The development of a high speed 3D 2-photon microscope for neuroscience

• Main Author: Kirkby, P. A.
• Published: University College London (University of London) 2010
• Subjects: 578
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594293

The progress of neuroscience is limited by the instrumentation available to it for studying the brain. At present, there is a serious instrumentation gap between functional Magnetic Resonance Imaging (fMRI) of whole brains and the microscopic scale functional imaging possible with today’s optical microscopes and electrophysiology techniques, such as patch clamping of individual neurons. This thesis describes the development of a new extension to optical microscopy that enables refocusing within 25 microseconds rather than the large fraction of a second possible by moving the sample or objective. The system is capable of refocusing a laser beam that is monitoring activity in 3D samples of live brain tissue 300 times faster than previously possible. This will make practical a new type of optical functional imaging for studying small sub-networks of neurons containing up to about 30,000 neurons at up to 30,000 sub micrometre sized monitored points of interest per second. The thesis describes the development of a detailed design for a new type of 3D scanner that uses Acousto-Optic Deflectors (AODs) to diffractively deflect and focus an intense laser beam beneath a conventional microscope objective. The fluorescence of calcium sensitive dyes in live neurons is used to monitor action potentials conveying signals between neurons. The optical and systems engineering problems and design trade-offs involved are discussed in detail. The results of extensive computer modelling are described and innovative solutions to several key optical physics based engineering problems are explained. The practical problems found in building a prototype machine incorporating these innovations are described and the encouraging first operational results from the machine reported.