Fast Multi-Functional 3D Imaging of Cellular Activities in Deep Tissue

项目摘要 我们建议开发和推广一种先进的2 p贝塞尔光束光片（2 pBLS）显微镜 用于深层组织中的高速亚微米分辨率体积成像的仪器。拟议的文书将 在大视场上具有显著更高的体积成像速度，保持亚微米衍射， 尽管存在组织散射和像差，但在深部组织中的分辨率有限， 使用FRET传感器的寿命成像和动态强度调制传感器的高速光片成像 用于深部组织的双功能成像。该系统将在斑马鱼和 老鼠分别在斑马鱼中，我们将展示遗传编码FRET传感器的实时FLIM成像 并对斑马鱼肾细胞内Ca ~（2+）和cAMP活性进行了成像研究。在小鼠中， 我们将展示用活性传感器（Ca 2+和GEVI）遗传标记的神经和神经胶质活动的成像， FLIM-FRET传感器（PKA）和离体和体内结构标记物。

PROJECT SUMMARY We propose to develop and disseminate an advanced 2p Bessel beam light sheet (2pBLS) microscopy instrument for high-speed sub-micron-resolution volumetric imaging in deep tissue. The proposed instrument will have a significantly higher volume imaging speed over a large field of view, maintain a sub-micron diffraction- limited resolution in deep tissue despite the presence of tissue scatter and aberration, and perform simultaneous lifetime imaging with FRET sensors and high-speed light sheet imaging of dynamics intensity-modulated sensors for dual functional imaging in deep tissue. The system will be tested with two imaging studies in zebrafish and mouse respectively. In zebrafish, we will demonstrate live FLIM imaging of genetically encoded FRET sensors in zebrafish kidney and perform an imaging study of Ca2+ and cAMP activities in zebrafish renal cells. In mice, we will demonstrate imaging of neural and glial activities genetically labeled with activity sensors (Ca2+ and GEVI), FLIM-FRET sensors (PKA) and structural markers ex vivo and in vivo.