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显微镜是近年来发展起来的一系列光学成像技术，它可以在远小于衍射极限(~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方面(诺贝尔化学奖，2014年)。这些技术的基础依赖于通过权衡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。尽管全细胞成像已经达到了~20 nm的分辨率，但这通常需要10秒的时间，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的高速单分子定位显微镜(HsSMLM)来克服基于低速单分子的超分辨率显微镜在动态生命系统中的局限性。PHD的主要目标将是开发用于hsSMLM的物理显微镜和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，用现有显微镜中使用的高灵敏度相机取代低灵敏度的高速相机。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。一旦实现这一点，就有可能将传统SMLM中已有的技术转移到hsSMLM版本中，例如3D双螺旋点扩展函数成像[1]光谱分辨[2，3]和极化分辨[4]超分辨率显微镜，以实现新的生物学研究。该项目将提供多学科培训，学生将在其中发展光子学、生物学、显微镜、信号处理和新型探针开发的技能。该项目允许以方法为导向和以应用为导向的开发，为一个引人入胜和非常成功的博士提供了最好的机会。应用：一旦开发，许多生物学问题的新前沿将成为可能。主要的生物学兴趣与人类健康有关，包括：(1)实时成像和直接可视化人类诱导的多潜能干细胞来源的皮质神经元中的突触传递，这在阿尔茨海默氏症和帕金森氏症等神经退行性疾病中非常重要。突触的传输跨越毫秒的时间尺度和纳米的长度尺度，因此从未被成像过。HsSMLM将使首次直接观察到这一已建立的机制。(2)获得性免疫的分子基础，在T细胞中的受体聚集，在风湿性关节炎等自身免疫性疾病中非常重要。目前，全细胞成像非常耗时：目前成像一个各向同性分辨率为22 nm的T细胞需要大约4个小时。HsSMLM将把这一时间缩短到不到一秒。行业参与：该项目将涉及与Owen Richards博士的合作，Owen Richards博士是显微镜公司的应用科学家，也是行业合作伙伴3i。我在学术发现的许可和商业化方面都有很好的记录。

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.