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.

最早的生物结构研究是由早期的显微镜先驱罗伯特·胡克和安东尼·范·列文虎克在17世纪进行的。1665年，当罗伯特·胡克用他开发的最早的光学复合显微镜(两个透镜：物镜和目镜)之一观察软木切片中的“盒子”时，他第一个引入了“细胞”这个术语。他可能并没有完全意识到这一发现的意义，因为直到很明显，绝大多数有机体都是由细胞组成的时候，细胞理论才诞生。细胞理论最早由M.J.Schleiden和Theodore Schwann于1839年提出，该理论认为细胞是普遍存在的，是生物体的基本单位。经过300多年的显微镜改进，细胞的功能得到了令人着迷的发现，现在荧光显微镜，一种用发光染料标记物体的光学显微镜形式，已经成为研究细胞生物学的重要工具。许多技术的发展极大地提高了成像质量和分辨率，然而，当成像细胞时，需要在成像速度、获得的体积和样品的寿命之间进行折衷，这限制了可以研究的生物学问题的类型。在活细胞中的动态观察通常局限于一个很小的亚细胞区域。晶格光片(LLS)技术解决了这些挑战，提供了成像数据集的一步变化可能和可以提出的生物学问题。LLS显微镜是第一种商业可用的技术，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)，而不会受到激光照射(光毒性)的损害。这为研究新的细胞生物学问题打开了可能性，这些问题包括形态、对压力的反应和发育过程。晶格光片独特地产生非常薄(500 Nm)的激光薄片，穿过样品以产生一系列图像(光学部分)，这些图像(光学部分)可以重建为样品的三维视图。使用LLS，这种薄片足够薄，可以清晰地解析单元格中的三维细节。对于其他显微镜技术，我们受到获取这些图像的速度和在样品因暴露在激光下而死亡之前可以观察的时间长度的限制。与其他非光片显微镜不同，样品的照明被限制在焦平面上，即样品中的一个切片。由于该平面上方和下方的样本区域不暴露在光中，因此提高了样本的生存能力，允许在更长的时间内对重要的生物过程进行温和观察，而不会牺牲图像采集的速度或图像分辨率。能够在整个细胞体积内详细观察这些过程确实是独一无二的。这项革命性的技术将使达勒姆内外的研究人员能够解决植物细胞生长和发育的基本问题，最终将导致对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的更多了解。这种LLS技术的新变种允许高速同时对两个通道进行成像，从根本上扩展了我们可以询问的关于蛋白质和结构相互作用的生物学问题。该仪器将得到专门的专家技术人员的支持，并与北方大学N8研究伙伴关系的科学家以及全国与英国植物细胞生物学社区共享。