Super-resolution Light-Addressable Potentiometric Sensors (LAPS)

体外细胞模型已经非常成功地用于研究疾病机制和药物的功效和毒性。它们有可能最终消除对动物实验的需求。研究细胞培养中复杂的生物过程需要复杂的测量工具，目前缺乏能够提供活细胞表面（基底）侧定量化学信息的工具。在这个项目中，将开发一种新型仪器，通过以纳米级横向分辨率定量测量离子浓度、细胞电信号和活细胞表面附着区域的运输特性，可以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寻址电位传感器（LAPS）。在这种技术中，光被调制，然后聚焦到电解质/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物体的局部阻抗。利用蓝宝石上硅（SOS）的薄半导体层和用于光电流激发的飞秒近红外激光器，实现了800 nm的分辨率，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）将荧光显微镜带入了超越衍射极限的活细胞成像的纳米尺度，为生物成像研究提供了活细胞细节的新水平。在这个项目中，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。在STED中，我们建议使用两束激光。然而，与STED相比，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照射LAPS衬底，用于光电流激发，并用一束相同波长的高恒定强度的甜甜圈形光束照射lap衬底，用于抑制调制光束中心焦点外的光电流。在STED中，这有望导致分辨率远低于小于50纳米的衍射极限，并将允许亚细胞特征的分辨率。与之前用于高分辨率LAPS的飞秒激光技术相比，我们设想将分辨率提高16倍（不会产生STED的任何缺点，如光漂白或需要专门的荧光染料），同时将光学装置的成本降低10倍。通过化学修饰EIS结构中的绝缘体表面，不同的离子种类可以特异性地诱导表面电荷的变化，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基侧（面向表面）的pH值。该仪器将用聚合物模式进行验证，以获得有关可以达到的分辨率的定量信息，然后将使用两种细胞模型进一步表征。(i)使用琼脂糖凝胶将酵母细胞固定在pH值敏感的表面。在葡萄糖存在的情况下，单个酵母细胞下和周围的pH值变化将使用新的测量系统进行监测。（ii）视网膜色素上皮细胞（RPE）已被用作研究年龄相关性黄斑变性（AMD）机制的细胞模型，AMD是老年人失明的最常见原因。我们将对视网膜色素上皮基底侧的pH值和阻抗变化进行成像，以获得有关局部沉积物形成机制的更多信息，这是AMD的标志。

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.