PhD: Real-time nanoscale imaging in live cells - High Speed Single Molecule Localisation Microscopy

背景：基于单分子的超分辨率显微镜是最近发展起来的一种光学成像技术，它允许在远小于衍射极限（~250 nm）的空间尺度上成像。近年来，这些技术取得了巨大的成功，特别是在研究重要的生物学问题（2014年诺贝尔化学奖）。这些技术的基础依赖于通过交换时间信息来获得更高的空间分辨率，因此它们仍然相对较慢。虽然全细胞成像已经达到了~ 20nm的分辨率，但这通常需要10分钟，并且显然与动态生命系统不兼容。目的：本项目旨在通过开发一种新型高速单分子定位显微镜（hsSMLM）来克服低速单分子超分辨率显微镜用于动态生命系统的局限性。博士学位的主要目标是开发用于hsSMLM的物理显微镜，以及在高速成像时从背景中提取荧光信号所需的数据分析技术。方法：这项工作将建立在现有的SMLM技术的基础上，用低灵敏度的高速相机取代现有显微镜中使用的高灵敏度相机。我们最初的目标是实现50纳米分辨率的实时（~25赫兹）细胞成像。一旦实现了这一点，就有进一步的潜力将传统SMLM中的既定技术转移到hsSMLM版本，例如3D双螺旋点扩展函数成像[1]光谱分辨率[2,3]和偏振分辨率[4]超分辨率显微镜，以实现新的生物研究。该项目将提供多学科培训，培养学生在光子学、生物学、显微镜学、信号处理和新型探针开发方面的技能。这个项目允许以方法为主导和以应用为主导的发展，为一个有吸引力和非常成功的博士提供最好的机会。应用：一旦开发成功，许多生物学问题将进入一个新的前沿。主要的生物学兴趣与人类健康有关，包括：(1)人类诱导多能干细胞衍生的皮质神经元突触传递的实时成像和直接可视化，这在阿尔茨海默病和帕金森病等神经退行性疾病中很重要。突触传输发生在毫秒级的时间尺度和纳米级的长度尺度上，因此从未被成像。hsSMLM将能够第一次直接观察这一已建立的机制。(2)适应性免疫的分子基础，在T细胞受体聚集中，在类风湿关节炎等自身免疫性疾病中起重要作用。目前，全细胞成像非常耗时：以22纳米各向同性分辨率成像T细胞目前需要约4小时。hsSMLM将把这个时间缩短到不到1秒。工业参与：该项目将与显微镜公司的应用科学家和工业合作伙伴3i的Owen Richards博士合作。我在授权和商业化学术发现方面都有出色的记录。

Background: Single-molecule based super-resolution microscopy is a recently developed family of optical imaging techniques that allow for imaging at spatial scales far smaller than the diffraction limit (~250 nm). These techniques have seen a great deal of success in recent years, particularly in investigating important biological questions (Nobel Prize Chemistry 2014). The basis of these techniques relies on gaining higher spatial resolution by trading off temporal information, therefore they remain relatively slow. Although whole-cell imaging has been achieved to ~20 nm resolution, this can typically take 10s of minutes and is clearly not compatible with a dynamic living systems. Aims: This project will aim to overcome the limitations of low-speed single-molecule based super-resolution microscopy for dynamic living systems by developing a novel high-speed Single-Molecule Localisation Microscope (hsSMLM). The primary aim of the PhD will be to develop both the physical microscope used for hsSMLM and the data analysis techniques necessary to extract the fluorescence signal from the background when imaging at high speed. Methodology: This work will build upon existing SMLM techniques, substituting the high sensitivity cameras used in existing microscopes for a lower sensitivity high speed camera. Our initial goal is to achieve real-time (~25 Hz) cellular imaging with 50 nm resolution. Once this is achieved, there is further potential to transfer established techniques in traditional SMLM to hsSMLM versions, such as 3D Double helix point spread function imaging[1] spectrally resolved[2,3] and polarisation resolved[4] super-resolution microscopy to enable novel biological investigations.This project will deliver multidisciplinary training where the student will develop skills in photonics, biology, microscopy, signal processing and novel probe development. This project allows for both method-led and application-led development, providing the best possible opportunity for an engaging and highly successful PhD.Application: Once developed, a new frontier of numerous biological questions will be accessible. The primary biological interests pertain to human health and include:(1) real-time imaging and direct visualisation of synaptic transmission in human induced pluripotent stem cell derived cortical neurons, important in neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Synaptic transmission occurs across time scales of milliseconds and length scales of nanometres, and as such have never been imaged. hsSMLM would enable the first direct observation of this established mechanism.(2) The molecular basis of adaptive immunity, in receptor clustering in T cells, important in auto immune diseases such as rheumatoid arthritis. At present whole-cell imaging is extremely lengthy: imaging a T cell with 22 nm isotropic resolution currently takes ~4 hours. hsSMLM would reduce this to less than a second. Industrial Engagement: The project will involve a collaboration with Dr Owen Richards, an applications scientist at the microscopy company and industrial partner 3i. 3i have an excellent track-record in both licencing and commercialising academic discoveries.