Development of a super-resolving STED FLIM microscope for biological applications

我们的目标是开发一种荧光显微镜，以显着提高空间分辨率的分子过程的图像，以便在分子水平上研究疾病的机制。通过使用荧光分子（“荧光团”）来标记与信号传导相关的蛋白质，例如在白色血细胞和被免疫系统询问的可疑细胞之间的界面（“免疫突触”）处，可以了解特定蛋白质的空间和时间组织及其相互作用。不幸的是，荧光显微镜的分辨率被衍射限制在~300 nm，并且感兴趣的分子要小得多。探测它们相互作用的一种方法是使用光谱技术，因为荧光发射的性质可以根据荧光团的局部环境而变化，并且还可以用于区分不同的分子种类。通过用发射不同波长的荧光团标记不同的蛋白质，然后比较不同的“颜色”图像，可以获得关于共定位的信息，从中可以推断相互作用-尽管受到空间分辨率的限制。从荧光寿命（荧光信号衰减的时间）中可以了解更多信息，这可能对其局部物理或化学环境以及其他分子的存在敏感。荧光寿命成像（FLIM）可用于绘制分子环境的变化，并通过利用Förster共振能量转移（FRET）检测蛋白质-蛋白质相互作用，其中一个荧光团标记的蛋白质的发射通过直接能量转移到附近合适的荧光团而猝灭，这些荧光团可以标记第二个分子。这种能量转移只有在荧光团在~10 nm范围内时才能发生，也会缩短荧光寿命，因此FLIM提供了一种方法来映射蛋白质对何时何地相互作用-精确度受显微镜空间分辨率的限制。在细胞间信号传导的研究中，我们使用FLIM-FRET来成像一个关键分子（KIR受体）的磷酸化（即“激活”），该关键分子在询问可疑细胞时参与确定白色血细胞的反应。出乎意料的是，我们观察到KIR受体的磷酸化发生在小的微簇中-但共聚焦显微镜没有足够的空间分辨率来检测它们的存在。这是一个典型的应用，我们正在开发一种新的显微镜，能够解决低于衍射极限的结构。其他包括信号分子的共定位和分离，信号复合物的组装和使用FLIM来阐明肌纤维中肌动球蛋白跨桥的状态和分布。我们的目标是利用S。受激发射损耗（STED）常规的共聚焦显微镜扫描聚焦的激发光束穿过样品并检测所产生的荧光以获取图像，其分辨率取决于样品上聚焦点的大小。在STED中，样品由两个共线光束扫描：第一个以通常的方式激发荧光，但第二个“STED”光束通过受激发射耗尽激发态粒子数来抑制荧光。该第二光束具有中间有孔的“甜甜圈”轮廓，使得激发光斑的外部被“关闭”，而中心保持不变，从而实现更小的有效光斑和超过衍射极限的分辨率。在演示了STED原型显微镜，将FLIM和自适应补偿的显微镜中的像差，我们现在的目标是建立一个系统，适合生物学家使用，研究细胞信号和其他分子的组织，分辨率超过衍射极限使用技术，如FLIM和FRET。

We aim to develop a fluorescence microscope to image molecular processes with significantly improved spatial resolution in order to study the mechanisms of disease at a molecular level. By using fluorescent molecules ('fluorophores') to label proteins associated with signalling, e.g. at the interface ('immune synapse') between white blood cells and suspect cells being interrogated by the immune system, it is possible learn about the spatial and temporal organisation of specific proteins and their interactions. Unfortunately the resolution of fluorescence microscopes is limited by diffraction to ~300 nm and the molecules of interest are much smaller. One way to probe their interactions is to use spectroscopic techniques because the properties of fluorescence emission can vary according to the local environment of the fluorophore and can also be used to distinguish different molecular species. By labelling different proteins with fluorophores emitting at different wavelengths and then comparing the different 'colour' images, one can obtain information about co-localisation, from which interaction can be inferred - albeit limited by the spatial resolution. More can be learned from the fluorescence lifetime - the time over which a fluorescence signal decays - which can be sensitive to its local physical or chemical environment and to the presence of other molecules. Fluorescence lifetime imaging (FLIM) can be used to map changes in molecular environment and to detect protein-protein interactions by exploiting Förster Resonant Energy transfer (FRET), where the emission of one fluorophore-labelled protein is quenched by direct energy transfer to nearby suitable fluorophores that can be labelling a second molecule. This energy transfer, which can only occur if the fluorophores are within ~10 nm, also reduces the fluorescence lifetime and so FLIM provides a means to map when and where pairs of proteins interact - to a precision limited by the spatial resolution of the microscope. In a study of inter-cell signalling, we used FLIM-FRET to image the phosphorylation (i.e. the 'activation') of a key molecule (the KIR receptor) involved in determining the response of a white blood cell when interrogating a suspect cell. Unexpectedly, we observed that the phosphorylation of the KIR receptor occurred in small microclusters - but the confocal microscope did not have sufficient spatial resolution to do more than detect their presence. This is an exemplar application for which we are developing a new microscope capable of resolving structures below the diffraction limit. Others include the colocalisation and segregation of signalling molecules, the assembly of signalling complexes and the use of FLIM to elucidate the state and distribution of actomyosin cross bridges in muscle fibres. We aim to build a super-resolving fluorescence microscope using the technique pioneered by S. Hell called stimulated emission depletion (STED). A regular confocal microscope scans a focussed excitation beam across a sample and detects the resulting fluorescence to acquire an image, the resolution of which depends on the size of the focussed spot on the sample. In STED, the sample is scanned by two collinear beams: the first excites fluorescence in the usual way but the second 'STED' beam suppresses it by depleting the excited state population through stimulated emission. This second beam has a 'doughnut' profile with a hole in the middle such that the outside of the excitation spot is 'switched off' while the centre remains, thereby realising a smaller effective spot and resolution beyond the diffraction limit. Having demonstrated a STED prototype microscope incorporating FLIM and adaptive compensation of aberrations in the microscope, we now aim to build a system suitable for use by biologists to study the organisation of cell signalling and other molecules with resolution beyond the diffraction limit using techniques like FLIM and FRET.