| Popis: |
In this thesis, experiments into artificially guiding neuronal growth cones using tightly focused laser beams were performed and evaluated. The experiments are performed by focusing a laser beam to the leading edge of a developing growth cone and attempting to change the direction of growth cone. These experiments were carried out using Gaussian, line and asymmetric line beam profiles. There was no noticeable change in the success rate with different beam profiles. Following this, I assisted my colleague Dr Michael Mazilu in the construction of a mathematical model of filopedia in an optical field in order to help explain the mechanism for optically guided neuronal growth which suggests that optical trapping forces on filopedia are responsible. Next, I set about implementing a system to automate the process of laser guided neuron growth by employing a spatial light modulator and a custom-built computer program. This allowed the computer to track a developing growth cone and automatically adjust the position of the laser beam as the growth cone developed. This program was successfully employed to artificially grow neuronal growth cones towards a user-inputted target point. The use of the spatial light modulator to beam shape was also demonstrated with the use of a Bessel beam being used to guide neurons for the first time. I also used a transgenic cell line of neurons to show for the first time that HSP70 is not involved in this phenomenon. This was accomplished by transfecting NG108’s with a plasmid containing HSP70 promoter tagged GFP. Under enough thermal or mechanical stress, the cells would express HSP70 which would produce a detectable GFP signal. No GFP was detected in cells after being exposed to laser irradiation of a power higher than would normally be used to guide neurons. Combined, these experiments show that the beam profile of the operating laser does not significantly affect the success of artificial growth and that the optical force on filopedia near the laser beam is likely to be the mechanism for this phenomenon. A possible heating effect of the laser has also been shown to not be strong enough to elicit a heat shock stress response from the cell. The demonstration of an automatic system which incorporates beam shaping has also been shown and such a system shows the potential to advance the investigation of artificial neuron growth using lasers. |
