4-Laser Cell Sorter

随着我被任命为弗里德里希-亚历山大-纽伦堡大学药物生物学的主席，我想启动研究重点的科学重新定位。在接下来的几年里，我们将加紧研究细菌传染病，这是人类面临的三大威胁之一。我们正在研究细菌如何相互交流，并通过细胞衍生的小泡来调节宿主细胞。这些胞外小泡是基本的细胞通讯工具，与各种病理生理学有关。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。教席上的实验室将在2022/23年进行大规模翻新，以建立最佳的研究基础设施。在这些整修之后，我计划建立一个独立的S2区，在那里将优化和评估病原体的感染模型。为此，我想为测量区域配备一个共焦荧光显微镜和一台其他用户也可以使用的流式细胞仪。我为这两个主要研究仪器提交单独但平行的申请，我要求进行联合审查，因为这些设备是将在主席会议上创建的S2基础设施的共同组成部分。共聚焦显微镜主要用于感染模型的时空可视化，而流式细胞术则用于对细胞样本进行量化。这两个设备的区域分离将导致细胞样本运输过程中的不确定性和不稳定性，因此将意味着科学结果的显著减少，并阻碍新的抗感染疗法的开发。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(侧面和前向散射)并行测量至少15种荧光颜色。激光覆盖405 nm(紫色)、488 nm(绿色)、561 nm(黄绿色)和638 nm(红色)的波长，以测量大多数典型的荧光团和补偿量。通过紫光激光可以直接测量约200 nm大小的囊泡，从而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(相当于可溶荧光分子)的分辨率可测量，才能获得最高的灵敏度。该装置应对井板中的活细菌提供稳定和无气泡的分选。除了FACS设备外，还需要一台功能强大的数据采集PC，以及一个可用于在单个工作站计算机上评估分析的软件加密狗。该设备安装在一个单独的黑暗房间里。

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.