MAPPING THE BRAIN: Sub-100nm resolution, large volume X-ray connectomics with near-field multislice ptychography

绘制大脑中单个神经元之间数千个连接的图谱，是一个被称为连接组学的领域，对于我们理解神经退行性疾病（如自闭症和精神分裂症）背后的机制，以及大脑对图像、气味和声音等刺激的复杂反应，至关重要。几十年来，电子显微镜（EM）一直是连接组学中占主导地位的成像技术，EM方法的最新进展现在使大脑区域的3D成像达到数百立方微米的体积。这足以捕获无脊椎动物或小型脊椎动物（如幼虫）的整个神经系统。不幸的是，这些EM技术的工作方式是从样品上削去非常薄的薄片，然后逐个成像。这意味着它们需要数月至数年的数据采集和处理时间，而切片过程本身具有破坏性且极易出错。因此，在提供最高分辨率的同时，单独使用EM很难获得更广泛的上下文信息，例如通过EM 3D图像中可见的突触连接在一起的神经元的身份。该项目旨在开发一种桥接方法，可以提供相关的，非破坏性的脑组织成像，在低于100纳米的分辨率，目标和背景EM连接组学。先进形式的同步加速器x射线显微镜已经在某种程度上提供了这种背景信息，而且有利的是，x射线的穿透能力意味着这些方法可以快速成像大量样品，而无需破坏性切片；问题是样品体积和分辨率必须相互权衡——更大、更厚的样品使x射线束散射，导致图像分辨率迅速下降。目前最先进的x射线显微镜可以在200微米厚的样品上实现约100纳米的分辨率；该项目将开发一种新的3D x射线工具，以低于100纳米的分辨率对立方毫米体积的脑组织进行成像。直到最近，考虑到所需的计算资源，我们在这个项目中探索的想法是不可能的。然而，今天，计算机硬件的惊人进步，特别是图形处理单元（gpu）上的并行计算，意味着十年前需要许多小时才能运行的进程现在可以接近实时。这正在改变研究人员对计算机在显微镜中的作用和潜力的看法。我们在这个项目中的工作是基于一种叫做ptychography的变革性技术，它使用迭代算法从一个非常简单的无透镜光学系统捕获的衍射数据中重建物体的图像。本质上，平面照相术用代码代替了x射线显微镜中的透镜。在过去的十年里，全息摄影领域呈指数级增长，世界上大多数同步加速器上都有专用的全息摄影光束线。英国处于这项研究的前沿，在算法开发和新颖实验方法方面有着良好的记录。我们的项目将补充这些正在进行的努力，并确保印刷术在英国仍然是一个活跃的、有竞争力的话题，并确保英国在这个令人兴奋和快速增长的领域保持世界领先地位。我们的计划汇集了谢菲尔德大学，钻石光源和克里克研究所在一个新的和令人兴奋的合作。调查小组在技术发展的每一步都拥有专业知识，从光学实验原理的证明，到同步加速器的实施，再到与克里克研究所的大脑专家合作进行的尖端、高影响力的应用研究。

Mapping the thousands of connections between individual neurons in the brain, a field called connectomics, is critical to our understanding of the mechanisms behind neurodegenerative conditions, such as autism and schizophrenia, and the brain's complex responses to stimuli, such as images, smells and sounds. For many decades, electron microscopy (EM) has been the dominant imaging technique in connectomics, and recent advances in EM methods now enable 3D imaging of regions of the brain up to several hundred micrometres cubed in volume. This is sufficient to capture the entire nervous systems of invertebrates or small vertebrate animals, such as larvae. Unfortunately, these EM techniques work by shaving very thin slices from the sample to be imaged one by one. This means they require months to years of data acquisition and processing times, whilst the slicing process itself is destructive and highly error-prone. Thus, while providing the highest resolution, using EM alone it is difficult to obtain wider contextual information, such as the identity of neurons that are linked together by the synapses visible in the EM 3D images. This project aims to develop a bridging method that can provide correlative, non-destructive imaging of brain tissue at sub-100nm resolution, to target and contextualise EM connectomics. Advanced forms of synchrotron X-ray microscopy already go some way toward providing this contextual information, and advantageously, the penetrative power of X-rays means these methods can image large sample volumes quickly and without destructive slicing; the problem is that sample volume and resolution must trade off against one another - larger, thicker samples scatter the X-ray beam leading to a rapid falloff in image resolution. Current state-of-the-art X-ray microscopy can achieve a resolution of approximately 100nm over a 200 micron thick sample; this project will develop a new 3D X-ray tool to image brain tissue at sub-100nm resolution over a cubic millimetre volume. Until recently, the ideas we explore in this project would have been impossible given the computing resources required. Today however, phenomenal advances in computer hardware, especially parallel computing on Graphic Processing Units (GPUs), mean processes that required many hours to run a decade ago are now possible in close to real time. This is transforming the way Researchers think about the role and potential of computing in microscopy. Our work in this project is based on one such transformative technique called ptychography, which uses iterative algorithms to reconstruct an image of an object from diffraction data captured by a very simple, lens-free optical system. Essentially ptychography replaces the lenses in an X-ray microscope with code. The field of ptychography has grown exponentially over the past decade and dedicated ptychography beamlines are now coming online at most synchrotrons around the world. The UK is at the forefront of this research, with a strong track record in algorithm development and novel experimental approaches. Our project will complement these on-going efforts and ensure ptychography remains an active, competitive topic within the UK, and that the UK remains a world-leader in this exciting and rapidly growing field.Our Programme brings together Sheffield University, the Diamond Light Source and the Crick Institute in a new and exciting collaboration. The Investigative team holds expertise at every step of the technique development journey, from optical bench proof of principle, through implementation at the synchrotron to cutting edge, high impact application studies in collaboration with the brain specialists at the Crick Institute.