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

项目总结 我们建议发展和传播先进的2p贝塞尔光束光片(2pBLS)显微镜 用于深部组织的高速亚微米分辨率体积成像的仪器。拟议的文书将 在大视场范围内具有明显更高的体积成像速度，保持亚微米级的衍射- 尽管存在组织散射和像差，但深层组织的分辨率有限，并同时执行 FRET传感器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。该系统将通过斑马鱼和斑马鱼的两项成像研究进行测试 分别为小鼠。在斑马鱼中，我们将演示遗传编码的FRET传感器的实时胶片成像 并对斑马鱼肾脏细胞中的钙离子和cAMP活性进行成像研究。在老鼠身上， 我们将演示用活性传感器(钙离子和GEVI)基因标记的神经和神经胶质活动的成像， 体外和体内的膜-FRET传感器(PKA)和结构标记。

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.