Multiplexed multiphoton fluorescence lifetime microscopy: Real time imaging of protein-protein interactions at the immune synapse

从最早发明照相机开始，人类就一直在寻求观察那些人眼和大脑无法判断的过快或过于复杂的过程。第一张奔跑的马的实时图像让我们了解了它的运动，子弹击穿苹果的瞬间——图像冻结了时间上的瞬间，这样我们就可以研究微小的细节。在细胞生物学中，我们对细胞功能的理解不断发展，因为我们在显微镜下观察复杂的动态过程，通过高速相机捕捉并慢慢揭示其隐藏的复杂性。随着生物学家提出越来越复杂的问题，我们必须开发更复杂的工具来合理化我们观察到的复杂数据。我们目前对细胞中蛋白质相互作用的理解主要是通过使用显微镜工具来描述细胞器（如线粒体）内信号事件的定位和区隔。利用所谓的Förster共振能量转移（FRET）技术可以获得关于蛋白质关联的进一步见解。FRET作为一个分子标尺，使我们能够测量蛋白质或蛋白质结构域在纳米长度尺度上的相对分离。我们的工作重点是通过FRET和高分辨率荧光寿命成像（FLIM）确定蛋白质-蛋白质相互作用。不幸的是，这些先进的技术在捕捉细胞事件方面相对较慢，因此我们想要实时观察细胞迁移（通常在化学梯度作用下的定向运动）等过程的愿望受到了阻碍。通过这个项目，我们寻求显著加快蛋白质相互作用数据的获取，使我们能够观察细胞信号传导的发生。这可以通过我们的激励和检测通道的多路复用来实现，我们设想在不损失图像空间分辨率的情况下，将成像提高1000倍。这项工作代表了最先进的功能成像技术，有机会以前所未有的细节观察复杂的细胞事件：捕捉t细胞在调查癌细胞时的图像，形成动态的三维接触，并观察驱动这些过程的蛋白质信号事件；观察细胞对单一蛋白质水平的化学刺激作出反应的瞬间。我们将开发的技术将极大地提高我们对细胞内动态事件的理解，为整个生命科学中不同应用的药物相互作用提供见解。

From the earliest invention of the camera, humans have been seeking to observe processes that are too fast or too complicated for the human eye and brain to determine. The first time-laspe images of a running horse allowing us to understand its motion, the moment a bullet ripped through an apple - images, freezing a moment in time so that we can examine minute details. In cellular biology our understanding of cellular function continues to evolve as we observe complex dynamic processes played out under a microscope, captured by a camera at high speed and slowly revealing its hidden intricacy. As biologists ask every more complex questions, so we must develop more sophisticated tools to rationalise the complex data that we observe. Our current understanding of protein interaction in cells is informed, principally, through the use of microscopical tools to delineate localisation and compartmentalisation of signalling events within cellular organelles such as mitochondria. Further insight can be gained regarding protein association utilising the so called Förster resonance energy transfer (FRET) technique. FRET acts as a molecular ruler, enabling us to measure the relative separation between proteins or protein-domains on the nanometer length scale. Our work has focused on the determination of protein-protein interactions by FRET and high-resolution fluorescent lifetime imaging (FLIM). Unfortunately, these advanced techniques are relatively slow in capturing cellular events so that our desire to observe real-time the processes involved in, for example cell migration (directed motion often under the action of chemical gradient) are stymied. With this project, we seek to significantly speed up the acquisition of protein-interaction data to allows us to observe cellular signalling as it happens. This can be achieved through multiplexing of our excitation and detection channels to such an extent that we envisage a 1000-fold improvement in imaging with no loss of spatial resolution in the image. This work represents the state-of-the-art in functional imaging with the opportunity to observe complex cellular events in unpreceded detail: capturing an image of a T-cell as it surveys a cancer cell, forming dynamic 3-dimensional contacts and observing the protein signalling events that drive these processes; observing the moment a cell responds to a chemical stimulus at the level of single-proteins. The technology we will develop will drammatically improve our understanding of dynamic events within cells offering insight into drug interactions in diverse applications throughout the lifesciences.