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Super-resolution Light-Addressable Potentiometric Sensors (LAPS)

超分辨率光可寻址电位传感器 (LAPS)

基本信息

批准号：
BB/P026788/1
负责人：
Steffi Krause
金额：
$ 19.25万
依托单位：
Queen Mary University of London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP026788%2F1
关键词：
Super resolution Light Addressable Potentiometric

项目摘要

In-vitro cell models have been used very successfully to investigate disease mechanisms and the efficacy and toxicity of drugs. They have the potential to eventually abolish the need for animal experiments. The investigation of complex biological processes in cell culture requires sophisticated measurement tools, and there is currently a lack of tools capable of providing quantitative chemical information on the surface-attached (basal) side of living cells. In this project, a novel instrument will be developed that can revolutionise our ability to understand ion fluxes and pH changes in living cells by quantitatively measuring the concentrations of ions, electrical cell-signals and the transport properties of living cells in the surface attachment area with nanoscale lateral resolution. The instrument proposed is based on light-addressable potentiometric sensors (LAPS). In this technique, light is modulated and then focused onto an electrolyte/ insulator/ semiconductor (EIS) structure. The light excites a local photocurrent, which depends on the local surface charge of the insulator and the local impedance of anything in contact with the insulator surface. Using thin semiconductor layers in silicon-on-sapphire (SOS) and a femtosecond, near-infrared laser for photocurrent excitation, a resolution of 800 nm was achieved, which allowed measurement of single cells, but was not sufficient for reliable sub-cellular resolution.Super-resolution microscopy or nanoscopy (Nobel Prize in Chemistry 2014) has brought fluorescence microscopy into the nanodimension of live-cell imaging beyond the diffraction limit, which provided a new level of detail of living cells in bioimaging research. In this project, the principle of Stimulated Emission Depletion (STED) nanoscopy will be applied to LAPS to obtain a novel instrument capable of imaging ion concentrations and cell impedance at the nanoscale. As in STED, we propose to use two laser beams. However, in contrast to STED, we will use these two beams to excite and inhibit photocurrent in EIS structures instead of fluorescence. We will illuminate the LAPS substrates with one focused, modulated light beam for photocurrent excitation and with a doughnut shaped light beam of high, constant intensity of the same wavelength for inhibition of the photocurrent outside the central focus of the modulated beam. As in STED, this is expected to result in a resolution well below the diffraction limit of less than 50 nm and will allow resolution of subcellular features. In contrast to the femtosecond laser technology employed previously for high-resolution LAPS, we envisage a sixteen-fold improvement in the resolution (without incurring any of the disadvantages of STED such as photobleaching or the necessity of specialised fluorescent dyes) while reducing the cost of the optical setup tenfold.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 this project, we will specifically measure pH on the basal (surface facing) side of cells. The instrument will be validated with polymer patterns to obtain quantitative information about the resolution that can be achieved and will then be further characterised using two cell models. (i) Yeast cells will be immobilised on the pH sensitive surfaces using agarose gel. The change of pH under and around a single yeast cell in the presence of glucose will be monitored using the new measurement system. (ii) 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. We will image pH and impedance changes at the basal side of the retinal pigment epithelium to gain more information about the mechanism of the formation of local deposits, which are the hallmark of AMD.

体外细胞模型已被非常成功地用于研究疾病机制以及药物的疗效和毒性。它们有可能最终废除动物实验的需要。研究细胞培养中的复杂生物过程需要复杂的测量工具，目前还缺乏能够提供活细胞表面附着(基底)侧的定量化学信息的工具。在这个项目中，将开发一种新的仪器，通过以纳米级的横向分辨率定量测量离子浓度、细胞电信号和活细胞在表面附着区的传输特性，来彻底改变我们了解活细胞中离子通量和pH变化的能力。提出的仪器是基于光可寻址电位传感器(LAPS)。在该技术中，光被调制，然后聚焦到电解液/绝缘体/半导体(EIS)结构上。光激发局部光电流，这取决于绝缘体的局部表面电荷和与绝缘体表面接触的任何东西的局部阻抗。利用蓝宝石上硅(SOS)中的薄层半导体和飞秒近红外激光进行光电流激发，实现了800 nm的分辨率，这使得可以测量单个细胞，但不足以实现可靠的亚细胞分辨率。超分辨率显微镜或纳米显微镜(2014年诺贝尔化学奖)将荧光显微镜引入活细胞成像的纳米维度，超出了衍射极限，这为生物成像研究中活细胞的细节提供了一个新的水平。在本项目中，受激发射耗尽(STED)纳米显微镜的原理将应用于LAPS，以获得一种能够在纳米尺度上成像离子浓度和细胞阻抗的新型仪器。正如在STED中一样，我们建议使用两束激光。然而，与STED不同的是，我们将使用这两束光来激发和抑制EIS结构中的光电流，而不是荧光。我们将用一束聚焦的调制光束来激发光电流，并用一束相同波长的高、恒定强度的甜甜圈形状的光束来照射LAPS基板，以抑制调制光束中心焦点外的光电流。正如在STED中一样，这预计将导致远低于50 nm的衍射极限的分辨率，并将允许亚细胞特征的分辨率。与以前用于高分辨率LAPS的飞秒激光技术相比，我们预计分辨率将提高16倍(不会招致任何STED的缺点，如光漂白或特殊荧光染料的必要性)，同时将光学设置的成本降低10倍。通过对EIS结构中的绝缘体表面进行化学修饰，表面电荷的变化可以由不同的离子物种特定地诱导，从而产生定量的浓度依赖信号。在这个项目中，我们将专门测量细胞底部(面向表面)的pH值。该仪器将用聚合物图案进行验证，以获得有关可实现的分辨率的定量信息，然后将使用两个细胞模型进一步表征。(I)用琼脂糖凝胶将酵母细胞固定在对pH敏感的表面。新的测量系统将监测单个酵母细胞在葡萄糖存在下及其周围的pH值变化。(Ii)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。我们将成像视网膜色素上皮基底部的pH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。