Development of a single channel hyperspectral fluorescence lifetime instrument

荧光寿命成像(FLIM)提供了一种强大的光学成像方式，可用于对比不同类型的荧光分子(称为‘荧光团’)或提供有关当地荧光团环境的信息。虽然传统的荧光强度成像被广泛用于分子生物学，通过将蛋白质附着在方便的荧光团上来可视化已被标记的蛋白质的分布，但要获得关于影响荧光过程效率的因素的定量数据相对困难。这一点很重要，因为原则上，荧光效率是当地荧光团环境的函数，可以提供有关荧光团正在发生的事情的信息，而传统的荧光强度成像只报告荧光团的位置。由于衰减、荧光团浓度或光程长度等因素的变化，基于强度的荧光效率测量可能不可靠，这些因素可能非常难以量化。荧光寿命测量对这些因素不敏感。这使得薄膜对于福斯特共振能量转移(FRET)的新技术很有用，在这种技术中，荧光被相邻的荧光团猝灭(减弱)。这种分子间能量转移导致的荧光猝灭要求它们在~10 nm范围内，因此这为生物学家提供了一种当蛋白质对相互作用时成像的方法。薄膜是有用的，因为猝灭的荧光团表现出较短的荧光寿命。在Imperial，我们有一系列跨学科的研究项目，利用FLIM，包括细胞间信号和细胞内信号通路的Flim-FRET成像，这对于了解癌症等疾病的机制非常重要。成功的FRET实验需要仔细优化荧光团标记，这必须通过对照实验进行测试。其他薄膜实验需要对荧光探测器发射的辐射进行表征。我们目前选择的Flim&FRET工具是共焦显微镜，但由于许多生物学家和其他合作者在这台昂贵的仪器上争夺时间，我们有一个主要的瓶颈，限制了研究进展。拟议中的新工具将允许对蛋白质或荧光团进行FRET和控制实验，这些实验可以离线完成。这对于描述许多其他薄膜实验的荧光团也是有用的。此外，它将是用户友好的，并且相对容易复制，所以我们可以设想将这项技术推广给帝国理工学院生命科学部门和癌症研究所(ICR)的合作者。这将对建立新的FILM和FRET实验的进展产生非常积极的影响，并将极大地缓解我们共焦显微镜系统的拥堵。我们通过用短脉冲光激发分子，并观察它们如何通过发射光子来损失这种能量来测量荧光寿命。发射的光子的波长和荧光信号衰减的时间尺度是不同荧光团分子的特征。对于这个项目，我们的目标是开发一种自动的高光谱荧光寿命测量系统，该系统将根据波长和时间衰减(寿命)曲线同时记录荧光发射，以提供荧光团发射的完整光谱-时间特征。这一新工具将适用于多孔板阵列中的试管测量和均质分析，在高通量筛选中用于药物发现。它还将配置光纤探头，用于现场点测量。

Fluorescence lifetime imaging (FLIM) provides a powerful optical imaging modality that may be used to contrast different types of fluorescent molecule (called 'fluorophores') or to provide information concerning the local fluorophore environment. While 'conventional' fluorescence intensity imaging is widely used for molecular biology to visualise distributions of proteins that have been 'labelled' by attaching them to convenient fluorophores, it is relatively difficult to obtain quantitative data concerning factors that affect the efficiency of the fluorescence process. This is important because, in principle, the fluorescence efficiency is a function of the local fluorophore environment and can give information concerning what is happening to the fluorophore, whereas conventional fluorescence intensity imaging merely reports where it is located. Intensity-based measurements of fluorescence efficiency can be unreliable because of variations in factors such as attenuation, fluorophore concentration or optical pathlength, which can be very difficult to quantify. Fluorescence lifetime measurements are insensitive to these factors. This makes FLIM useful for the new technique of Forster Resonant Energy transfer (FRET) where fluorescence is quenched (diminished) by adjacent fluorophores. This quenching of fluorescence by energy transfer between molecules requires them to be within ~ 10 nm and so this provides a means for biologists to image when pairs of proteins are interacting. FLIM is useful because the quenched fluorophores exhibit a shorter fluorescence lifetime. At Imperial we have a range of interdisciplinary research programmes exploiting FLIM including FLIM-FRET imaging of inter-cell signalling and signal pathways within cells, which are important to understand the mechanism underlying diseases such as cancer. Successful FRET experiments require careful optimisation of fluorophore labelling that must be tested by control experiments. Other FLIM experiments require characterisation of the radiation emitted by fluorescence probes. Our current tool of choice for FLIM & FRET is a confocal microscope but, with many biologists and other collaborators competing for time on this expensive instrument, we have a major bottleneck that is limiting research progress. The proposed new tool would permit FRET and control experiments on protein or fluorophores to be done 'off-line'. It would also be useful to characterise fluorophores for many other FLIM experiments. Furthermore it would be user-friendly and relatively easy to replicate so we could envisage spreading this technique to our collaborators within Imperial's life science departments and at the Institute of Cancer Research (ICR). This would have a very positive impact on progress towards establishing new FLIM and FRET experiments and would also greatly relieve the congestion on our confocal microscope system. We measure fluorescence lifetime by exciting molecules with a short pulse of light and observing how they lose this energy by emitting photons. The wavelength of the photons emitted and the timescale over the fluorescence signal decays are characteristic of the different fluorophore molecules. For this project we aim to develop an automatic hyperspectral fluorescence lifetime measurement system that would simultaneously record the fluorescence emission in terms of the wavelength and temporal decay (lifetime) profiles to provide a full spectro-temporal characterisation of the fluorophore emission. This new tool will be applicable to cuvette measurements and homogenous assays in multiwell plate arrays, which in high-throughput screening are used for drug discovery. It will also be configurable with fibre-optic probes for in situ point measurements.