Integrating confocal microscopy and cryo plasma FIB milling for tomography

生命科学行业是英国经济的重要组成部分：它拥有超过22万名员工，对gdp和英国贸易平衡做出了重大贡献，对于为患者开发领先的治疗方法至关重要。它以英国在健康和生命科学方面世界领先的研究基地为基础。反过来，工程和物理科学（EPS）研究的进步使许多关键的研究突破成为可能，这些研究提供了越来越复杂的仪器和方法，以支持对活生物体（从微生物到植物、动物和人体）和生物过程（包括疾病病理学和药物作用）的研究。整个生态系统的研发——从基础认识到应用研究再到产品开发——对于实现长期经济增长和农业、食品安全、医疗保健和公共卫生领域的持续进步至关重要。历史上的创新模式往往是线性的，涉及一定程度的意外发现。如果物理科学家、工程师、生命科学家和工业界大规模合作，颠覆性技术和科学突破将会加速。富兰克林在哈维尔科学与创新校区有一个中心（Hub），与英国领先的高等教育机构的正式辐条相连，它将整合学术界和工业界的互补专业知识，创建一个国家卓越中心，在物理科学和生命科学的融合中开发方法。它将创造高价值的就业机会，保护和吸引外来投资，推动长期增长；并为政府的创新、工业和区域战略作出贡献。可见显微镜与电子成像的相关性，当然是RFI的一个关键目标，它支撑了跨尺度成像。因此，对于细胞和组织来说，精确定位待加工区域是至关重要的。由于拥挤的细胞环境，由于样品受到电子剂量的破坏，在测量过程中几乎不可能确认感兴趣的区域在EM体积内。利用相关光和电磁（CLEM）方法指导铣削和成像。从广义上讲，荧光标签被放置在感兴趣的蛋白质上；这在技术上极具挑战性，而且容易出错。我们建议与Thermo Fischer Scientific合作，将荧光成像和等离子FIB铣削结合在一台机器上，从而改变断层扫描的工作流程。该设备在相关成像主题和质谱主题中都有应用。

The Life Sciences sector forms a key part of the UK economy: it employs over 220,000 people, contributes significantly toGDP and UK balance of trade, and is crucial for developing leading-edge treatments for patients. It is underpinned by the UK's world-leading research base in the health and life sciences. Many key research breakthroughs are, in turn, enabled by advances in engineering and physical sciences (EPS) research - which provide ever more sophisticated instrumentation and methods to support the study of living organisms (from microbes to plants, animals and the human body) and biological processes (including both disease pathology and drug action). R&D across all parts of this ecosystem - from fundamental understanding to applied research to product development - is crucial for the delivery of long-term economic growth and continued advances in agriculture, food security, healthcare and public health. Historic models of innovation have often been linear, involving a degree of serendipity. Disruptive technologies and scientific breakthroughs will be accelerated if physical scientists, engineers, life scientists and industry work together, and at scale. The Franklin has a focal point (Hub) at Harwell Science and Innovation Campus, linked to formal Spokes in leading HEIs across the UK, it will integrate complementary expertise from academia and industry to create a national centre of excellence for methods development at the convergence of the physical and life sciences.It will create high-value jobs, protect and attract inward investment, and drive long-term growth; and contribute to the delivery of the Government's innovation, industrial and regional strategies.The correlation of visible microscopy with electron imaging, is of course a critical goal of the RFI, it underpins imaging across scales. Thus for cells and tissue, the accurate targeting of the zone of interest to be milled is critical. Due to the crowded cellular environment, it is, as a practical matter, almost impossible to confirm the region of interest is within the EM volume during measurement, since the sample is damaged by electron dose. Correlative Light and EM (CLEM) methods are used to guide the milling and imaging. In broad terms fluorescent tag(s) are put onto the protein(s) of interest; this is extremely technically challenging and prone to error. We propose, in a collaboration with Thermo Fischer Scientific , to combine fluorescence imaging and plasma FIB milling in a single machine, thus transforming the workflow for tomography. This equipment has applications in both correlative imaging themes and in the mass spectrometry theme.