PROJECT SUMMARYWe propose to develop and disseminate an advanced 2p Bessel beam light sheet (2pBLS) microscopyinstrument for high-speed sub-micron-resolution volumetric imaging in deep tissue. The proposed instrument willhave 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 simultaneouslifetime imaging with FRET sensors and high-speed light sheet imaging of dynamics intensity-modulated sensorsfor dual functional imaging in deep tissue. The system will be tested with two imaging studies in zebrafish andmouse respectively. In zebrafish we will demonstrate live FLIM imaging of genetically encoded FRET sensorsin zebrafish kidney and perform an imaging study of Ca2+ and cAMP activities in zebrafish renal cells. In micewe 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.