BioLaser: Establishing a High-Resolution Laser Ablation Tomography Platform for UK Bioimaging Research

背景概述生物材料的二维和三维(断层)图像提供了亚细胞结构和功能之间的关系的洞察力。然而，特别是对于坚硬或不透明的组织，目前能够做到这一点的仪器都很大，而且极其昂贵。此外，样品制备和/或成像速度很慢，阻碍了对生物学研究通常所需的许多样品进行经济有效的分析。因此，迫切需要新的方法以高分辨率和高速度对生物样本进行成像和定量描述，从而能够分析目前被认为难以进行高通量调查的特征。在组件激光和成像技术存在的情况下，让它们协同工作，并且至关重要的是，在生物样本上实现高分辨率的亚细胞成像，需要进行修改和早期的概念探索和验证研究。我们建议对现有的第一代激光消融(高功率、超快激光脉冲)技术进行新的修改，使人们能够对以前使用传统技术难以解决的生物问题有新的生物学见解和理解。项目摘要“生物激光：激光消融断层扫描的生物研究”是一个泵启动项目，将激光技术(剑桥大学制造研究所，IFM)、植物研究、生理学(NIAB，剑桥)和图像分析(两个中心)的世界级专业知识结合在一起。这种方法的核心是使用精密激光(基于一种名为激光消融断层扫描(LAT)的技术)。这将与过程监测相结合，根据样品的成分和形貌调整消融过程(“计量学”)，为显微镜和提供高分辨率2D和3D图像的“化学成像”平台生成质量前所未有的精密消融生物样品，从而能够对细胞和亚细胞结构进行定量分析。使用植物组织阵列，我们将评估和验证设计精密LAT生物成像系统所需的参数。该项目围绕关键方法构建，所有这些方法都必须解决，才能成功开发基于LAT的高分辨率生物成像方法：1.基于激光制造技术的专业知识：IMF为该项目提供世界一流的精密激光研究、工程和制造专业知识。这样的现场专业知识是项目成功的基础。精确的消融控制：要实现高分辨率成像，需要一个均匀的平面。这需要精确地了解激光的位置和功率，同时测量每个激光脉冲后的烧蚀表面，通过过程中测量提供，以确保真正实现高分辨率成像。先进的成像和分析工具：将使用一套最先进的2D和3D成像和分析方法，利用UofC.4的成像专业知识，对生产的精密加工样品进行研究。图像验证：将要开发的工具和方法是强大和精确的。然而，如果不进行参数设置和验证，所生成的图像的科学价值可能有限。生成的图像数据将使用传统方法进行验证，为参数优化提供反馈回路。该项目将为未来通过IFM交付的台式样机的制造奠定基础。实现该项目的目标将导致第一代LAT系统与目前工作在低倍率下的一步一步的变化。最终，我们的目标是在剑桥建立一个LAT生物成像中心，并促进为英国和其他地区生产类似的平台。这种能力建设方法将促进充分实现精密LAT生物成像的潜在影响所需的技术吸收的临界量。

Background summaryTwo- and three-dimensional (tomographic) images of biological materials provide insight into the relationships between subcellular structure and function. However, particularly for hard or opaque tissues, instruments that can do this are currently large and extremely expensive. Furthermore, sample preparation and/or imaging is slow, preventing cost effective analysis of the many samples commonly required for biological studies. Thus, there is urgent need for new methods to image and quantitatively describe biological samples at high resolution and speed, allowing analysis of traits currently considered intractable to high-throughput investigation. While the component laser and imaging technologies exist, getting them to work together, and crucially, to achieve high-resolution sub-cellular imaging on biological samples, requires modifications and early concept exploratory and validation investigations. We propose novel modifications to existing, first-generation laser ablation (high-powered, ultrafast laser pulses) technology, enabling new biological insight and understanding into biological problems that were previously intractable using conventional techniques.Project summary'BioLaser: Biological Investigations by Laser Ablation Tomography' is a pump-priming project that aligns world-class expertise in laser technology (Institute for Manufacturing, IFM, at the University of Cambridge), plant research, physiology (NIAB, Cambridge) and image analysis (both centres). The approach centers around the use of precision lasering (based on a technique called 'Laser Ablation Tomography', LAT). This will be combined with in-process monitoring to adjust the ablation process according to the composition and topography of the sample ('metrology'), to generate precision ablated biological samples of unprecedented quality for microscopy and 'chemical imaging' platforms that render high-resolution 2D & 3D images, allowing quantitative analysis of cellular and subcellular structures. Using an array of plant tissues, we will evaluate & validate the parameters required to design a precision LAT bioimaging system. This project is structured around for key approaches, all of which must be addressed to successfully develop LAT-based methodologies for high-resolution bioimaging:1. Expertise in laser-based manufacturing technologies: IMF provides this project with world-class expertise in precision laser research, engineering and manufacturing. Such on-site expertise is the foundation for project success.2. Precise ablation control: To achieve high-resolution imaging, a uniform plane is required. This needs precise knowledge of the position and power of the laser alongside measurement of the ablated surface after each laser pulse, provided via in-process metrology to ensure high-resolution imaging is truly achievable.3. Advanced tools for imaging and analysis: The precision machined samples produced will be investigated using a suite of state-of-the-art 2D & 3D imaging and analysis approaches, exploiting imaging expertise at the UofC.4. Image validation: the tools and approaches to be developed are powerful and precise. However, used without parameterisation and validation, the resulting images could be of limited scientific value. The image data generated will be validated using conventional approaches, providing a feedback loop for parameter optimisation.This project will lay the foundation for future manufacture of a benchtop prototype, delivered via IFM. Achieving the project aims would result in a step-change over first generation LAT systems that currently work at low magnification. Ultimately, we aim to establish a LAT bioimaging hub based in Cambridge, and to facilitate the production of similar platforms for the UK and beyond. This capacity-building approach will promote the critical-mass in technology uptake necessary for the potential impact of precision LAT bioimaging to be fully realised.