High content analysis of 3-D cell cultures with multidimensional fluorescence imaging

细胞对刺激的反应取决于细胞信号传导过程，这是分子相互作用的组合。疾病与正常信号传导过程的偏离有关，因此读出细胞中的分子相互作用有助于阐明疾病的机制，也为评估潜在的治疗药物提供了一种手段。细胞中的许多信号分子是蛋白质，它们之间的相互作用被广泛研究，使用显微镜将感兴趣的蛋白质标记为荧光分子（“荧光团”）。对于药物发现，细胞阵列的荧光成像是自动化的，因此可以“分析”许多化合物对细胞信号传导过程的影响。荧光团被它们吸收的波长的辐射“激发”，由此产生的特征发射（荧光）被成像探测器记录下来。通过用不同的荧光团标记不同种类的蛋白质，并在各自的发射波长下比较图像，可以通过观察它们何时同时出现在同一位置来了解蛋白质的相互作用。不幸的是，这种“共定位”通常受到光衍射的限制，分辨率只有几100纳米——比典型信号蛋白的尺寸（~1-10纳米）大得多。然而，有可能使用Förster共振能量转移（FRET）来确认相互作用，这需要用不同的荧光团标记蛋白质并观察能量何时在它们之间转移。这种能量传递只能发生在它们之间距离小于10纳米的情况下。使用FRET读出相互作用的最定量的方法是通过减少“供体”荧光团的荧光寿命，因为它由于能量转移而失去能量。荧光寿命测量可以在每个像素上进行，这种荧光寿命成像（FLIM）使蛋白质相互作用能够在空间和时间上进行映射。荧光寿命还可用于区分参与调节细胞能量消耗的不同分子种类或天然荧光分子的不同状态，这也可被疾病改变。对于细胞生物学研究和药物发现，基于荧光的研究通常涉及成像薄的（因此是透明的）细胞层。不幸的是，这不是细胞的正常生理环境，它们的行为往往与正常的3-D组织环境不同。然而，在天然生物组织中成像细胞是极具挑战性的，在药物测试的高通量模式下实现这一点更是如此。相反，人们对合成三维培养物越来越感兴趣，这些培养物由许多相互作用的细胞组成，表现出与天然组织相似的行为，具有更大的光学可及性——尽管不幸的是，它们比薄层细胞更强烈地散射和吸收光。然而，这种3-D细胞培养物可以排列以进行快速成像，我们建议开发一个自动化平台来提供这种细胞培养物的3-D图像，利用FLIM读取分子相互作用。为此，我们将优化用于荧光成像的3-D细胞培养和标记方法，并将开发和评估用于基于flm的检测的自动显微镜。对于较大的样品，我们将利用多光子激发，这需要以两倍于通常所需的激发波长照射荧光团，这样它们就需要两个光子同时到达。这两个光子的吸收是强度依赖的，因此可以被安排为只发生在扫描激光束的焦平面上，这样发射的光子都来自样品中的特定深度。然后扫描焦平面就可以实现三维成像。波长较长的光毒性较小，被样品散射的较少，因此能够进行更深的成像。

The response of cells to stimuli depends on cell signalling processes, which are combinations of molecular interactions. Disease is associated with deviations from normal signalling processes and so reading out molecular interactions in cells can help elucidate mechanisms of disease and also provide a means to evaluate potential therapeutic drugs. Many of the signalling molecules in cells are proteins and their interactions are widely studied using microscopy with proteins of interest being labelled with fluorescent molecules ("fluorophores"). For drug discovery, fluorescence imaging of arrays of cells is automated so that the effect of many compounds on cell signalling processes can be "assayed". Fluorophores are "excited" by radiation at a wavelength at which they absorb and the resulting characteristic emission (fluorescence) is recorded using an imaging detector. By labelling different kinds of protein with different fluorophores and comparing the images at their respective emission wavelengths, it is possible to learn about protein interactions by observing when they appear in the same place at the same time. Unfortunately this "co-localisation" is usually limited by diffraction of light to a resolution of a few 100 nm - much larger than the size of typical signalling proteins (~1-10 nm). It is possible, however, to confirm interactions using Förster Resonant Energy transfer (FRET), which entails labelling the proteins with different fluorophores and observing when energy is transferred between them. This energy transfer can only occur if they are within ~10 nm of each other. The most quantitative way to read out interactions using FRET is through the reduction in the fluorescence lifetime of the "donor" fluorophore as it loses energy due to the energy transfer. Fluorescence lifetime measurements can be made in every pixel and this fluorescence lifetime imaging (FLIM) enables protein interactions to be mapped in space and time. Fluorescence lifetime can also be used to distinguish different molecular species or different states of naturally fluorescent molecules that are involved in regulating the consumption of energy in the cell, which can also be altered by disease. For cell biology research and for drug discovery, fluorescence-based studies typically involve imaging thin - and therefore transparent - layers of cells. Unfortunately this is not a physiologically normal environment for cells and they often behave differently compared to when they are in their normal 3-D tissue context. However, it is highly challenging to image cells in native biological tissue and even more so to realise this in a high throughput mode for drug testing. Instead, there is increasing interest in assaying synthetic 3-D cultures comprising many cells that interact with each other, presenting behaviour reminiscent of that in native tissue with greater optically accessibility - although unfortunately they can scatter and absorb light more strongly than thin layers of cells. Such 3-D cell cultures can be arrayed for rapid imaging, however, and we propose to develop an automated platform to provide 3-D images of such cell cultures, utilising FLIM to read out molecular interactions. For this we will optimise the 3-D cell culture and labelling methodologies for fluorescence imaging and will develop and evaluate automated microscopes for FLIM-based assays. For larger samples we will utilise multiphoton excitation, which entails illuminating the fluorophores at twice the wavelength usually required for excitation, such that they need two photons arriving simultaneously. This two photon absorption is intensity-dependent and so can be arranged to occur only in the focal plane of a scanning laser beam such that the emitted photons all originate from a specific depth in the sample. Scanning the focal plane then enables 3-D imaging. The longer wavelength light is less phototoxic and is scattered less by the sample, thus enabling deeper imaging.