Super-resolution multiphoton imaging of synaptic transmission

近年来，几项技术进步导致了显微镜的发展，这种显微镜可以比以往任何时候都更详细地观察物体。我们可以分辨出比之前认为的更近的物体，这催生了各种不同的方法，使得现在被称为“超分辨率”的显微镜成为可能。我已经开发了一种独特的原型显微镜，它使用红外线激光操作，可以可视化组织内部的荧光分子。目前，与现有的类似类型的显微镜相比，它的空间分辨率提高了两倍以上，并且它还可以在不同的模式下以超过商业系统100倍的速度运行。在此，我们希望继续开发这种显微镜，以进一步提高速度和分辨率，并使其足够方便用户在活体脑组织中进行实验。我们将开发显微镜，以期将该技术商业化，使其易于为其他研究人员所用。然后，我们想用它来研究为什么大脑中可兴奋细胞(突触)之间的一些特殊通信点的行为与其他细胞不同。我们将比较相同细胞形成的不同突触中钙信号的差异，因为钙既触发并控制大脑中化学交流的敏感性。突触前终末内钙的绝对水平可能很好地代表了短期记忆保持的机制。然后，我们将使用电活动的测量以及可视化释放的光学方法来研究钙信号和递质释放之间的联系。最后，我们将使用一种经过基因改造的小鼠，在突触前终端表达钙传感器，以确定导致突触钙信号和递质释放差异的机制是什么。

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.