High-speed correlative live imaging microscope for biomedical applications

疾病可影响人类、牲畜和农作物，构成重大风险之一，并造成重大经济负担。心血管疾病目前是人类死亡人数最多的疾病(全世界每年有1,450万人死亡)，其次是传染病(下呼吸道感染、腹泻、艾滋病毒/艾滋病、肺结核--900万人)、癌症(880万人)等。因此，全球目前有近3800万人感染了人类免疫缺陷病毒1型(HIV)。还有许多其他虽然不会危及生命的疾病和状况，特别是影响全球10亿人的神经性疾病(癫痫、阿尔茨海默病和痴呆症)。在分子和细胞水平上了解疾病进展和感染传播机制是成功开发有效药物、疫苗和治疗方法的关键。这需要开发能够在模拟生理条件的溶液中进行非侵入性、高分辨率、实时、多模式生命细胞成像的显微技术，以便保留细胞功能。几乎所有类型的细胞都有高度结构化的表面，由脂膜和支持细胞骨架结构组成，这些结构定义了为执行特定功能而定制的微域。尽管单分子荧光成像使研究人员能够研究单个蛋白质如何发挥其功能，但这些数据缺乏与细胞表面形态有关的空间信息。这是因为荧光显微镜不能用于长时间的细胞形貌成像，因为由于细胞膜的循环，脂质荧光染料被带入细胞。基于扫描电子显微镜和荧光显微镜的相关成像只能在化学固定和干燥的制剂中产生细胞表面和荧光标记分子的组合图像，因此不能用于研究活体过程。在这里，我们建议开发一种新的相关生物医学研究的活体成像技术。成像技术将基于高速扫描离子电导显微镜(HS-SICM)和光片荧光显微镜的组合，HS-SICM可以产生几个纳米分辨率的活生物细胞膜表面的无标签3D图像，而光片荧光显微镜可以提供单分子分辨率和每秒高达800个平面的采集速率。这种成像技术将使研究人员能够找出单个分子在细胞膜上的哪些位置发挥其功能。我们将通过成像来验证我们的新显微技术，方法包括：在活的T细胞中组装人类免疫缺陷病毒(HIV)样颗粒；在β细胞中释放单个胰岛素小泡，负责产生胰岛素-胰岛素是一种控制血糖水平并与糖尿病有关的激素；在活的巨噬细胞中形成与炎症和阿尔茨海默病有关的Myddosome。这种仪器一旦建成，将在基础和应用生物医学科学中得到广泛应用，因为它将使研究人员能够进行目前不可能进行的实验。

Diseases can affect humans, life stock and crops, posing one of the major risks and causing significant economic burdens. Cardiovascular diseases are currently the deadliest in humans (14.5 million deaths per year worldwide), followed by infectious diseases (lower respiratory infections, diarrhoea, HIV/AIDS, tuberculosis - 9M), cancers (8.8M) etc. For example, despite the fact that HIV was first isolated in 1983 and is one of the most well studied viruses, there is still no prospect of an HIV vaccine. As a result, there are nearly 38 million people worldwide currently living with human immunodeficiency virus type-1 (HIV). There are many other, although not life threatening, hugely debilitating diseases and conditions, particularly neurological (epilepsy, Alzheimer's disease and dementias) that affect 1 billion people worldwide. Understanding disease progression and infection spreading mechanisms at molecular and cellular level is key to the successful development of effective drugs, vaccines and therapies. This requires development of microscopy techniques capable of non-invasive, high resolution, real-time, multi-modal life cellular imaging in solutions that mimic physiological conditions so that cell functionality is retained. Almost all cell types have highly structured surfaces composed of lipid membrane and supporting cytoskeletal structures, which define microdomains tailored to perform specific function. Although single molecule fluorescence imaging enables researchers to study how individual proteins perform their function, the data lacks spatial information which could be linked to cell surface morphology. This is because fluorescence microscopy cannot be used for imaging of the cell topography over long periods of time since lipid fluorescent dyes are taken into cells due to the cell membrane recycling. Correlative imaging based on Scanning Electron Microscopy and Fluorescence Microscopy can produce combined images of cell surfaces and fluorescently labelled molecules only in chemically fixed and dried preparation, hence can not be used to study live processes. Here we propose to develop a new correlative live imaging technique for biomedical research. The imaging technique will be based on a combination of high-speed Scanning Ion Conductance Microscopy (HS-SICM) that can produce label-free 3D images of living biological cell membrane surfaces with several nanometers resolution and a light sheet fluorescence microscopy that can deliver single molecule resolution and acquisition rates up to 800 planes per second. This imaging technique will enable researchers to find out at what locations at the cell membrane individual molecules perform their functions. We will validate our new microscopy technique by imaging the assembly of human immunodeficiency virus (HIV) -like particles in living T-cells; the release of individual insulin vesicles in beta cells that are responsible for the production of insulin - the hormone that controls blood sugar levels and is implicated in diabetes mellitus; Myddosome formation in living macrophage that is linked to inflammation and Alzheimer's disease. Such an instrument, once built, will find a widespread application in fundamental and applied biomedical sciences, as it would enable researchers to perform experiments that are impossible at present.