3D 6DOF Manipulation of Micro-object Using Laser Trapped Microtool

Proceedings of the 2006 IEEE International Conference on Robotics and Automation, Orlando, Florida, May 200

PLANNING FOR AUTOMATED OPTICAL MICROMANIPULATION OF BIOLOGICAL CELLS

Optical tweezers (OT) can be viewed as a robot that uses a highly focused laser beam for precise manipulation of biological objects and dielectric beads at micro-scale. Using holographic optical tweezers (HOT) multiple optical traps can be created to allow several operations in parallel. Moreover, due to the non-contact nature of manipulation OT can be potentially integrated with other manipulation techniques (e.g. microfluidics, acoustics, magnetics etc.) to ensure its high throughput. However, biological manipulation using OT suffers from two serious drawbacks: (1) slow manipulation due to manual operation and (2) severe effects on cell viability due to direct exposure of laser. This dissertation explores the problem of autonomous OT based cell manipulation in the light of addressing the two aforementioned limitations. Microfluidic devices are well suited for the study of biological objects because of their high throughput. Integrating microfluidics with OT provides precise position control as well as high throughput. An automated, physics-aware, planning approach is developed for fast transport of cells in OT assisted microfluidic chambers. The heuristic based planner employs a specific cost function for searching over a novel state-action space representation. The effectiveness of the planning algorithm is demonstrated using both simulation and physical experiments in microfluidic-optical tweezers hybrid manipulation setup. An indirect manipulation approach is developed for preventing cells from high intensity laser. Optically trapped inert microspheres are used for manipulating cells indirectly either by gripping or pushing. A novel planning and control approach is devised to automate the indirect manipulation of cells. The planning algorithm takes the motion constraints of the gripper or pushing formation into account to minimize the manipulation time. Two different types of cells (Saccharomyces cerevisiae and Dictyostelium discoideum) are manipulated to demonstrate the effectiveness of the indirect manipulation approach

Magnetically Driven Micro and Nanorobots

Manipulation and navigation of micro and nanoswimmers in different fluid environments can be achieved by chemicals, external fields, or even motile cells. Many researchers have selected magnetic fields as the active external actuation source based on the advantageous features of this actuation strategy such as remote and spatiotemporal control, fuel-free, high degree of reconfigurability, programmability, recyclability, and versatility. This review introduces fundamental concepts and advantages of magnetic micro/nanorobots (termed here as "MagRobots") as well as basic knowledge of magnetic fields and magnetic materials, setups for magnetic manipulation, magnetic field configurations, and symmetry-breaking strategies for effective movement. These concepts are discussed to describe the interactions between micro/nanorobots and magnetic fields. Actuation mechanisms of flagella-inspired MagRobots (i.e., corkscrew-like motion and traveling-wave locomotion/ciliary stroke motion) and surface walkers (i.e., surface-assisted motion), applications of magnetic fields in other propulsion approaches, and magnetic stimulation of micro/nanorobots beyond motion are provided followed by fabrication techniques for (quasi)spherical, helical, flexible, wire-like, and biohybrid MagRobots. Applications of MagRobots in targeted drug/gene delivery, cell manipulation, minimally invasive surgery, biopsy, biofilm disruption/eradication, imaging-guided delivery/therapy/surgery, pollution removal for environmental remediation, and (bio)sensing are also reviewed. Finally, current challenges and future perspectives for the development of magnetically powered miniaturized motors are discussed

Characterization of CD93 Diffusion in Human Monocytes and Chinese Hamster Ovary Cells.

The diffusion of CD93, a putative efferocytic receptor, was studied in the membrane of human monocytes and CHO cells using single particle tracking. We found that of CD93 molecules were confined in compartments consistent with actin corrals, while moved freely. Cage effect analysis showed short-lived caging of free CD93 and longer-lasting caging of confined CD93, with smaller corrals resulting in stronger caging. The motion of both free and confined CD93 was found to be consistent with a subdiffusive continuous time random walk, suggesting that CD93 diffusion is affected by several processes. We also sought to develop a total internal reflection fluorescence compatible traction force microscopy substrate intended for use in force characterization in frustrated efferocytosis. TIRF-compatible silicone substrates of a uniform thickness were manufactured successfully, but were found to be unsuitable for reliable force measurements due to their susceptibility to the non-specific adsorption of quantum dots

Microbial biodeterioration of outdoor stone monuments: Assessment methods and control strategies

Biodeterioration is the least understood decay mechanisms of outdoor stone monuments. Microbial colonisation is largely determined by the properties of the stone and environmental conditions. The literature on microorganisms on outdoor stone monuments and their decay mechanisms was reviewed. For the assessment and quantification of microbial deterioration, methods that can be carried out by cultural heritage conservators with limited microbiological skills were selected and adjusted for the application on outdoor stone monuments. To this end, the total biomass was quantified by a protein assay (Folin-Lowry method), its phototrophic contribution through chlorophyll a absorbance and the amount of extracellular substances (EPS) were assessed by carbohydrate quantification (phenol method). Microbial activity was measured through two different enzyme assays: fluorescein diacetate cleavage and dehydrogenase activity (INT reduction). In order to develop a long-term monitoring strategy, these parameters were tested in the morning (8 am) and in the afternoon (4 pm) on biofilms from a sunny and a shady sampling site on a limestone wall in the south of Mexico. The experiments were performed in the dry season and the rainy season. Changes in biofilm composition and activity during the day were very small, while seasonal changes were more pronounced. The largest differences could be seen in samples from the different sampling sites (sun and shade), where the microbial population had established over years of distinct environmental conditions. Variations in biofilm composition and activity exceeding such natural variation may indicate the necessity for an antimicrobial treatment. The choice of an antimicrobial agent is difficult and the ideal treatment does not exist. Of the various chemical antimicrobial agents tested (Mergal K14, Parmetol DF12, Troysan S97, Preventol R50 hydrogen peroxide and ethanol) on microbial biofilms on stone, ethanol (70%) was the most effective, as revealed by ATP measurements. A flexible, non-invasive in vivo system, employing the bioluminescent bacterium Vibrio fischeri, was developed to assess sub-lethal effects of antimicrobial treatments and to test combined treatments for synergy. Various biocides and ultrasound (267 kHz, 20 kHz), alone and in combination, were tested for their effect on V. fischeri (Mergal K14, Parmetol DF12, Troysan S97, Preventol R50 hydrogen peroxide and ethanol) and a microbial biofilm on stone (Troysan S97, Preventol R50 and ethanol). The tests did not reveal synergistic effects however, a systematic, comprehensive study on chemical and/or physical methods might reveal an innovative approach towards a more environmentally friendly microbial eradication method for outdoor stone monuments. Long-term monitoring of the composition and activity of a microbial biofilm may provide data to determine if an antimicrobial treatment is necessary. If an antimicrobial intervention cannot be avoided, low-toxic substances, such as ethanol, should be considered first. For the evaluation of the success of an antimicrobial treatment, ATP measurement has proven to be a reliable and simple method that does not require specialised skills

Enzymatic activity and mechanical stability of cellulosomal components

The work presented in this thesis consists of two lines of research, both inspired by the intricate multi-enzyme complexes called cellulosomes. Cellulosomes are extracellular machines produced by anaerobic bacteria to efficiently degrade plant cell wall polysaccharides. To this end they employ an arsenal of specialized enzymes arranged on hierarchal, multi-domain protein scaffolds by means of non-covalent receptor-ligand interactions. Cellulosomes are highly effective tools for cellulose degradation due to a range of evolutionary adaptations, including targeted substrate adhesion, intelligent substrate-adjusted enzyme composition, efficient assembly mechanisms, and enhanced mechanical properties. The first part of this thesis describes development of the novel assay for the determination of cellulolytic activity of multi-component enzyme mixtures on lignocellulosic substrates. The crucial feature of the assay is a polymerization-based amplification scheme that effectively integrates and localizes the signal in the form of an insoluble hydrogel. Quantitative readout of the amount of polymer formed is achieved in both bulk and microscale implementations, including fluorescence microscopy, turbidity measurements and scanning microscopy. When bulk readout modalities are employed, the assay enables the use of natural biomass substrates in screening applications, addressing a shortcoming of the currently used methods. Insight into cellulose degradation at the microscale is enabled by combining the assay with time-resolved imaging techniques, specifically TIRF microscopy. The second part of the work concentrates on the unique mechanical properties of cellulosomal components. Particularly, highly specific protein-protein complexes responsible for the assembly of cellulosomes are investigated. These cohesion-dockerin non-covalent links bridge bacterial host cell and cellulosic carbon sources in turbulent environments, and therefore are subject to mechanical forces in vivo. One of the strongest known receptor-ligand pairs is reported as part of this thesis. First, the complex is characterized using single molecule force spectroscopy. To this end, an improved experimental protocol was developed and implemented. Next, the mechanisms behind the exceptional mechanostability of the interaction were elucidated employing full-atom steered molecular dynamic simulations, in collaboration with the group of prof. Klaus Schulten from University of Illinois, USA. A new network based analysis of simulation trajectories is developed to visualize the force propagation paths through the protein complexes.In der vorliegenden Arbeit werden zwei verschiedene Forschungsprojekte vorgestellt, die bei- de von komplizierten Multienzymkomplexen, auch Cellulosome genannt, inspiriert wurden. Cellulosome sind extrazelluläre Maschinen, die von manchen Bakterien zur Zersetzung von Polysacchariden aus den Zellwänden von Pflanzen eingesetzt werden. Zu diesem Zweck verwenden sie eine Vielzahl von spezialisierten Enzymen, die mittels nicht-kovalenter Rezeptor Ligand Wechselwirkungen auf hierarchisch aufgebauten Protein Gerüsten angeordnet werden. Durch eine Reihe evolutionärer Anpassungen wie gezielter Substratanbindung, substratspezifischer Enzym Zusammensetzungen, effizienter Assemblierungsmechanismen, enzymatischer Synergieeffekte und hochangepassten mechanischen Eigenschaften sind Cellulosome hocheffektive Werkzeuge für den Celluloseabbau. In ersten Teil dieser Arbeit wird die Entwicklung eines neuartigen Assays zur Bestimmung der cellulytischen Aktivität von mehrkomponentigen Enzym Mischungen auf lignocellulosischen Substraten beschrieben. Das Kernelement dieses Assays ist ein polymerisationsbasierter Amplifikationsmechanismus der das Signal mittels eines unlöslichen Hydrogels integriert und lokalisiert. Dabei wird eine quantitative Auslese des produzierten Polymers für Makro- wie Mikroimplementationen erreicht. Dabei werden unter anderem Fluoreszenzmikroskopie, Trübungsmessungen und Rastersondenmikroskopie verwendet. Für Ensemble Auslesemethoden ermöglicht das Assay den Einsatz natürlicher Biomasse als Substrat und greift damit eine der Schwächen herkömmlicher Methoden auf. Weiter wird ein zusätzlicher Erkenntnisgewinn über die Zersetzungen von Celluolse auf der Mikroskala durch die Kombination des Assays mit Bildgebungsverfahren wie Totalreflexionsfluoreszenzmikroskopie (TIRF) ermöglicht. Der zweite Teil der Arbeit beschäftigt sich mit den einzigartigen mechanischen Eigenschaften cellulosomaler Komponenten. Insbesondere werden hochspezifische Proteinkomplexe, die für die Assemblierung von Cellulosomen verantwortlich sind, untersucht. Diese Komplexe formen eine nicht-kovalente Brücke zwischen bakteriellen Wirtszellen und deren cellulosischen Kohlenstoffquellen. Durch die turbulenten Umbgebungen, in denen diese Bakterien zu finden sind, unterliegen diese Bindungen in vivo hohen externen Kräften. Im Rahmen dieser Arbeit wird eine der stärksten bekannten Rezeptor Ligand Wechselwirkungen beschrieben. Zunächst wird der Komplex mittels Einzelmolekülkraftspektroskopie charakterisiert. Dazu wird ein verbessertes experimentelles Protokoll vorgestellt. Anschließend werden die zugrunde liegenden Mechanismen für die extreme mechanische Stabilität der Wechselwirkung mittels atomarer Moleküldynamik Simulationen im Rahmen einem Kollaboration mit der Gruppe von Prof. Klaus Schulten von der University of Illinois, USA beleuchtet. Dabei wird ein netzwerkbasiertes Analyseverfahren der Simulationen zur Visualisierung von Kraftpropagationspfaden durch Proteinkomplexe entwickelt