Super-resolution multiphoton imaging of synaptic transmission

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.

By combining structured illumination with localisation and processing algorithms developed for stochastic imaging techniques, I have developed a super-resolution multiphoton microscope with a more than two fold improvement in lateral (xy) and axial (z) resolution. By using an acousto-optic deflector (AOD) and an acousto-optic modulator (AOM) system that allows wavelength dependent compensation of temporal and spatial aberration associated with pulsed lasers passing through AOD devices, it is possible to illuminate specimens with a sequence of overlapping grid patterns. Each diffraction-limited spot is identified and located and then all spots are processed to produce a super-resolution image. The microscope can also be used in a high-speed mode where fluorescent light from individual points of interest can be measured at rates up to 50 KHz.

In this application, we would first like to rebuild the microscope using optical components that are optimised for multiphoton imaging and then to characterise the improvement in spatial and temporal performance that is gained. This will enable us to speak with companies with a view towards commercialisation. The microscope will then be used in both high speed and super-resolution modes to examine the role of presynaptic calcium signalling on the release characteristics of synapses formed between cerebellar granule cells and Purkinje cells. We have previously shown that synapses formed by different segments of the same axon have very different release properties. We intend to establish whether spatial or temporal differences in calcium signalling are responsible for these differences. Finally, we will use a transgenic mouse developed in my laboratory that expresses a calcium sensor selectively in presynaptic terminals to find the mechanisms responsible for differences in synaptic release.

This project benefits significantly upon the prior development of a series of tools and technologies designed to facilitate visualisation of dynamic events in living tissue. The prototype multiphoton microscope described here is unique and has a great potential for commercial success because it is faster and operates at a higher resolution than any other currently available. I have previous experience of microscope development and I am already working with a company (Prior Scientific) to commercialise a high-speed, digital confocal microscope. Knowing that this can be a fairly long-winded process, I intend to contact companies at a very early stage of this project and to apply for further funding to assist in commercialisation within the first 12 months. I will apply for Pathfinder funding for a market report so that we can assess the most appropriate way to take the technology to market. It is likely that the intellectual property will be protected through a patent application and I am already in discussions with the enterprise office at Leicester University in this regard.

Preliminary to this application, we received BBSRC funding to develop a series of sensors that allow aspects of synaptic activity to be measured optically in real time. One of the calcium sensors we made has been use to generate two strains of transgenic mouse. One of these strains will be used in this application. Sensor expression is under the control of the thy1.2 promotor. In the first mouse, expression is observed in most regions of the brain we have so far studied but it is particularly high in hippocampus. This provides a tool that is potentially very useful to the entire neuroscience community because we can use these mice to look at presynaptic activation in models ranging from dissociated cultures, to organotypic cultures to in vivo measurements in awake, behaving animals. The mice can be used to detect synaptic connectivity and measure signalling strength and, as such, should be hugely useful to the entire neuroscience community because it will be possible to examine precisely where and when presynaptic inputs to neurones and non-neuronal cells in the brain are activated and how they are modified.

The transgenic mouse can also be interbred with other animals and so we envisage it will be possible to make transgenic mice that have both a model of disease such as Alzheimer's disease as well as a sensor that allows a direct measurement of potential changes in synaptic transmission during disease progression.

In order to publicise the importance of these sensors, we need to demonstrate their use in situ and this is one of the corollary aims of this application. We have now characterised the sensors in model systems and used them in hippocampal cultures to examine synaptic signalling and we are in the process of submitting this work for publication. Academic beneficiaries will be informed through the usual means of publication and conference attendance but we will also bring this to the attention of our enterprise office, members of which actively inform various companies about technologies developed at Leicester University.

Other beneficiaries include the PDRA who will receive training in a range of disciplines including optics, electrophysiology and imaging as well as generic skills such as data analysis.