A novel frequency domain FLIM microscope for the dynamic study of protein function in live cells

光学技术是探测活细胞中蛋白质功能的有力工具。蛋白质可以用高特异性的荧光标记，这使得人们能够以高的空间和时间分辨率研究它们在活细胞内何时何地产生或降解。荧光特征还可以报告标记蛋白质的分子环境的细微变化。例如，蛋白质之间的相互作用可能导致报告荧光团发射的细微强度、颜色和寿命变化。因此，在生命科学中不断需要新的，更好的，更灵活的仪器来测量活细胞内的荧光。荧光寿命尤其是这种努力中的关键参数。分子邻近可以淬灭荧光，这反过来又导致寿命的减少。然而，从细胞中的分子测量寿命是一项艰巨的任务：在精确度和可以执行寿命测量的速度之间总是存在权衡。在目前的建议中，我们寻求改进一种称为频域寿命成像显微镜（FD FLIM）的技术：这种技术与宽场荧光显微镜结合使用很受欢迎，因为它在应用中简单灵活，但它有缺点，因为在其传统形式中，测量速度和精度是有限的。在目前的工作中，我们的目标是克服这些局限性，并大大提高FD FLIM的能力，在生活系统中的蛋白质-蛋白质相互作用的测量。这里开发的仪器将用于研究调节细胞分裂的分子过程，我们将能够比以前更详细地做到这一点：我们研究的特征是高度动态的，发生在小的空间尺度上，提高的时间分辨率和精度将量化我们迄今为止无法观察到的事件。我们在这里开发的能力将可供来自不同部门的研究人员使用，并且在需要快速监测分子尺度事件（如细胞中的信号传导事件）或需要高测量精度的情况下（例如记录可能报告聚合单体的不同寡聚状态的微妙寿命变化）非常有用。

Optical techniques are powerful tools to probe the function of proteins in living cells. Proteins can be fluorescently labelled with high specificity and this permits one to study where and when they are produced or degraded within living cells with high spatial and temporal resolution. The fluorescence signatures may also report on subtle changes in the molecular environment of the tagged proteins. For example interactions between proteins may lead to subtle intensity, colour and lifetime changes in the emission of the reporting fluorophores. There is therefore a constant demand in the life sciences for novel, better, and more flexible instrumentation to measure fluorescence from within living cells. The fluorescence lifetime in particular is a key parameter in such efforts. Molecular proximity can quench fluorescence and this in turn results in a reduction of the lifetime. Measuring lifetime from molecules in cells is however a difficult task: There is always a trade-off between the precision and the speed at which a lifetime measurement can be performed. In the current proposal we seek to improve on a technique called frequency domain lifetime imaging microscopy (FD FLIM): This technique is popular in conjunction with widefield fluorescence microscopy because it is simple and flexible in application but it has drawbacks, because in its conventional form, the speed of measurement and the precision are limited. In the present work we aim to overcome these limitations and vastly improve on the capability of FD FLIM for measurements of protein-protein interactions in living systems. The instrument to be developed here will be used in the study of the molecular processes, which regulate cell division and we will be able to do this in much more detail than before: The features we study are highly dynamic and occur over small spatial scales and the improved temporal resolution and precision will quantify events that we have not been able to observe so far. The capability we are developing here will be available for researchers from various departments and be useful in situations where either molecular scale events need to be monitored at rapid speed, such as signalling events in cells, or in situations where high measurement precision is required, for example to record subtle lifetime changes that may report on different oligomeric states of aggregating monomers.