4D Multiplexed Functional Imaging in Deep Tissue

Abstract

We present a deep tissue multiplexed functional imaging method that probes multiple cellular conditions in live model organisms. The method uses FRET-based biosensors to sense cellular conditions such as calcium and cAMP concentration levels, and uses fluorescence lifetime quantification to interpret the cellular conditions reported by the FRET biosensors. The method is based on Fourier lifetime excitation-emission matrix (FLEEM) spectroscopy [1] that simultaneously measures fluorescence lifetimes at multiple excitation and emission wavelengths within 23 microseconds, allowing quantifications on multiple FRET biosensors simultaneously in live model organisms. Samples are imaged in 3D by combining FLEEM spectroscopy with scanning laser optical projection tomography (SLOT) [2]. We demonstrate the method in zebrafish embryos transiently expressing cAMP FRET biosensor, which showed an increase in cAMP concentration upon physiological stimulus with forskolin and IMBX. Calcium and cAMP concentration levels during zebrafish embryonic development were monitored with time lapse 3D functional imaging of the embryo from 12 hours to 22 hours post fertilization. The method opens the door to multiplexed functional imaging of cellular biochemistries in whole live organisms.[1] M. Zhao and L. Peng, Optics Letters. 35,2910. (2010).[2] R. A. Lorbeer, et al., Optics Express. 19,5419. (2011).View Large Image | View Hi-Res Image | Download PowerPoint Slide