Correlation of imaging and impedimetric data to predict cell state and changes in bioreactors

监测和影响细胞（群体）生理状态的能力不仅对学术界的可重复性结果至关重要，而且对制药业尤为重要。在这里，细胞的生理状态与生物制品的生产直接相关，从而与利润和质量直接相关。有多种方法可以测量单元状态，但大多数方法只关注单个参数。整合和关联不同类型的数据会提供更多信息。相关光电子显微镜（CLEM）是一种将（活）光显微镜（LM）的优点与电子显微镜（EM）的纳米空间分辨率相结合的最强大的成像技术之一。利用这项技术，关键的生物学问题已经得到了解答。细胞结构和代谢活动影响细胞的电特性，通过采用宽带阻抗谱方法可以研究多种细胞特性。一般来说，高频与细胞质/内部结构的变化有关（10- 60mhz），中频与细胞膜有关（2-10MHz），低频与细胞大小有关（0.1-2 MHz）。我们已经证明，当暴露于毒性挑战时，细胞培养阻抗谱的剂量依赖性特征与毒素的作用模式b[2]有关。从单个实验中提取不仅仅是LM和EM数据，还包括其他类型的信息，将CLEM领域扩展到一种称为相关多模态成像（CMI）的方法，这是一种需要和动力。原则上，CMI方法可以从单个事件中提取任何类型的信息，并将数据关联和集成。该项目首次能够理解并将显微镜下检测到的细胞结构的形态学和解剖学变化与阻抗谱特征的变化联系起来。阻抗谱可用于监测生物反应器中的细胞生长，通过了解与细胞应激相关的阻抗谱变化，将能够早期发现和减轻细胞应激。我们建议开发一种CMI方法，将控制和应激（例如毒素）条件下的实时成像与阻抗测量相结合，以便更好地了解对应激源的不同反应。我们将使用活钙和/或ATP成像结合阻抗测量。随后，我们也将致力于将应力受体- gfp成像和EM形态学引入到这一相关方法中，以提高技术的能力。

The ability to monitor and influence the physiological state of a cell (population) is of critical importance not just for reproducible results in academia but especially in the pharmaceutical industry. Here, the cell physiological state is directly correlated with the production of biologicals and consequently profitability and quality.There are multiple ways to measure the cell state but most look at a single parameter. It is much more informative to integrate and correlate different types of data. Correlative Light Electron Microscopy (CLEM) is one of the most powerful imaging technologies combining the advantages of (live) light microscopy (LM) with the nanometer spatial resolution of electron microscopy (EM) into one experiment. Using this technology, key biological questions have been answered [1].Cell structures and metabolic activity affect the electrical properties of the cell and by employing a broadband approach to impedance spectroscopy multiple cell properties can be investigated. In general terms, high-frequencies are associated with changes in the cell cytoplasm/internal structures (10-60Mz), middle-frequencies with the cell membrane (2-10MHz) and lower-frequencies with cell size (0.1-2 MHz). We have shown that dose-dependent characteristics of the impedance spectra of cell culture when exposed to a toxic challenge are related to the toxin's mode of action [2].There is a need and drive to extract more than just LM and EM data from a single experiment and to include other types of information, expanding the CLEM field to an approach called Correlative Multimodal Imaging (CMI). In principle the CMI approach would extract any type of information from a single event and correlate and integrate the data.This project enables, for the first time, to understand and correlate morphological and anatomical changes in the cell structure, detected microscopically, to changes in characteristics of an impedance spectrum. Impedance spectroscopy can be employed to monitor cells growing in a bioreactor and by understanding the changes in the impedance spectra related to cell stress will enable the early detection and mitigation of cell stress.We propose to develop a CMI approach that combines live imaging with impedance measurements in control and stressed (e.g. toxins) conditions in order to better understand the different responses to stressors. We will use live calcium and / or ATP imaging in combination with impedance measurements. Subsequently we will also aim to introduce stress receptor-GFP imaging and EM morphology into this correlative approach to increase the power of the technology.