Unravelling the Working Mechanisms of Fluorescent Molecular Rotors for Bioimaging

本文的总体目标是详细了解荧光分子转子的工作机制，特别是基于BODIPY的荧光分子转子的工作机制。这些转子可以作为探针来评估细胞膜和脂质双分子层的局部粘度。我们的目标将通过结合多尺度原子模拟和先进的荧光成像来实现。所获得的见解将允许优化荧光分子转子作为荧光显微镜中快速可靠的粘度传感器的使用，以及其他生物技术相关的粘度传感和成像分析。我的项目的第一阶段可以总结为三个问题：是否有可能准确地描述基于bodipy的原型转子（BODIPY-Phe）的势能面？我们能了解到它的激发态特性吗？哪种模拟方法是研究BODIPY-Phe最有效的工具？因此，这一阶段工作的目的是完善和比较模拟技术，这可能需要对转子进行详细的未来研究，同时获得对控制其光物理特性的辐射衰变机制的定性洞察。该项目的第二阶段将在第一阶段的基础上，在真空和溶剂中进行更复杂的分子动力学模拟。这些将与实验吸收、发射和BODIPY转子在不同粘度溶剂中的各向异性测量一起进行。这些任务的目的是获得对分子在真实环境中的行为的更有效的理解。这一切都将由另外两个分子转子：DCVJ和ThT的基态和激发态的计算特征来补充。这些转子被认为与BODIPY具有不同的工作机制，因此应该是很好的比较工具。

The overall aim of the thesis will be to gain a detailed understanding of the working mechanism of fluorescent molecular rotors, in particular those based on BODIPY. These rotors can be used as probes to evaluate the local viscosity in cell membranes and lipid bilayers. Our goal will be achieved by a combination of multiscale atomistic simulations in combination with advanced fluorescence imaging. The insight gained will allow to optimise the use of fluorescent molecular rotors as rapid and reliable viscosity sensors in fluorescence microscopy, and other biotechnologically relevant viscosity-sensing and imaging assays.The first stage of my project can be summarised by three questions: Is it possible to accurately characterise the potential energy surface of a prototypical BODIPY-based rotor (BODIPY-Phe)? What can we learn about its excited state characteristics? Which simulation methods are the most efficient and effective tools for studying BODIPY-Phe? The aim of this stage of work is, therefore, to refine and compare the simulation techniques which will likely be needed for detailed future studies of the rotor, whilst gaining a qualitative insight into the radiative decay mechanisms that control its photophysical properties.The second stage of the project will build on the first by performing more complex molecular dynamics simulations, both in vacuo and in solvents. These will be done in conjunction with experimental absorption, emission and anisotropy measurements of the BODIPY rotor in solvents of varying viscosities. The aim of these tasks is to gain a more validated understanding of the behaviour of the molecule in real environments. This will all be complemented by computational characterisation of the ground and excited state of two other molecular rotors: DCVJ and ThT. These rotors are thought to have different working mechanisms to BODIPY, so should be good comparative tools.