4-Laser Cell Sorter

随着我被任命为埃尔兰根-新<s:1>伦堡大学（Friedrich-Alexander-University erlangen - nrnberg）的药物生物学主席，我想对研究重点进行科学的重新定位。在今后几年中，我们将加紧研究细菌传染病，这是对人类的三大威胁之一。我们正在研究细菌如何通过细胞衍生的囊泡相互交流和调节宿主细胞。这些细胞外囊泡是基本的细胞通讯和相关的各种病理生理。我们的基本发现有助于开发细菌感染和炎症性疾病领域迫切需要的新治疗方案。椅子上的实验室将在2022/23年进行大规模翻新，以创造最佳的研究基础设施。在这些翻新之后，我计划建立一个独立的S2区域，对病原体的感染模型进行优化和评估。为此，我想在一个测量区域配备一台共聚焦荧光显微镜和一台流式细胞仪，其他用户也可以使用。我正在为这两种主要研究仪器提交单独但并行的申请，我要求进行联合审查，因为这些设备这些设备是S2基础设施的共同组成部分，将在主席处创建。共聚焦显微镜主要用于感染模型的时空可视化，而流式细胞术用于量化细胞样本。这两种装置的面积分离将在细胞样品的运输过程中带来不确定性和不稳定性，因此将意味着科学成果的显著减少，并损害新的抗感染疗法的发展。计划中的台式流式细胞仪具有广泛的适用性，配备了四个激光器，因此可以通过两个散射光探测器（侧面和正向散射）平行测量至少15种荧光颜色。激光覆盖波长405 nm（紫色），488nm（绿色），561nm（黄绿色）和638nm（红色）来测量最典型的荧光团和补偿。通过紫色激光直接测量大小在200纳米左右的囊泡的可能性，可以将它们与细胞和细胞碎片区分开来。所有荧光必须以10-30 MESF（等效可溶性荧光染料分子）的分辨率测量，以达到最高灵敏度。该设备应对孔板中的活菌提供稳定且无气泡的分选。除了FACS设备，还需要一个功能强大的数据采集PC，以及一个软件加密狗，可用于评估个人工作站计算机上的分析。这个装置被安装在一个单独的暗室里。

With my appointment at the chair of Pharmaceutical Biology at the Friedrich-Alexander-University Erlangen-Nürnberg, I would like to initiate a scientific reorientation of the research focus. In the coming years, we will be working intensively on bacterial infectious diseases, which represent one of the top three threats to humanity. We are investigating how bacteria communicate with each other and modulate host cells by means of cell-derived vesicles. These extracellular vesicles are fundamental cell communicators and relevant in various pathophysiologies. Our fundamental findings contribute to the development of urgently needed new therapeutic options in the field of bacterial infections and inflammatory diseases. The laboratories at the chair will be extensively renovated in 2022/23 to create an optimal research infrastructure. Following these refurbishments, I plan to establish an independent S2 area in which infection models on pathogens will be optimised and evaluated. For this purpose, I would like to equip a measuring area with a confocal fluorescence microscope and a flow cytometer that is also accessible to other users. I am submitting separate but parallel applications for these two major research instruments and I am asking for a joint review, as the devices as these devices are a common integral part of the S2 infrastructure to be created at the chair. The confocal microscope is primarily used for the spatial-temporal visualisation of infection models, while flow cytometry is used to quantify the cellular samples. An areal separation of these two devices would entail uncertainties and instabilities during the transport of the cell samples and would therefore mean a significant reduction in scientific results and impair the development of new anti-infective therapies. The planned benchtop flow cytometer has a wide applicability and is equipped with four lasers and thus the possibility to measure at least 15 fluorescent colours in parallel via two scattered light detectors (side and forward scattering). The lasers cover the wavelengths 405 nm (violet), 488 nm (green), 561 nm (yellow-green) and 638 nm (red) to measure most typical fluorophores and compensations. The possibility to directly measure vesicles in the size range around 200 nm via a violet laser allows to distinguish them from cells and cell debris. All fluorescence must be measurable with a resolution of 10-30 MESF (Molecules of Equivalent Soluble Fluorochromes) to achieve the highest sensitivity. The device shall provide stable and bubble-free sorting of live bacteria in well plates. In addition to the FACS device, a powerful data acquisition PC is also required, as well as a software dongle that can be used to evaluate the analyses on individual workstation computers. The device is installed in a separate, dark room.