Super-resolution multiphoton imaging of synaptic transmission

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In recent years, several technological advances have led to the development of microscopes that can observe objects in greater detail than ever before. We can resolve objects that are closer together than previously thought possible and this has spawned a variety of different methods that allow what is now called "super-resolution" microscopy. I have developed a unique prototype microscope that operates with an infra-red laser which allow visualisation of fluorescent molecules deep within tissues. It presently offers a more than two fold improvement in spatial resolution compared to existing microscopes of a similar type and it can also operate in a different mode at speeds over 100 times faster than commercial systems.Here, we would like to continue the development of this microscope to improve the speed and resolution further and to make it sufficiently user friendly for experimentation in living brain tissue. We will develop the microscope with a view towards commercialising the technology to make it readily available to other researchers. We would then like to use it to examine why some of the specialised points of communication between excitable cells in the brain (synapses) behave differently from others. We will compare how calcium signalling differs in different synapses formed by the same cells as calcium both triggers and controls the sensitivity of chemical communication in the brain. The absolute level of calcium within the presynaptic terminals may well represent the mechanism for the retention of short-term memory. We will then examine the link between calcium signalling and transmitter release using measurements of electrical activity as well as optical methods of visualising release. Finally, we will use a strain of mouse that has been genetically modified to express a calcium sensor in presynaptic terminals to establish what are the mechanisms that give rise to differences in calcium signalling and transmitter release at synapses.