances in the field neurogenomics and high-throughput screening.