Developing a new bio-imaging tool for correlative light electron microscopy

开发一种用于相关光电子显微镜的新型生物成像工具

• 批准号: 2749963
• 金额: --
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2749963
• 关键词: Developing; new; bio; imaging; tool

Correlative Light Electron Microscopy (CLEM) is one of the most powerful imaging technologies as it combines the advantages of live cell imaging from light microscopy (LM) with the sub-nanometer spatial resolution of electron microscopy (EM). Using this technology, key biological questions have been answered.CLEM is, however, seriously hampered by the availability of robust probes. It is highly questionable whether most bimodal probes, using a fluorophore (for LM) and an electron-dense gold nanoparticle (for EM) attached to the protein of interest, actually show the same protein pool. This is due to limitations such as photobleaching or quenching of the fluorophore, and/or detachment of the probes while trafficking inside the cell or after sample processing for EM. These are serious drawbacks that need to be addressed.To do so, the aim of this project is to develop a novel CLEM method where a gold nanoparticle (AuNP) is used as the same probe for both LM and EM. AuNP will be visualised in LM in living cells using a novel nonlinear optical microscopy technique developed at Cardiff University. The technique uses electronically-resonant Four Wave Mixing (FWM) and exploits the strong and photostable absorption and scattering of light of a AuNP at the localised surface plasmon resonance (LSPR). Using a combination of short optical pulses to generate and detect changes in the AuNP transmission or scattering at the LSPR, the technique is uniquely sensitive to single small AuNPs which are detected background free with high spatial resolution in 3D. Notably, the technique is also uniquely sensitive to the AuNP shape, which opens up the prospect of probe multiplexing via shape recognition.In this project, spherical AuNPs and gold nanorods of various sizes will be conjugated to proteins of interest (for example the iron-binding protein transferrin) and internalised in mammalian cell lines. The aim will be to demonstrate the full CLEM workflow starting from imaging living cells with FWM (at Cardiff), to fixation, sectioning and correlative imaging of the same AuNP within the cell ultrastructure revealed by EM (at Bristol).The project will also explore the pioneering concept of assembling AuNPs directly inside living cells using proteins expressed in the cytoplasm which have the capability to bind metals and concentrate these to form electron dense particles. This could open the way to genetically tag cytoplasmic proteins with metallic NPs. [1] DOI: 10.1038/nature14503. [2] DOI: 10.1039/c9nr08512b.

相关光电子显微镜（CLEM）是最强大的成像技术之一，因为它结合了光学显微镜（LM）的活细胞成像与电子显微镜（EM）的亚纳米空间分辨率的优势。利用这项技术，关键的生物学问题已经得到了解答。然而，CLEM却受到了强大探针的严重阻碍。这是非常值得怀疑的大多数双峰探针，使用荧光团（LM）和电子致密的金纳米粒子（EM）连接到感兴趣的蛋白质，实际上是否显示相同的蛋白质池。这是由于限制，如荧光团的光漂白或淬灭，和/或探针在细胞内运输时或EM样品处理后的分离。这些都是需要解决的严重缺点。为此，本项目的目的是开发一种新的CLEM方法，其中金纳米颗粒（AuNP）用作LM和EM的相同探针。金纳米粒子将可视化在LM活细胞使用一种新的非线性光学显微镜技术开发的卡迪夫大学。该技术使用电子共振四波混频（FWM），并利用AuNP在局部表面等离子体共振（LSPR）下对光的强且光稳定的吸收和散射。使用短光脉冲的组合来产生和检测在LSPR处的AuNP透射或散射的变化，该技术对单个小AuNP独特地敏感，所述单个小AuNP在3D中以高空间分辨率无背景地检测。值得注意的是，该技术也是唯一敏感的金纳米粒子的形状，这开辟了前景探针复用通过形状recognization.In这个项目中，球形金纳米粒子和金纳米棒的各种大小将结合到感兴趣的蛋白质（例如铁结合蛋白转铁蛋白）和内在的哺乳动物细胞系。其目的是展示完整的CLEM工作流程，从FWM活细胞成像开始（在卡迪夫），固定，电镜显示细胞超微结构内相同AuNP的切片和相关成像（在布里斯托）。该项目还将探索使用细胞质中表达的蛋白质直接在活细胞内组装AuNP的开创性概念，这些蛋白质具有结合金属并将其集中形成的能力。电子致密粒子。这可能为用金属纳米颗粒遗传标记细胞质蛋白开辟了道路。[1]DOI：10.1038/nature14503. [2]DOI：10.1039/c9nr08512b。