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）是目前最强大的成像技术之一，它结合了光学显微镜（LM）的活细胞成像和电子显微镜（EM）的亚纳米空间分辨率的优点。利用这项技术，关键的生物学问题得到了解答。然而，CLEM受到可靠探针可用性的严重阻碍。大多数双峰探针，使用荧光团（LM）和电子致密金纳米粒子（EM）附着在感兴趣的蛋白质上，是否实际上显示出相同的蛋白质池，这是非常值得怀疑的。这是由于一些限制，如荧光团的光漂白或猝灭，和/或探针在细胞内运输或EM样品处理后脱落。这些都是需要解决的严重缺陷。为此，该项目的目的是开发一种新的CLEM方法，其中将金纳米颗粒（AuNP）用作LM和EM的相同探针。使用卡迪夫大学开发的新型非线性光学显微镜技术，AuNP将在活细胞中的LM中可视化。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）下对光的强吸收和散射。该技术使用短光脉冲组合来产生和检测LSPR中AuNP传输或散射的变化，对单个小AuNP具有独特的敏感性，这些小AuNP可以在3D中以高空间分辨率检测到无背景的AuNP。值得注意的是，该技术对AuNP形状也非常敏感，这为通过形状识别进行探针多路复用开辟了前景。在这个项目中，球形aunp和各种大小的金纳米棒将与感兴趣的蛋白质（例如铁结合蛋白转铁蛋白）结合，并在哺乳动物细胞系中内化。目的是演示完整的CLEM工作流程，从使用FWM成像活细胞（在卡迪夫）开始，到EM显示的细胞超微结构中相同AuNP的固定，切片和相关成像（在布里斯托尔）。该项目还将探索利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浓缩形成电子致密颗粒的能力。这可能为用金属NPs对细胞质蛋白进行遗传标记开辟道路。DOI: 10.1038/nature14503。[2] DOI: 10.1039/c9nr08512b。