Combined LAPS and SICM for multimodal live cell imaging

上皮和内皮组织排列在器官的腔或表面，如眼睛、肺、胃肠道和尿路或血管。这些组织的功能受损与许多健康问题有关，包括失明、动脉粥样硬化和糖尿病。这些组织中的细胞是强烈极化的，在顶端和底端表现出不同的特性。这种两极分化的性质对它们的功能至关重要，一方的事件直接影响另一方。因此，迫切需要能够同时研究根尖和基底部的工具。除了通过营养刺激或氧化应激引起的环境变化外，细胞通常还会对局部因素做出反应，比如光感受器外节断裂。这可能只在单个细胞内引起反应，但也会通过改变分泌影响邻近细胞。在这个项目中，将开发一种新的仪器，通过同时对细胞顶端和底部进行成像来提供有关顶端细胞形态和基础离子浓度以及电信号(如细胞表面电荷和阻抗)的同步信息，从而彻底改变我们监测细胞过程和极化细胞中细胞通信的能力。将首次建造一种仪器，将以前独立使用的两种电化学成像技术结合在一起来测量电池响应。光寻址电位传感器(LAPS)是基于电解液-绝缘体-半导体(EIS)结构的光电流测量。LAPS对表面电荷、离子浓度和阻抗很敏感。如果细胞培养在LAPS底物上，所有这些参数都可以在细胞的底物面上、基底部和侧面进行测量。扫描离子电导显微镜(SICM)测量通过扫描电池表面的纳米管的离子电流。SICM已被用来成像细胞外部(顶端)的详细细胞形态，并通过将试剂局部释放到单个细胞来诱导微环境的变化。通过结合LAPS和SICM，我们可以同时进行顶端和底端的功能电化学成像。在根尖侧局部释放试剂，同时连续成像离子浓度和细胞阻抗，将以前所未有的细节为我们提供对上皮和内皮组织传输机制的新见解，并有助于理解生理过程和疾病机制。通过化学修饰EIS结构中的绝缘体表面，不同的离子物种可以特异性地引起表面电荷的变化，从而在LAPS中产生定量的浓度依赖信号。在这个项目中，我们将具体测量pH、钙、锌和钠离子。该仪器将用聚合物模式进行验证，以确保LAPS和SICM的适当同步，然后将使用两个细胞模型进一步表征。(I)视网膜色素上皮(RPE)细胞已被用作研究老年性黄斑变性(AMD)机制的细胞模型。AMD是老年人最常见的致盲原因，锌、钙和pH的基础变化与沉积物的形成有关。我们将使用SICM纳米管通过顶端释放离子和诱导氧化应激的试剂来刺激RPE细胞，同时成像基底侧的离子浓度和阻抗变化，以获得更多关于局部沉积形成机制的信息，这是AMD的标志。(2)血管内皮细胞排列在血管的管腔表面，在循环血液和血管壁的其余部分之间起到屏障作用。新仪器将用于详细研究血管对钠和钙离子的通透性机制，这种通透性在动脉粥样硬化、糖尿病和肾脏疾病等疾病中会增加。

Epithelial and endothelial tissues line the cavities or surfaces of organs such as the eyes, lungs, gastrointestinal and urinary tract or blood vessels. Impaired function of these tissues is linked to many health issues including blindness, atherosclerosis and diabetes. The cells in these tissues are strongly polarised and display different properties on the apical and basal sides. This polarised nature is crucial for their function, and events on one side directly affect the other side. Hence, there is a great need for tools capable of investigating apical and basal sides simultaneously. Apart from environmental changes through nutritional stimuli or oxidative stress, cells often respond to localised factors like that of broken off photoreceptor outer segments. This may elicit response only in a single cell, but also affect neighbouring cells via changed secretion. In this project, a novel instrument will be developed that will revolutionise our ability to monitor cellular processes, and cell communication in polarised cells by simultaneously imaging cells apically and basally providing simultaneous information about apical cell morphology and basal ion concentrations and electrical signals such as cell surface charge and impedance. An instrument will be built that, for the first time, combines two electrochemical imaging techniques that have been used previously independently to measure cell responses. Light-addressable potentiometric sensors (LAPS) are based on the photocurrent measurements at electrolyte-insulator-semiconductor (EIS) structures. LAPS is sensitive to surface charges, ion concentrations and impedance. If cells are cultured on LAPS substrates, all these parameters can be measured on the substrate facing, basal, side of the cells. Scanning Ion Conductance Microscopy (SICM) measures ion currents through a nanopipette scanning the surface of the cell. SICM has been used to image detailed cell topography on the outer (apical) side of cells and to induce changes in the microenvironment by releasing reagents locally to a single cell. By combining LAPS and SICM, we can carry out functional electrochemical imaging simultaneously both apically and basally. The release of reagents locally on the apical side while continuously imaging ion concentrations and cell impedance basally will provide us with new insights into the transport mechanisms through epithelial and endothelial tissues with unprecedented detail and contribute to the understanding of physiological processes and disease mechanisms. By chemically modifying the insulator surface in the EIS structure, a change of surface charge can specifically be induced by different ionic species resulting in quantitative concentration dependent signals in LAPS. In this project, we will specifically measure pH, calcium, zinc and sodium ions. The instrument will be validated with polymer patterns to ensure proper synchronisation of LAPS and SICM and will then be further characterised using two cell models. (i) Retinal pigment epithelial (RPE) cells have been used as a cell model for the investigation of the mechanisms of age related macular degeneration (AMD) - the most prevalent cause of blindness in the elderly where basal changes in zinc, calcium and pH are implicated in deposit formation. We will stimulate RPE cells by apical release of ions and reagents inducing oxidative stress using the SICM nanopipette and simultaneously image ion concentrations and impedance changes at the basal side to gain more information about the mechanism of the formation of local deposits, which are the hallmark of AMD. (ii) The vascular endothelium lines the luminal surface of blood vessels performing a barrier function between circulating blood and the rest of the vessel wall. The new instrument will be used to study in detail the mechanism of vascular permeability for sodium and calcium ions, which is increased in diseases such as atherosclerosis, diabetes and renal diseases.