Guidance of neuronal growth with DEP J. James Jun Presenter University of Calgary Dr. Rudolf Potucek Supervisor January 25, 2007 Hi. My name is James and I would like to present my research project on guidance of neuronal growth with DEP.

Application of dielectrophoresis(DEP) for directing neuronal growth Neuronal Guidance Theory of DEP I will talk about three things during this talk. First I will talk about the goal of my research and second I will talk about the related theories and finally I will talk about the experiments and modelling. Modeling & Experiment

Neuronal growth: random vs. directed Neurons generally do not regenerate once damaged. This might explain why paraplegic and quadriplegic patients almost never regain their ability to control their body. However neurons can exhibit growth in abundance of neurotrophic factors. Under this environment, neurons undergo rapid growth in random directions. When one wants to achieve neuronal regeneration of damaged nerve connection, neuronal growth must be directed since random growth is slow and requires a lot more energy. Directional growth can be realized by applying a force in a certain direction to a point of growth called growthcone. There are several ways to apply a directional force to a growthcone. One way is to physically pull the growthcone but this method can damage the cell by rupturing the membrane. Thus non-contact force is preferred for guiding a neuronal growth. Random Directed

Electric forces: Electrophoresis vs Dielectrophoresis Any electric fields Charged object DC Dielectrophoresis Spatially varying field Polarizable object DC or AC Electric force is a non-contact based force that can be easily generated by creating an electric field with electrodes. There exists two types of electric force: electrophoresis or EP and dielectrophoresis or DEP. Electrophoresis is a simple coulombic force and all charged particle in an electric field undergoes electrophoresis. Dielectrophoresis is a force caused by a polarization of a particle in spatially non-uniform field. Dielectrophoresis requires the gradient of the electric field to be non-zero in order to have non-zero net force. Electrophoresis requires DC current to create directional force because if polarity of the field reversed as in AC, the force reverses and the time averaged force becomes zero. Dielectrophoresis however can work in both DC and AC current because if polarity of the field reversed, the polarization of the particle also reverses thus the direction of the force remains the same. In a conductive medium with ion species like the biological medium used in this experiment, DC current produces biochemical reaction that can damage both electrodes and cells. But AC current at high frequency has little or no biochemical impact except producing minor Joule heating which is amplitude-dependent. Thus DEP with AC current is an ideal way to exert a force on a growthcone.

Modeling and Experiment Pond snail Brain Neuron My experiment is mainly divided into three parts due to a multidisciplinary nature of this research. Biological experiment, physical modeling, and software development are these three main parts. My research group works with snail neurons and I had to acquire basic dissection skill to take out a brain from a snail and to collect neurotrophic factors from these brains. Due to extremely time consuming nature of the biological preps, I decided to do initial experiments with polymer beads whose size is about the size of the snail neuron which is about 100 microns. Microsphere Field Modeling Vision Tracking

Experiment with polymer bead The initial experiment will be performed with polymer bead with a similar size to the actual snail neuron which is about 100 microns. A polymer bead requires a lot less effort to prepare compare to the actual snail. There is no need for a dissection and other biological preperation efforts. Thus I decided to carry out an initial experiment with a polymer bead with similar electric properties to a real snail neuron. The bead will be located in between two electrodes. The electrode has an insulated tip. The electric field modeling is done with similar setup.

Electric field modeling Quadruple electrodes configuration top Here is an electric field modeling of a quadruple electrodes configuration is done with finite element modeling software called Coulomb. The four spatially arranged electrode create electric fields. In order to create a directionality, one electrode is set to have an antiparallel phase with respect to other three phases. This field diagram shows an electric field when one electrode has potential of 1v and other electrodes have potential of -1v. A bead is located at the center and the electric field through the bead is spatially varying thus creating dielectrophoresis. The actual experiment will be performed with two arm motorized micromanipulator which resembles robotic arms. side

Computer vision tracking I have developed a technique of tracking a growthcone during the last summer. I worked for Rudolf to develop a technique of visually tracking a growthcone. I have developed various computer vision algorithms for noise suppression and optimized tracking methods. This software vision tracking technique will be used for my experiment for following the growthcone and guiding until the desired destination is reached.

Experiment with a live snail neuron Challenges More complicated than the experiment with beads Long term experiment (3 days) Need an evaporation control with perfusion system Need a neurotrophic factor enriched medium Once the experiment with polymer bead is successful and provided that I still have enough time left, I will carry out experiments with a real snail neuron. There lies several challenges in experimenting with a live snail neuron. The experiment need to run for long term in order to see the growth of a snail neuron. This means there need to be means of controlling evaporation of medium. This is typically done with a perfusion system which circulates medium. Also the growth factors need to be stuck to the bottom of the dish so that the growth factors are not lost during the perfusion. If this experiment with a live snail neuron takes longer than this term, this project will be continued the summer term.

Conclusions DEP can be used to direct a neuronal growth with electric field modeling and computer vision tracking DEP is a non-contact force and bio-chemically safe Electric field modeling to optimizes DEP parameters Computer vision tracking to track a growthcone Experiments planned with beads then snail neurons Conclusion slide. Use the headline (no more than two lines) to state your most important conclusion. Begin the headline with In summary or In conclusion to ensure that the audience knows they have come to the presentation’s end. Support that headline with an image and parallel points. This slide should be your last slide. Audiences lose patience when they believe that they have come to the end, but other slides follow. Notice that the word Questions appears at the bottom of this slide. That strategy is much more effective than burning a slide with just the word Questions. This slide allows the audience to look at the most important slide of the presentation during the question period. See CSP, pages 65, 150, 151-152. Questions?