Rapid super-resolution optical imaging of immunological synapse formation initiated by holographic optical tweezers

全息光镊启动免疫突触形成的快速超分辨率光学成像

• 批准号: 415832635
• 负责人: Professor Dr. Thomas Huser
• 金额: --
• 依托单位: Arbeitsgruppe Biomolekulare Photonik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/415832635?language=en
• 关键词: Rapid; super; resolution; optical; imaging

This research proposal aims at developing a fast, super-resolving structured illumination microscope (SR-SIM) to resolve the structure and dynamics of signalling microclusters in living T cells. This is accomplished by implementing the SR-SIM image reconstruction process on fast parallel-processing graphics processor platforms to enable the imaging and displaying of reconstructed super-resolved images of living cells at video rate. The system will further integrate holographic optical tweezers that enable the manipulation (immobilization) of living, non-adherent immune cells within the focal plane of the microscope, and to initiate immunological synapse formation between living immune cells. To permit the three-dimensional reconstruction of images of living immune cells, the rapid acquisition of images from multiple image planes simultaneously, as well as compensation of spherical aberrations induced by the microscope optics will be included in the final optical system. The performance of this system will then be utilized to image an immunologically highly relevant targt, i.e. the vesicular trafficking of signalling molecules within T cells. The high temporal and spatial resoluton will enable us to determine if signalling molecules, such as TCR, CD4, and Lck are transported in the same or different vesicles. This system will allow us to image docking and fusion of individual vesicles delivered to the T cell plasma membrane, and it wil enable us to determine how vesicle transfer and clustering change between resting and activated T cells. The ability to control the position and orientation of non-adherent cells at will by optical tweezers, as well as to perform high-speed volumetric imaging will ultimately also enable us to determine the vesicular trafficking routes, rates, and degrees of clustering between interacting immune cells.

该研究计划旨在开发一种快速，超分辨率的结构照明显微镜（SR-SIM），以解决活T细胞中信号微簇的结构和动力学。这是通过在快速并行处理图形处理器平台上实现SR-SIM图像重建过程来实现的，以使得能够以视频速率成像和显示活细胞的重建超分辨率图像。该系统将进一步集成全息光镊，使活的，非粘附的免疫细胞的显微镜的焦平面内的操作（固定），并启动活的免疫细胞之间的免疫突触形成。为了允许活的免疫细胞的图像的三维重建，同时从多个图像平面的图像的快速采集，以及由显微镜光学引起的球面像差的补偿将包括在最终的光学系统。然后，该系统的性能将被用于成像免疫学上高度相关的目标，即T细胞内信号分子的囊泡运输。高的时间和空间分辨率将使我们能够确定信号分子，如TCR，CD 4和Lck是否在相同或不同的囊泡中转运。该系统将使我们能够对递送到T细胞质膜的单个囊泡的对接和融合进行成像，并且它将使我们能够确定囊泡转移和聚集在静息和活化T细胞之间如何变化。通过光镊随意控制非粘附细胞的位置和方向以及进行高速体积成像的能力最终也将使我们能够确定相互作用的免疫细胞之间的囊泡运输路线、速率和聚集程度。