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

ISOFLIM：3D 神经元培养物的各向同性分辨率荧光寿命成像

基本信息

批准号：
BB/T014318/1
负责人：
Simon Ameer-Beg
金额：
$ 73.12万
依托单位：
King's College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT014318%2F1
关键词：
ISOFLIM Isotropic resolution fluorescence lifetime

项目摘要

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.

这个多学科的研究团队站在显微镜发展的前沿，旨在探索活神经元内的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专门创建的通道中生长。神经元在这些通道中建立联系，使我们能够像在大脑中一样实时研究细胞间的交流--以一种完全可控的方式。一旦我们培育出这些“湿式人工神经网络”，我们就可以用先进的显微镜来描绘它们复杂的信号行为。在这项提议中，一个新的显微镜概念将被开发出来，它将推动在细胞水平上所能看到的东西的极限。通过在样品中创建光学焦点的三维晶格，并并行地读出它们，我们可以创建样品的3D表示。利用主要用于汽车行业3D检测和测距(LIDAR)的超精密相机技术，称为SPAD传感器阵列，我们将使用一种名为荧光寿命成像显微镜(FLIM)的技术来测量能量代谢等生物过程发生的速度。Flim在检测荧光分子的变化方面非常强大，可以用来测量蛋白质-蛋白质相互作用或蛋白质构象的变化--控制细胞行为的基本过程。通过在蛋白质上添加荧光标签并用激光照射它们，我们可以使用SPAD传感器阵列在细胞中可视化它们。当其中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，从而改变它们发出的光量。这种荧光共振能量转移(FRET)可以被测量来检测蛋白质的相互作用。Flim测量荧光寿命在FRET过程中如何变化，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。测量移动细胞中的FRET的第二个困难是，许多成像技术太慢，来自激光的光量可能会损害细胞。我们的新显微镜方法ISO-Flim(因为它生成了各向同性的分辨率图像)在一张照射到样品上的光线中产生光束，由灵敏的相机记录下来，使其快速且对细胞没有损害。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、快速地测量活着的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“实时”机制。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.

基于 FRET 的原位心肌细胞中肌球蛋白结合蛋白 C 的 N 末端定位。

DOI：
10.1073/pnas.2222005120
发表时间：
2023-03-21
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Chandler, Jessica;Treacy, Conor;Ameer-Beg, Simon;Ehler, Elisabeth;Irving, Malcolm;Kampourakis, Thomas
通讯作者：
Kampourakis, Thomas

FRET Sensor-Modified Synthetic Hydrogels for Real-Time Monitoring of Cell-Derived Matrix Metalloproteinase Activity using Fluorescence Lifetime Imaging