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

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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.