A Dual Camera Lattice Lightsheet Microscope To Deliver Transformative Multi-Channel Volume Imaging

双摄像头晶格光片显微镜可提供变革性的多通道体积成像

• 批准号: BB/X019578/1
• 负责人: Tim Hawkins
• 金额: $ 103.71万
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX019578%2F1
• 关键词: Dual; Camera; Lattice; Lightsheet; Microscope

The first studies of biological structures were by the early pioneers of microscopy, Robert Hooke and Antoni van Leeuwenhoek, in the 17th century. Robert Hooke, in 1665, was the first to introduce the term "cell" when he was viewing the "boxes" he saw in slices of cork using one of the earliest optical compound microscopes (two lenses: an objective lens and an eyepiece) that he developed. He probably didn't quite realise the significance of this discovery, as it was only when it became apparent that the great majority of organisms are composed of cells that Cell Theory was born. Cell Theory, first proposed by M.J. Schleiden and Theodore Schwann in 1839, states that cells are of universal occurrence and are the basic units of an organism. Over 300 years of microscope improvements have led to fascinating discoveries of how cells function and now fluorescence microscopy, a form of light microscopy where objects are tagged with light-emitting dyes, has become an essential tool to study the biology of the cell. Many technical developments have led to greatly improved image quality and resolution however when imaging cells a compromise between imaging speed, volume acquired and longevity of the sample is required which restricts the type of biological question that can be investigated. Dynamic observations in living cells are usually limited to a small subcellular area. Lattice Lightsheet (LLS) technology addresses these challenges providing a step change in the imaging datasets possible and the biological questions that can be asked. LLS microscopy is the first commercially available technology that will allow full volumetric imaging (3 cell volumes per second) at high resolution over long time periods without the damaging effects of laser illumination (phototoxicity). This opens up the possibility to study new cell biological questions about morphology, responses to stress, and in development. A lattice light-sheet uniquely creates a very thin (500nm) sheet of laser light which is passed across the sample to generate a series of images (optical sections) that can be reconstructed into a three-dimensional view of the sample. With LLS this sheet is thin enough to resolve the three-dimensional details within cells clearly. With other microscopy techniques, we are limited by the speed at which we can acquire these images and the length of time we can observe before the sample dies due to exposure to the laser light. Unlike other non-lightsheet microscopes, the illumination of the sample is restricted to the focal plane, one slice within the sample. Because the areas of the sample above and below this plane are not exposed to the light, the sample viability is improved allowing the gentle observation of important biological processes over longer periods of time without sacrificing the speed of image acquisition or image resolution. To be able to observe these processes in detail across the full volume of the cell is truly unique. This transformative technology will allow researchers in Durham and beyond to address fundamental questions in plant cell growth and development which will ultimately lead to a greater understanding of how plants adapt to different stresses (biotic and abiotic), and how animal cells divide and grow in normal and disease states. This new variant of LLS technology allows the simultaneous imaging of two channels at high speed fundamentally expanding the biological questions we can ask about where proteins and structures interact. The instrument will be supported by dedicated expert technical staff and shared with scientists in the N8 research partnership of Northern universities and nationally with the UK Plant Cell Biology Community.

对生物结构的第一次研究是由显微镜学的早期先驱罗伯特·胡克和安东尼·范·列文虎克在17世纪完成的。1665年，罗伯特·胡克（Robert Hooke）是第一个引入“细胞”一词的人，当时他正在使用他发明的最早的光学复合显微镜（两个镜头：一个物镜和一个目镜）观察他在软木薄片中看到的“盒子”。他可能并没有完全意识到这一发现的重要性，因为只有当绝大多数生物体是由细胞组成的这一事实变得很明显时，细胞理论才诞生了。1839年，M.J. Schleiden和Theodore Schwann首先提出细胞理论，认为细胞是普遍存在的，是生物体的基本单位。300多年来，显微镜的改进已经导致了对细胞功能的迷人发现，现在荧光显微镜，一种用发光染料标记物体的光学显微镜，已经成为研究细胞生物学的重要工具。许多技术的发展已经大大提高了图像质量和分辨率，但是当成像细胞需要在成像速度、获得的体积和样本的寿命之间做出妥协时，这限制了可以研究的生物问题的类型。活细胞的动态观察通常局限于一个小的亚细胞区域。Lattice Lightsheet （LLS）技术解决了这些挑战，为成像数据集提供了可能的步骤变化，并可以提出生物学问题。LLS显微镜是第一个商业上可用的技术，它将允许在长时间内以高分辨率进行全体积成像（每秒3个细胞体积），而不会产生激光照射的破坏性影响（光毒性）。这为研究细胞形态学、应激反应和发育过程等新的细胞生物学问题提供了可能性。晶格光片独特地创造了一个非常薄（500纳米）的激光片，该激光片穿过样品产生一系列图像（光学部分），可以重建成样品的三维视图。使用LLS，该薄片足够薄，可以清晰地分辨细胞内的三维细节。使用其他显微镜技术，我们可以获得这些图像的速度和时间长度的限制，我们可以在样品因暴露在激光下而死亡之前观察。与其他非光片显微镜不同，样品的照明仅限于焦平面，即样品内的一个切片。由于该平面上下的样品区域没有暴露在光线下，因此样品活力得到改善，可以在较长时间内温和地观察重要的生物过程，而不会牺牲图像采集速度或图像分辨率。能够在细胞的整个体积上详细观察这些过程确实是独一无二的。这种变革性的技术将使达勒姆大学和其他地方的研究人员能够解决植物细胞生长和发育的基本问题，最终使人们更好地了解植物如何适应不同的压力（生物和非生物），以及动物细胞如何在正常和疾病状态下分裂和生长。这种LLS技术的新变体允许同时对两个通道进行高速成像，从根本上扩展了我们可以问的关于蛋白质和结构在哪里相互作用的生物学问题。该仪器将由专门的专家技术人员提供支持，并与北方大学的N8研究伙伴关系中的科学家以及英国植物细胞生物学社区的全国科学家共享。