Focus enhanced single molecule super-resolution microscopy - correlative confocal and nanoscale imaging in thick tissues

荧光显微镜被广泛用作研究细胞和组织的生物学和生物物理性质的灵敏工具，包括作为一种保健技术用于组织样本的诊断病理学。共聚焦显微镜已经成功地研究了复杂三维细胞和组织中的较大结构，它通过有效地滤除聚焦外的光来产生清晰的图像。然而，在细胞生物体中发现的许多结构比光的波长小得多，因此很难用荧光显微镜观察到。在这个项目中，我们计划将共焦显微镜的原理与一种新的“超分辨率”显微镜方法相结合，这种方法克服了传统光学显微镜的限制，可以分辨出精细到~20 nm的细节。然而，到目前为止，在较厚的细胞制剂(例如&gt；5um)中很难实现如此高的分辨率，因为许多超分辨率方法依赖于在荧光显微镜中激发时发出光的单个分子的精确定位。在这个项目中，我们提出了一种易于实现的成像过程的改进，它将传统共焦显微镜的思想与基于单分子定位的超分辨率成像相结合，同时最大化了光的收集。新方法的关键是使用数字微设备(DMD)阵列对样品进行图案化照明。采用这种新的方法也允许我们将建造的新设备来实现标准的共焦显微镜。因此，它允许我们将传统的3D显微镜和我们新的有效的超分辨率模式结合起来，并从样本中获得更多的信息。这意味着我们可以将传统共焦显微镜的高通量与超分辨率提供的局部高分辨率相结合，换言之，在为病理测试获得的复杂生物样本中进行有效成像是两全其美的。我们的方法的实用性增加了，因为我们将采用最近证明的改进的共焦模式。最重要的是，我们将能够在复杂的软件控制下在各种模式之间无缝切换。为了提高在厚组织样本中提供扩展3D超分辨率数据的能力，我们将在新方法的工作流程中引入一项最近演示的技术。这项技术被称为DNA-Paint，它利用现代对DNA相互作用的理解来构建新的超分辨率成像标记。DNA-PAINT提供了一种在同一样本中组合许多不同标记类型的通用方法，还提供了一种方便的标记分子定位方法，因为互补的DNA链彼此瞬间结合在一起。与我们的超分辨率改进相结合，这将提供一种方法来记录整个厚样品深度的图像，并构建非常高分辨率的3D体积图像。新的超分辨率方法中的“共焦原理”是避免背景光的关键，否则将极大地损害组织中的DNA-Paint。为了展示我们新的组合超分辨率、共聚焦和相关显微模式的影响，我们将进行试点研究，以确立我们的新方法作为一种医疗保健技术，用于诊断心脏组织的病理、脑内成像和在复杂的3D排列中类似于肿瘤组织的细胞“块”。我们的新显微镜的新功能、其高效实施和复杂而直观的软件界面的结合将使这种多功能的新方法对许多不同领域的学术和商业用户具有高度的相关性。

Fluorescence microscopy is widely used as a sensitive tool to investigate the biology and biophysical properties of cells and tissues, including its use as a healthcare technology in diagnostic pathology of tissue samples. Larger structures in complex three-dimensional cells and tissues have been successfully investigated with confocal microscopy which produces sharp images by effectively rejecting out of focus light. However, many of the structures found within cellular organisms are much smaller than the wavelength of light and have therefore been difficult to observe with fluorescence microscopy. In this project we plan to combine principles from confocal microscopy with a new "super-resolution" microscopy method which overcomes the limits of conventional light microscopy and can resolve detail down to ~20 nm. To date, however, such high resolution has been difficult to achieve in thicker cell preparations (e.g. > 5 um), as many super-resolution methods rely on the precise localisation of individual molecules that emit light when excited in the fluorescence microscope. This "single molecule localisation microscopy" approach suffers from extensive background light that is generated in thick samples such as tissues and limits the achievable resolution.In this project we propose an improvement to the imaging process that can be easily implemented and which combines ideas from conventional confocal microscopy with super-resolution imaging based on single molecule localisation while maximising the collection of light. Key to the new approach is the use of a digital micro device (DMD) array for patterned illumination of the sample. Adopting this new approach also allows the new device that we will build to implement standard confocal microscopy. It therefore allows us to combine both conventional 3D microscopy and our new effective super-resolution modes and obtain increased information from the samples. This means we can combine the high throughput of conventional confocal microscopy with local high resolution provided by super-resolution, in other words the best of both worlds for effective imaging in complex biological samples obtained for pathology testing.The utility of our approach is increased because we will adopt an improved confocal mode that has been recently demonstrated. Most importantly, we will be able to seamlessly switch between the various modes under sophisticated software control.To improve the ability to provide extended 3D super-resolution data in thick tissue samples we will introduce a recently demonstrated technique into the workflow of our new approach. This technique, called DNA-PAINT, uses the modern understanding of DNA interactions to construct new markers for super-resolution imaging. DNA-PAINT provides a versatile way to combine many different marker types in the same sample and also introduces a convenient way to localise marker molecules as complementary DNA strands transiently bind to each other. In combination with our super-resolution improvements this will provide a way to record images throughout the depth of a thick sample and construct very high-resolution 3D volume images. The "confocal principle" in the new super-resolution approach is critical to avoid the background light that otherwise would greatly impair DNA-PAINT in tissue.To demonstrate the impact of our new combined super-resolution, confocal and correlative microscopy modes we will conduct pilot studies that establish our new approach as a healthcare technology for diagnostic pathology in heart tissue, imaging in the brain and in cell "clumps" that resemble tumour tissue in their complex 3D arrangement.The combination of new capabilities of our new microscope, its efficient implementation and sophisticated but intuitive software interface will make this a versatile new approach that will be highly relevant for academic and commercial users in many different fields.