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Two-photon microscopy has become an invaluable tool for imaging neural activity at the cellular level. The non-linear excitation process of two-photon imaging has enabled neuroscientists to observe multiple brain cell dynamics at a higher depth, while causing lower photo-damage in comparison to confocal microscopy. However, conventional galvanometer-mirror based two-photon microscopes are not agile enough to follow the action-potential path between firing neurons. A fast scanning and refocusing 3D two-photon microscope has been developed by the Department of Neuroscience, Physiology and Pharmacology of University College London capable of rapid accessing hundreds of points of interest. The instrument achieves this by employing a new type of 3D scanner that uses four Acousto-Optic Deflectors (AODs) to form a high performance Acousto-Optic Lens. The aim of this research is to develop a highly sophisticated system capable of generating the necessary signals to drive the four AODs. Such a system would enable arbitrary 3D scanning and compensate for inherent aberration of the op-tical systems as well as tissue-induced aberrations, and revolutionise neural ac-tivity imaging. Different paths to developing the control system and synthesiser were investi-gated in depth and evaluated, in order to yield a confident selection of tools, technologies, platforms and techniques. Since the early research stages, it was realised that the system should be developed around a Field Programmable Gate Array (FPGA) device, a new technology but ideal for fast parallel systems. The resulted system is capable of generating amplitude modulated, non-linear frequency ramps at a very high precision. Using several experimental setups, it was shown that with this driver the AOL is not only capable of rapid access 3D focusing, but also rapid scanning and correcting for aberrations, making it a via-ble adaptive optics device. A fast scanning, rapidly focusing lens, which can compensate for specimen and/or optical path aberrations, can revolutionise functional imaging and open new horizons in the exploration and understanding of the human brain. |
