TIRF Microscope with laser illumination and two EMCCD cameras

我们的研究试图了解质膜信号通路的空间组织和组装动力学如何控制T细胞的激活和功能。这项研究是基于我们的发现，即质膜蛋白定位于纳米尺寸的区域。我们的研究表明，T细胞的激活是由不同纳米结构域的空间关系、信号复合体在其中的组装以及信号分子在它们之间的移位动态控制的。我们使用多学科策略研究潜在的分子机制，这些策略将单分子显微镜(即单粒子跟踪和超分辨率)与生化、生物物理和细胞生物学方法相结合。特别是，我们感兴趣的是T细胞受体(TCR)和抑制程序细胞死亡蛋白(PD-1)在T细胞激活和抗肿瘤反应中的途径。我们的最终目标是识别T细胞信号通路中的新控制原理，并利用它们来改变T细胞对未来免疫疗法的反应。我们需要先进的全内反射(TIRF)显微镜，这对我们在阿尔伯特-路德维希-弗赖堡大学新建立的实验室以及实现我们当前和未来的研究目标至关重要。所要求的显微镜具有以下能力：(I)全内反射显微镜，其中照明仅限于细胞和玻璃表面之间的接触点。(2)单分子检测，用于超分辨率成像和粒子跟踪，以分析蛋白质的分布和动力学。通过两个EMCCD摄像机采集图像，以获得最高的灵敏度和速度。(Iii)用于光操纵的局部激光控制，以测量蛋白质的动力学和分散性。四台高功率激光器覆盖了对我们的研究和方法开发最重要的照明波长。(4)宽场照明，用于快速采集和钙分析。(V)活体细胞成像的环境控制。

Our studies seek to understand how the spatial organization and assembly dynamics of plasma membrane signaling pathways control T cell activation and functions. The research is based on our findings that plasma membrane proteins are localized in nanometer-sized domains. Our studies revealed that T cell activation is dynamically controlled by the spatial relationships of distinct nanodomains, the assembly of signaling complexes within them and translocation of signaling molecules between them. We study the underlying molecular mechanisms using multidisciplinary strategies that combine single molecule microscopy (i.e. single particle tracking and super-resolution) with biochemical, biophysical, and cell biological approaches. Specifically, we are interested in T cell receptor (TCR) and the inhibitory program cell death protein (PD-1) pathways during T cell activation and anti-tumor responses. Our ultimate goal is to identify novel control principles in T cell signaling pathways and use them to modify T cell responses for future immunotherapies.We request an advanced Total Internal Reflection (TIRF) microscope, which is essential to our newly established laboratory at the Albert-Ludwigs-Universität Freiburg and for realizing our current and future research goals. The requested microscope has the following capabilities: (i) Total Internal Reflection Microscopy, where illumination is limited to the contact site between cells and glass surfaces. (ii) Single molecule detection for super resolution imaging and particle tracking to analyze protein distributions and dynamics. Images are acquired with two EMCCD cameras for maximal sensitivity and speed. (iii) Localized laser control for photo-manipulation to measure protein dynamics and dispersion. Four high powered lasers covering the illumination wavelengths most significant for our research and method development. (iv) Widefield illumination for fast acquisition and calcium analyses. (v) Environmental control for live cell imaging.