Optical sectioning for 3D super-resolution microscopy

几个世纪以来，显微镜一直是促进我们对生物学理解的重要工具。最近的发展使显微镜分辨近距离物体的能力提高了20倍（2014年获得诺贝尔奖），使得在纳米尺度上对薄样品进行成像成为可能，纳米尺度是单个蛋白质分子的大小。然而，对厚样品（如组织和器官）中的细胞结构进行超分辨率成像仍然具有挑战性，因为来自成像平面上下的大量失焦光以及当光穿过样品时引入的像差。我们最近建造了一个最先进的超分辨率显微镜，它使用自适应反射镜技术来消除像差，这种技术最初是为哈勃太空望远镜开发的。我们已经证明，这种4Pi-SMS显微镜可以在分子分辨率下对细胞进行成像，但如果样品太厚，其灵敏度和分辨率会急剧下降。我们现在建议使用另一种称为时空聚焦的新方法来解决厚样品中背景光失焦的问题。它使用一种特殊的激光器，产生两倍波长（和一半能量）的光脉冲，以激发显微镜检测到的荧光分子。通过塑造这些脉冲，我们可以创造一种条件，在这种条件下，唯一被激发的荧光分子位于薄片上（1-2微米厚），来自激光的两个光子同时激活它们。这意味着我们只激发我们想要检测的分子，并将背景失焦光减少约100倍。将时空聚焦整合到4pi SMS系统中，将使对更复杂的样品（如组织或类器官）进行定量超分辨率成像成为可能，这将允许解决一系列全新的问题。作为原理证明，我们将测试这台显微镜在果蝇卵巢和小鼠肠道类器官中的单分子成像效果如何，因为这些样本很容易获得，厚度超过50微米。更具体地说，我们计划研究一组保守的极性蛋白质的分子组织，这些蛋白质使细胞的一侧与另一侧不同。这将为时空聚焦4Pi-SMS提供一个具有挑战性的测试，因为这些蛋白质中的一些位于苍蝇和哺乳动物上皮细胞的顶端侧，因此位于样品中超过10微米深。以20纳米的精度可视化这些蛋白质，并能够计算复合物中的分子数量，将使我们能够回答该领域的主要开放性问题。例如，我们计划研究上皮细胞的顶端和外侧之间的边界是如何被指定的，以及关键的顶端极性因子——非典型蛋白激酶C是如何被募集到顶端膜上的。4Pi-SMS显微镜允许人们看到组织内部的结构，这些结构在其他光学显微镜方法中是不可见的，因此很难预测我们可能会发现什么新特征。

Microscopy has been an essential tool in advancing our understanding of biology for centuries. Recent developments have improved the ability of microscopes to resolve close objects by a factor of >20 (recognised with the Nobel Prize in 2014), making it possible to image thin samples at the nanometre scale, which is the size of individual protein molecules. It has remained challenging, however, to perform super-resolution imaging of cellular structures in thick samples, such as tissues and organs, because of the large amount of out of focus light from above and below the image plane and the aberrations that are introduced as the light passes through the sample. We have recently built a state of the art super-resolution microscope that removes the aberrations using adaptive mirror technology that was first developed for the Hubble space telescope. We have demonstrated that this 4Pi-SMS microscope can image cells at molecular resolution, but its sensitivity and resolution deteriorate dramatically if the samples are too thick.We now propose to address the problem of out of focus background light in thick samples using another new approach called spatio-temporal focusing. This uses a special laser that produces pulses of light at twice the wavelength (and half of the energy) needed to excite the fluorescent molecules that the microscope detects. By shaping these pulses, we can produce conditions where the only fluorescent molecules to be excited lie in a thin sheet (1-2 micrometres thick) where two photons from the laser activate them simultaneously. This means that we only excite the molecules that we want to detect and reduces the background out of focus light by a factor of ~100. Incorporating spatio-temporal focusing into the 4pi SMS system will make it possible to perform quantitative super-resolution imaging on more complex samples, such as tissues or organoids, which will allow a whole new range of questions to be addressed. As a proof of principle, we will test how well this microscope can image single molecules in the ovaries of the fruitfly, Drosophila, and in mouse intestinal organoids, as these samples are readily available and are more than 50 micrometres thick. More specifically, we plan to investigate the molecular organisation of a conserved set of polarity proteins that make one side of a cell different from the other. This will provide a challenging test for the spatio-temporal focusing 4Pi-SMS, because several of these proteins are localised on the apical side of epithelial cells in both flies and mammals and therefore lie more than 10 micrometres deep in the sample. Visualising these proteins with 20 nanometre precision and being able to count the number of molecules in a complex will allow us to answer major open questions in the field. For example, we plan to investigate how the boundary between the apical and lateral sides of epithelial cells is specified and how the key apical polarity factor, atypical protein kinase C, is recruited to the apical membrane. The 4Pi-SMS microscope allows one to see structures inside tissues that are not visible with other light microscopy methods, and it is therefore hard to predict what new features we may find.