20 research outputs found

    Single Cell Deposition and Patterning with a Robotic System

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    Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques

    Three dimensional optofluidic devices for manipulation of particles and cells

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    Optical forces offer a powerful tool for manipulating single cells noninvasively. Integration of optical functions within microfluidic devices provides a new freedom for manipulating and studying biological samples at the micro scale. In the pursuit to realise such microfluidic devices with integrated optical components, Ultrafast Laser Inscription (ULI) fabrication technology shows great potential. The uniqueness and versatility of the technique in rapid prototyping of 3D complex microfluidic and optical elements as well as the ability to perform one step integration of these elements provides exciting opportunities in fabricating novel devices for biophotonics applications. The work described in this thesis details the development of three dimensional optofluidic devices that can be used for biophotonics applications, in particular for performing cell manipulation and particle separation. Firstly, the potential of optical forces to manipulate cells and particles in ULI microfluidic channels is investigated. The ability to controllably displace particles within a ULI microchannel using a waveguide positioned orthogonal to it is explored in detail. We then prototype a more complex 3D device with multiple functionalities in which a 3D optofluidic device containing a complex microchannel network and waveguides was used for further investigations into optical manipulation and particle separation. The microfluidic channel network and the waveguides within the device possess the capability to manipulate the injected sample fluid through hydrodynamic focusing and optically manipulate the individual particles, respectively. This geometry provided a more efficient way of investigating optical manipulation within the device. Finally, work towards developing a fully optimised 3D cell separator device is presented. Initial functional validation was performed by investigating the capability of the device to route particles through different outlet channels using optical forces. A proof of concept study demonstrates the potential of the device to use for cell separation based on the size of the cells. It was shown that both passive and active cell separation is possible using this device

    A Bio-Assembly, Mosaic Building, and Informatics System for Cell Biology

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    In the field of regenerative medicine, there is a need to develop technologies that can increase the overall efficiency of imaging and expanding cells in culture and in complex heterogeneous arrangements necessary for tissue construction. Long-term live cell imaging has the potential to significantly enhance our understanding of intercellular signaling pathways and the dependence of phenotype on cell arrangement. A transdisciplinary approach has been taken to bridge the fields of cell biology, robotics, and photonics to create a long-term live cell imaging system capable of single cell handling as well as the acquisition of multiple types of data needed for data mining and a general informatics approach to cell culture. A Bio-Assembly Mosaic Builder and Informatics (BAMBI) system was designed and developed using custom software to control a 3-axis stage manufactured by Galil Inc, and custom 1-axis micromanipulator for robotic operations. The software also employs a Sony charged-coupled device sensor for real-time image feedback and data acquisition. The system is mounted on a Carl Zeiss Axiovert 200 inverted microscope. Custom-built environmental controls are used to maintain the temperature, humidity, and gas conditions for extended live cell work. The software was designed using Visual C++ for the Windows PC platform using an object orientated and modular design methodology to allow the BAMBI software to continue to grow with new tasks and demands as needed. The modular approach keeps functional groups of code within context boundaries allowing for easy removal, addition, or changes of functions without compromising the usability of the whole system. BAMBI has been used to image cells within a novel cell culture chamber that constricts cell growth to a true monolayer for high-resolution imaging. In one specific application, BAMBI was also used to characterize and track the development of individual Colony Forming Units (CFU) over the five-day culture period in 5-day CFU-Hill colony assays. The integrated system successfully enabled the tracking and identification of cell types responsible for the formation of the CFU-Hill colonies (a putative endothelial stem cell). BAMBI has been used to isolate single hematopoietic stem cell (HSC) candidate cells, accumulate long-term live cell images, and then return these cells back to the in-vivo environment for further characterization. From these results, further data mining and lineage informatics suggested a novel way to isolate and purify HSCs. Studies such as these are the fundamental next step in developing new therapies for regenerative medicine in the future

    Rapid prototyping of soft bioelectronic implants for use as neuromuscular interfaces

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    Neuromuscular interfaces are required to translate bioelectronic technologies for application in clinical medicine. Here, by leveraging the robotically controlled ink-jet deposition of low-viscosity conductive inks, extrusion of insulating silicone pastes and in situ activation of electrode surfaces via cold-air plasma, we show that soft biocompatible materials can be rapidly printed for the on-demand prototyping of customized electrode arrays well adjusted to specific anatomical environments, functions and experimental models. We also show, with the monitoring and activation of neuronal pathways in the brain, spinal cord and neuromuscular system of cats, rats and zebrafish, that the printed bioelectronic interfaces allow for long-term integration and functional stability. This technology might enable personalized bioelectronics for neuroprosthetic applications

    Investigating the Use of Streaming and Robotic Dielectrophoresis to Enable Continuous Cell Sorting and Automatic Cell Transfer in Sample Preparation

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    The sorting of targeted cells or particles from a sample is a crucial step in the sample preparation process used in medical diagnosis, environmental monitoring, bio-analysis and personalized medicine. Current cell sorting techniques can be broadly classified as label based or label-free. Label-based techniques mostly rely on fluorophores or magnetic nanoparticles functionalized to bind with targeted cells. Although highly specific, this approach can be expensive and suffers from limitations in the availability of suitable markers. Label-free techniques exploit properties inherent to the cell, such as density and size, to simplify the sorting protocol and reduce cost by eliminating the need to incubate samples with labels. However, the specificity of separation is low due to minor differences between the density and size of many cells of interest. In this work, the use of dielectrophoresis (DEP) is emphasized as a label-free technique that exploits the combination of size and membrane capacitance of a cell as a marker. DEP is the movement of dielectric particles in the presence of a non-uniform electric field, which can be towards the electrode (positive DEP) or away from electrode (negative). The cell membrane capacitance used in this project can distinguish between cells based on their type, age, fate, and circadian rhythm to provide higher specificity than other label free sorting techniques. DEP has been demonstrated to separate various bio particles including viruses, plant and animal cells, biomolecules as well as stem cells. The work presented here integrates fabrication, numerical simulations, analysis and experimentation to focus on three main objectives 1) addressing the gaps of knowledge in electrode fabrication 2) developing analytical system for rapid cell sorting of cell population 3) demonstrating feasibility of automated single cell sorting. These are addressed in the following paragraphs in that order. Carbon electrodes are excellent alternatives for DEP because of the ease of fabrication of 3D electrode geometries and low voltages involved. Previous works have used these electrodes for applications like DEP, electrochemical bio sensing applications, fuel cells and micro-capacitors. The fabrication process involves photo patterning of SU-8 posts followed by carbonization in an inert atmosphere. During pyrolysis, the structures retain their shape, but show shrinkage. Though the fabrication process is reproducible, limited knowledge is available about the shrinkage process. Shrinkage affects the design of devices where these structures are used because the electrode dimensions after pyrolysis vary from the design and resulting electric field in the domain is affected. Previous works observed dependence of shrinkage on structure height and width, but a defining relation between shrinkage and the geometry was lacking. In this work, shrinkage is studied as an effect of degassing through the lateral and top surface area of the electrodes. Empirical relations are to enable prediction of shrinkage in the design stage StreamingDEP refers to focusing particles into narrow streams with a proper play of positive DEP and drag force. This is important in continuous sorting because of the high throughput, limited exposure of cells to electric field and ability of integration to further analysis steps. Though streamingDEP has been demonstrated previously, the dependence of the particle focusing on system parameters has not been studied. In this work, an analytical model is built to study the effect of electrode geometry, flow and electric field parameters as well as cell properties. The analytical expression developed here is validated by experiments and simulations. Robotic transfer is required for efficient handling of cells and integration with analysis steps. Liquid handling robots are currently used in laboratories to transfer cells between different steps. Though they have precise control over the transfer of cells, the sorting ability is limited. To address these limitations, a proof-of-concept of roboticDEP device was innovated to enable transfer of targeted cells. The development of this system required studying the influence of DEP parameters in pick up and transfer of cells. The device was studied for elimination of contamination by using flow and electric field. The robotic DEP platform demonstrates a novel and unique approach to automated cell sorting with potential applications for single cell analysis, cell sorting and cell patterning

    Development of Advanced Optics and High Resolution Instrumentation for Mass Spectrometry Based Proteomics

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    Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain spatial and chemical information of analytes on a wide variety of surfaces. The ability to image biological analytes is an important tool in many areas of life science research, including: the ability to map pharmaceutical drugs in targeted tissue, to spatially determine the expression profile of specific proteins in healthy vs. diseased tissue states, and to rapidly interrogate biomolecular microarrays. However, there are several avenues for improving the imaging MS experiment for biological samples. Three significant directions this work addresses include: (1) reducing chemical noise and increasing analyte identification by developing sample preparation methodologies, (2) improving the analytical figures of merit (i.e., spatial resolution, analysis time) by implementing a spatially dynamic optical system, and (3) increasing both mass spectral resolution and ion detection sensitivity by modifying a commercial time-of-flight (TOF) MS. Firstly, sample methodology schemes presented in these studies consist of obtaining both ?top-down? and ?bottom-up? information. In that, both intact mass and peptide mass fingerprinting data can be obtained to increase protein identification. This sample methodology was optimized on protein microarrays in preparation for bio tissue analysis. Other work consists of optimizing novel sample preparation strategies for hydrated solid-supported lipid bilayer studies. Sample methods incorporating nanomaterials for laser desorption/ionization illustrate the ability to perform selective ionization of specific analytes. Specifically, our results suggest that silver nanoparticles facilitate the selective ionization of olefin containing species (e.g., steroids, vitamins). Secondly, an advanced optical design incorporating a spatially dynamic optical scheme allows for laser beam expansion, homogenization, collimation, shaping, and imaging. This spatially dynamic optical system allows user defined beam shapes, decreases analysis times associated with mechanical movement of the sample stage, and is capable of increasing the MS limits of detection by simultaneously irradiating multiple spots. Lastly, new data acquisition strategies (multiple anode detection schemes) were incorporated into a commercial time-of-flight mass spectrometer to increase both sensitivity and resolution in a matrix assisted laser desorption/ionization mass spectrometer. The utility of this technique can be applied to many different samples, where high mass spectral resolution allows for increased mass measurement accuracy

    The Boston University Photonics Center annual report 2006-2007

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    This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2006-2007 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This annual report summarizes activities of the BUPC over the period of July, 2006 through June, 2007, corresponding to the University’s fiscal year. These activities span the Center’s complementary missions in research, education, technology development, and commercialization. This reporting period included a milestone, as BUPC completed its tenth year of operation in its landmark building in the heart of the University’s Charles River Campus. Faculty research activity reached an all time high when evaluated by the usual metrics of external funding, scholarly publications, honors and awards. The Center’s educational programs were bolstered by two summer programs hosting more than 40 undergraduate interns, and by the launch of a competitive graduate fellowship program sponsoring ten BUPC graduate fellowships. In technology development, the prototype RedOwl sniper detection system pioneered by Center faculty, staff, and industry partners was fieldtested by the US Department of Defense, and has been handed off to industry partners for further pre-commercial development. Three new defense/security prototypes were developed by BUPC to address critical national defense needs in the past year and 13 faculty development projects were supported in collaboration with the Army Research Laboratory to fill the technology pipeline for our future defense-related prototyping efforts. The Center’s business incubator had a transformative year. After revising its core mission and operational strategy in the summer of 2006, the incubator generated significant demand for the intellectual environment, facilities, and expertise available to participating companies. New companies attracted by this revised value proposition now occupy all available space

    Design and Modeling of Multi-Arm Continuum Robots

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    Continuum robots are snake-like systems able to deliver optimal therapies to pathologies deep inside the human cavity by following 3D complex paths. They show promise when anatomical pathways need to be traversed thanks to their enhanced flexibility and dexterity and show advantages when deployed in the field of single-port surgery. This PhD thesis concerns the development and modelling of multi-arm and hybrid continuum robots for medical interventions. The flexibility and steerability of the robot’s end-effector are achieved through concentric tube technology and push/pull technology. Medical robotic prototypes have been designed as proof of concepts and testbeds of the proposed theoretical works.System design considers the limitations and constraints that occur in the surgical procedures for which the systems were proposed for. Specifically, two surgical applications are considered. Our first prototype was designed to deliver multiple tools to the eye cavity for deep orbital interventions focusing on a currently invasive intervention named Optic Nerve Sheath Fenestration (ONSF). This thesis presents the end-to-end design, engineering and modelling of the prototype. The developed prototype is the first suggested system to tackle the challenges (limited workspace, need for enhanced flexibility and dexterity, danger for harming tissue with rigid instruments, extensive manipulation of the eye) arising in ONSF. It was designed taking into account the clinical requirements and constraints while theoretical works employing the Cosserat rod theory predict the shape of the continuum end-effector. Experimental runs including ex vivo experimental evaluations, mock-up surgical scenarios and tests with and without loading conditions prove the concept of accessing the eye cavity. Moreover, a continuum robot for thoracic interventions employing push/pull technology was designed and manufactured. The developed system can reach deep seated pathologies in the lungs and access regions in the bronchial tree that are inaccessible with rigid and straight instruments either robotically or manually actuated. A geometrically exact model of the robot that considers both the geometry of the robot and mechanical properties of the backbones is presented. It can predict the shape of the bronchoscope without the constant curvature assumption. The proposed model can also predict the robot shape and micro-scale movements accurately in contrast to the classic geometric model which provides an accurate description of the robot’s differential kinematics for large scale movements

    Studies in biological surface science: microfluidics, photopatterning and artificial bilayers

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    Herein is presented the collective experimental record of research performed in the Laboratory for Biological Surface Science. These investigations are generally classified under the category of bioanalytical surface science and include the following projects. Chapters III and IV describe the creation of a microfluidic device capable of generating fixed arrays of concentration gradients. Experimental results were matched with computational fluid dynamics simulations to predict analyte distributions in these systems. Chapters V and VI demonstrate the discovery and utility of photobleaching fluorophores for micropatterning applications. Bleached fluorophores were found to rapidly attach to electron rich surfaces and this property was used to pattern enzymes inside microfluidic channels in situ. Finally, Chapter VII exhibits a method by which solid supported lipid bilayers can be dried and preserved by specifically bound proteins. The intrinsic property of lateral lipid mobility was maintained during this process and a mechanism by which the protein protects the bilayer was suggested
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