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

荧光显微镜作为一种灵敏的工具被广泛用于研究细胞和组织的生物学和生物物理特性，包括它在组织样本病理诊断中的应用。复杂的三维细胞和组织中较大的结构已经成功地用共聚焦显微镜进行了研究，该显微镜通过有效地抑制失焦光来产生清晰的图像。然而，在细胞生物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，我们计划将共聚焦显微镜的原理与一种新的“超分辨率”显微镜方法结合起来，这种方法克服了传统光学显微镜的限制，可以分辨到~ 20nm的细节。然而，迄今为止，如此高的分辨率在较厚的细胞制剂（例如bbb50 μ m）中很难实现，因为许多超分辨率方法依赖于在荧光显微镜下激发时发光的单个分子的精确定位。这种“单分子定位显微镜”的方法受到在厚样品（如组织）中产生的大量背景光的影响，限制了可实现的分辨率。在这个项目中，我们提出了一种可以很容易实现的成像过程的改进，它结合了传统共聚焦显微镜的思想和基于单分子定位的超分辨率成像，同时最大限度地收集光。新方法的关键是使用数字微器件（DMD）阵列对样品进行图案照明。采用这种新方法还允许我们将构建的新设备实现标准共聚焦显微镜。因此，它使我们能够结合传统的3D显微镜和我们新的有效的超分辨率模式，并从样品中获得更多的信息。这意味着我们可以将传统共聚焦显微镜的高通量与超分辨率提供的局部高分辨率结合起来，换句话说，这是对病理检测中获得的复杂生物样品进行有效成像的两全其美。我们的方法的效用增加，因为我们将采用一种改进的共聚焦模式，最近已经证明。最重要的是，我们将能够在复杂的软件控制下无缝切换各种模式。为了提高在厚组织样本中提供扩展的3D超分辨率数据的能力，我们将在我们的新方法的工作流程中引入最近演示的技术。这项技术被称为DNA- paint，它利用对DNA相互作用的现代理解来构建用于超分辨率成像的新标记。DNA- paint提供了一种在同一样品中组合许多不同标记类型的通用方法，并且还引入了一种方便的方法来定位标记分子，因为互补的DNA链会短暂地相互结合。结合我们的超分辨率改进，这将提供一种方法来记录整个厚样本深度的图像，并构建非常高分辨率的3D体图像。新的超分辨率方法中的“共聚焦原理”对于避免背景光至关重要，否则背景光会极大地损害组织中的DNA-PAINT。为了证明我们的新组合超分辨率、共聚焦和相关显微镜模式的影响，我们将进行试点研究，将我们的新方法作为一种医疗保健技术，用于心脏组织的病理诊断、大脑成像和细胞“团块”，这些细胞“团块”在复杂的3D排列中类似于肿瘤组织。我们的新显微镜的新功能，其高效的实施和复杂但直观的软件界面的组合将使这一多功能的新方法，将高度相关的学术和商业用户在许多不同的领域。

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.