Massively parallel nanofluidic systems are lab-on-a-chip devices where solution phase biochemical and
biological analyses are implemented in high density arrays of nanoliter holes micro-machined in a thin
platen. Polymer coatings make the interior surfaces of the holes hydrophilic and the exterior surface of the
platen hydrophobic for precise and accurate self-metered loading of liquids into each hole without cross-contamination.
We have created a "nanoplate" based on this concept, equivalent in performance to standard
microtiter plates, having 3072 thirty-three nanoliter holes in a stainless steel platen the dimensions of a
microscope slide. We report on the performance of this device for PCR-based single nucleotide
polymorphism (SNP) genotyping or quantitative measurement of gene expression by real-time PCR in
applications ranging from plant and animal diagnostics, agricultural genetics and human disease research.
We have developed a novel microarray technology for performing very large numbers of biochemical, chemical and cell-based nanoliter volume synthesis, storage and screening operations in a massively parallel manner. The Living Chip is an array of precisely machined through-holes retaining nanoliters of fluid by capillary action. Sample loading, washing and recovery are operations that can be performed manually or with simple automation. Mixing between co- registered through-holes is achieved by stacking two or more precision aligned arrays and optical assay read-outs are in parallel with a CCD imaging system. An automated picker system transfers hits into lower density microtiter plates for further analysis. We will present result demonstrating massively parallel implementation of both homogeneous and inhomogeneous fluidic and cell-based assay systems and applications of the chip for drug compound library storage and management.
To improve the effectiveness of microsurgical techniques, we have developed a semi-autonomous robotic surgical tool (called the 'Smart Scalpel') as an alternative approach to the treatment of vascular lesions. The Smart Scalpel employs optical reflectance spectroscopy and computer vision to identify and selectively target blood vessels with a focused treatment laser. Since the laser only heats along the diseased blood vessels, collateral damage to adjacent healthy tissue is substantially minimized. The Smart Scalpel also employs rapid real-time feedback analysis for on-line modification of the treatment parameters, quantification of treatment efficacy and compensation for motion tremor. These capabilities allow precise control over the energy dosimetry to achieve optimal treatment result. This paper presents the design of a prototype instrument, methods of the image analysis, and preliminary results with animal models.
Of increasing interest is miniature and portable instrumentation for non- or minimally-invasive functional microscopy. Two-photon fluorescence confocal microscopy is one optical image modality not yet exploited in this context primarily because of the large physical size of the laser light sources needed to stimulate the two-photon absorption process. As a possible alternative, we report on the successful application of light at 1064 nm from a passively, Q-switched Nd:YAG microchip laser for two-photon fluorescence microscopy of biological samples. The high peak power in a small optomechanical package makes the microchip laser an attractive starting point for development of portable two-photon fluorescence imaging system for medical endoscopy and other remote sensing applications.
We have developed novel microarray technology for performing large numbers (up to 106) of chemical and cell-based assays in parallel. This technology is particularly relevant to the high-throughput screening methods used by the pharmaceutical industry to identify potential drug candidates. In this paper we provide an overview of the system and its enabling technologies, including an economical manufacturing process for creating these microarrays, a fluorescence imaging system for detecting `hits', a fluid delivery system for loading arrays, and a method for mixing reagents.
Biological applications of MEMS technology (bioMEMS) is of increasing interest in the development of miniature and portable instrumentation for cell-based microassays and sensor applications. A major bioMEMS challenge is the physical incorporation of living cells into sensors and diagnostic devices and creation of the environmental conditions conducive for organization of differentiated cells into tissue-like structures. Our work towards these goals is illustrated by a tissue-based bioassay system we are developing based on a miniature cross-flow bioreactor constructed from of an array of cell-filled microchannels integrated into an environmentally-controlled polymer microfluidics manifold. We describe our microchannel array and manifold manufacturing methods and report on the in vitro culture of cell populations in the bioreactor.
New sensor technologies with the sensitivity and specificity capable of detecting biological and chemical agents at low concentration are of increasing importance for many environmental monitoring applications. We propose a potentially new class of microsensors that exploits the mechanical dynamics of a micrometer-sized particle held in a 3D optical force trap formed by a focused laser beam. Modulation of the laser trapping power axially perturbs the microparticle from its equilibrium position and permits measurement of the mechanical compliance transfer function (force input, displacement output) characterizing the particle micromechanical dynamics. In a mechanically homogeneous and isotropic environment, the particle motion is readily modeled as a forced harmonic oscillator; however, physico-chemical interactions between the particle and its surroundings impose external forces that modify the compliance transfer function. Our preliminary measurements indicate < 10 ppm changes in mass of a trapped microparticle can be detected with this method, suggesting possible applications as a chemical/biological sensor or for solubility measurements of microparticles.
While feedback control is widespread throughout many engineering fields, surgical instruments with embedded feedback control systems are uncommon. To improve the effectiveness of microsurgical techniques, we are presently developing a semi-autonomous robotic surgical tool as an alternative approach to treatment of skin hemangiomas like nevus flammus. Current PWS phototherapy relies on selective absorption of optical radiation by the ectatic blood vessels in a PWS resulting in thermally-mediated vessel necrosis. Although shown to be effective,heating of the surrounding tissue by photon absorption results in unacceptable collateral damage. The 'Smart Scalpel' approach employs optical reflectance spectroscopy to selectively target blood vessels in a PWS for heating with a focused laser beam. Collateral damage to adjacent tissue is substantially minimized and continuous imaging throughout the procedure allows modification of the delivered therapy to optimize therapeutic outcomes. Our work reported here involves optical system design and construction, initial quantification of imaging system resolution and contrast, and preliminary verification of the imaging and targeting strategies.
While feedback control is widespread throughout many engineering fields, there are almost no examples of surgical instruments that utilize a real-time detection and intervention strategy. This concept of closed loop feedback can be applied to the development of autonomous or semi- autonomous minimally invasive robotic surgical systems for efficient excision or modification of diseased tissue. Spatially localized regions of the tissue are first probed to distinguish pathological from healthy tissue based on differences in histochemical and morphological properties. Energy is directed to only the diseased tissue, minimizing collateral damage by leaving the adjacent healthy tissue intact. Continuous monitoring determines treatment effectiveness and, if needed, enables real-time treatment modifications to produce optimal therapeutic outcomes. The present embodiment of this general concept is a microsurgical instrument we call the Smart Scalpel, designed to treat skin angiodysplasias such as port wine stains. Other potential Smart Scalpel applications include psoriasis treatment and early skin cancer detection and intervention.
The Fourier transform Confocal Raman Microscope (FT-CRM) enables non-invasive 3D Raman spectroscopic analysis and visualization of chemically heterogeneous preparations. The instrument combines a confocal optical microscope with a visible light Fourier transform Raman spectrometer to acquire and analyze the Raman spectrum of light scattered from a voxel in the sample defined by the confocal optics. Scanning the sample relative to the confocal voxel and recording the Raman spectrum at each scan position generates a multi- dimensional data set encoding the spatially-varying compositional properties of the sample. We report here on the spatial and spectral FT-CRM image properties that includes recent work on correlation-based Raman spectroscopic imaging and application of parametric spectral estimators for robust Raman spectrum estimation.
Traditional approaches in confocal microscopy have focussed on techniques that use elastically, or Rayleigh, scattered photons to generate volumetric intensity or phase images of an object. Common to these imaging modes is an inability to discriminate between optically similar but chemically distinct materials. We report in this paper on a new class of confocal microscope which uses inelastically, or Raman, scattered light to generate volumetric chemical images of a material. We designed and built a prototype instrument, called a confocal scanning laser FT-Raman microscope, which combines a confocal scanning laser microscope with a FT Raman spectrometer. The high depth and lateral spatial resolution of the confocal optics design defines a volume element from which the Raman scattered light is collected and then analyzed by the spectrometer for its spectral content. The sample is scanned through the microscope focal volume and a 3D chemical image is generated based on the content of the Raman spectrum measured at each scan position. The results to be presented include instrument characterization measurements and examples of volumetric chemical imaging.
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