High-speed correlative live imaging microscope for biomedical applications

疾病可影响人类、牲畜和作物，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最致命的疾病（全世界每年死亡1450万人），其次是传染病（下呼吸道感染、腹泻、艾滋病毒/艾滋病、结核病——900万人）、癌症（880万人）等。例如，尽管艾滋病毒于1983年首次被分离出来，并且是研究得最充分的病毒之一，但仍然没有研制出艾滋病毒疫苗的前景。因此，目前全世界有近3 800万人感染了1型人类免疫缺陷病毒（艾滋病毒）。还有许多其他虽然不危及生命，但严重使人衰弱的疾病和病症，特别是影响全世界10亿人的神经系统疾病（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。这就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、多模态生命细胞成像的显微镜技术，以保留细胞的功能。几乎所有的细胞类型都具有由脂质膜和支持细胞骨架结构组成的高度结构化的表面，这些结构定义了适合执行特定功能的微域。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，但数据缺乏与细胞表面形态相关的空间信息。这是因为由于脂质荧光染料由于细胞膜的再循环而进入细胞，因此荧光显微镜不能用于长时间的细胞地形成像。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备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过程。在此，我们提出了一种新的生物医学研究相关的实时成像技术。成像技术将基于高速扫描离子电导显微镜（HS-SICM）的结合，该技术可以产生几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高达每秒800个平面的采集速率。这种成像技术将使研究人员能够发现细胞膜上单个分子发挥作用的位置。我们将通过成像人类免疫缺陷病毒（HIV）样颗粒在活t细胞中的组装来验证我们的新显微镜技术；β细胞中单个胰岛素囊泡的释放，负责胰岛素的产生——胰岛素是控制血糖水平的激素，与糖尿病有关；巨噬细胞中Myddosome的形成与炎症和阿尔茨海默病有关。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.