ISOFLIM: Isotropic resolution fluorescence lifetime imaging of 3D neuron cultures

这个多学科的研究团队处于显微镜发展的最前沿，旨在探索活神经元内空间和时间的四个维度。利用生物工程，我们开发了新的方法来训练神经元在生物材料中特别创建的通道内生长。神经元在这些通道中建立连接，使我们能够像在大脑中一样实时研究细胞间的通信-以完全可控的方式。一旦我们培养出这些“湿件人工神经网络”，我们就可以使用先进的显微镜来成像它们复杂的信号行为。在这项提案中，将开发一种新的显微镜概念，推动在细胞水平上可以看到的内容。通过在样品中创建光学焦点的三维晶格，并并行地将它们阅读出来，我们可以创建样品的3D表示。使用主要为汽车行业的3D检测和测距（LIDAR）开发的超精密相机技术（称为SPAD传感器阵列），我们将使用称为荧光寿命成像显微镜（FLIM）的技术测量生物过程（如能量代谢）发生的速度。FLIM在检测荧光分子的变化方面非常强大，可用于测量蛋白质-蛋白质相互作用或蛋白质构象的变化-这是控制细胞行为的关键过程。通过将荧光标签添加到蛋白质上，并用激光照射它们，我们可以使用SPAD传感器阵列在细胞中可视化它们。当两个不同颜色的标签彼此相距一定距离时，就会发生能量转移，从而改变它们发射的光量。这种荧光共振能量转移（FRET）可以被测量以检测蛋白质相互作用。FLIM测量FRET期间荧光寿命如何变化，并且不依赖于存在多少蛋白质，使其成为检测活细胞中蛋白质相互作用的可靠方法。在移动细胞中测量FRET的第二个困难是，许多成像技术太慢，激光的光量会损坏细胞。我们的新显微镜方法ISO-FLIM（因为它生成各向同性分辨率图像）在照射到样品上的一片光中生成光束，该光束由灵敏的相机记录，使其快速且对细胞无损伤。我们的新方法结合了这些技术，创造了一种新的显微镜，可以准确快速地测量活神经元中的蛋白质相互作用，使研究人员能够观察蛋白质功能的“真实的时间”机制。

Working at the very forefront of microscope development, this multidisciplinary research team aim to explore the four dimensions of space and time within live neurons. Using bioengineering, we have developed novel ways of training neurons to grow within specially created channels in biomaterials. The neurons make connections in these channels which enable us to investigate cell-to-cell communication in real-time as it would in the brain - in an entirely controllable way. Once we have grown these "wetware artificial neural networks" we can image their complex signalling behaviour using advanced microscopy. In this proposal, a new microscope concept will be developed which pushes the envelope of what can be seen at the cellular level. By creating a 3-dimensional lattice of optical foci in the sample and, in parallel, reading them out, we can create a 3D representation of the sample. Using ultra-sophisticated camera technology which was developed principally for 3D detection and ranging (LIDAR) in the automotive industry, called SPAD sensor arrays, we will measure the speed at which biological processes such as energy metabolism occur using a technique called fluorescence lifetime imaging microscopy (FLIM). FLIM is incredibly powerful for detecting changes in fluorescent molecules and can be used to measure protein-protein interactions or changes in protein conformation - essential processes for control of cellular behaviour.By adding fluorescent tags to proteins and illuminating them with a laser we can visualise them in a cell using SPAD sensor arrays. Energy transfer occurs when two of these tags with different colours come within a certain distance of each other, changing the amount of light that they emit. This Fluorescence Resonance Energy Transfer (FRET) can be measured to detect protein interactions. FLIM measures how the fluorescence lifetime changes during FRET and is not dependent on how much protein is present, making it a robust method for detecting protein interactions in live cells. The second difficulty in measuring FRET in moving cells, is that many imaging techniques are too slow and the amount of light from the laser can damage the cell. Our new microscopy method, ISO-FLIM (since it generates a isotropic resolution image), generates beams in a sheet of light that is shone onto the sample, which is recorded by a sensitive camera, making it fast and non-damaging to the cell. Our new method combines these techniques to create a new microscope to accurately and rapidly measure protein interactions in living neurons, allowing researchers to look at the 'real time' mechanics of protein function.