Optical sectioning for 3D super-resolution microscopy

用于 3D 超分辨率显微镜的光学切片

• 批准号: BB/P026486/1
• 负责人: Daniel St Johnston
• 金额: $ 19.21万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP026486%2F1
• 关键词: Optical; sectioning; 3D; super; resolution

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.

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倍以上（2014年获得诺贝尔奖），使其能够在纳米尺度上对薄样品进行成像，这是单个蛋白质分子的大小。然而，由于来自图像平面上方和下方的大量离焦光以及在光穿过样品时引入的像差，因此对厚样品（例如组织和器官）中的细胞结构进行超分辨率成像仍然具有挑战性。我们最近建立了一个国家的最先进的超分辨率显微镜，消除像差使用自适应镜技术，这是首次开发的哈勃太空望远镜。我们已经证明，这种4Pi-SMS显微镜可以成像细胞在分子分辨率，但其灵敏度和分辨率急剧恶化，如果样品太厚，我们现在建议解决的问题，在厚样品的焦点背景光使用另一种新的方法称为时空聚焦。这种方法使用一种特殊的激光器，它产生的光脉冲的波长是激发显微镜检测到的荧光分子所需的波长的两倍（能量的一半）。通过对这些脉冲进行整形，我们可以产生这样的条件，即唯一被激发的荧光分子位于一个薄片（1-2微米厚）中，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。将时空聚焦扩展到4pi SMS系统中将使对更复杂的样本（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。作为原理的证明，我们将测试这种显微镜在果蝇、果蝇和小鼠肠道类器官的卵巢中对单分子成像的能力，因为这些样品很容易获得，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的分子组织，这些蛋白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几种位于苍蝇和哺乳动物上皮细胞的顶侧，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问题。例如，我们计划研究上皮细胞的顶端和侧面之间的边界是如何指定的，以及关键的顶端极性因子，非典型蛋白激酶C，是如何被招募到顶端膜。4Pi-SMS显微镜允许人们看到其他光学显微镜方法不可见的组织内部结构，因此很难预测我们可能会发现什么新特征。

项目成果

A method for single molecule localization microscopy of tissues reveals non-random distribution of nuclear pores in Drosophila

组织单分子定位显微镜方法揭示果蝇核孔的非随机分布

• DOI: 10.1101/2021.05.24.445468
• 发表时间: 2021
• 作者: Cheng J

Single-Molecule Localization Microscopy Reconstruction Using Noise2Noise for Super-Resolution Imaging of Actin Filaments

使用 Noise2Noise 进行单分子定位显微镜重建以实现肌动蛋白丝的超分辨率成像

• DOI: 10.1109/isbi45749.2020.9098713
• 发表时间: 2020
• 作者: Lefebvre J

A single-molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila

组织的单分子定位显微镜方法揭示果蝇的非随机核孔分布

• DOI: 10.3929/ethz-b-000534433
• 发表时间: 2021
• 作者: Cheng, Jinmei

A single-molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila.

• DOI: 10.1242/jcs.259570
• 发表时间: 2021-12-15
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Cheng J;Allgeyer ES;Richens JH;Dzafic E;Palandri A;Lewków B;Sirinakis G;St Johnston D
• 通讯作者: St Johnston D

Protein Tracking By CNN-Based Candidate Pruning And Two-Step Linking With Bayesian Network

• DOI: 10.1109/mlsp.2019.8918873
• 发表时间: 2019-10
• 期刊: 2019 IEEE 29th International Workshop on Machine Learning for Signal Processing (MLSP)
• 作者: Mariia Dmitrieva;Helen L. Zenner;Jennifer H. Richens;D. Johnston;J. Rittscher