Spatio-temporal mapping of protein activity in live zebrafish using FRET FLIM OPT

Abstract

Optical projection tomography is a “mesoscopic” imaging technique, which fills a size gap in imaging, between techniques that provide subcellular resolution such as confocal laser scanning microscopy and cellular/whole body imaging techniques such as PET/MRI. OPT provides three dimensional (3D) whole body imaging utilising absorption or fluorescence contrast in mesoscopic (1-10 mm), transparent samples. This has the potential to be developed for use in biomedical research, for example in drug screening, developmental biology, and the study of disease mechanisms. To fully exploit the advantages of OPT, we image live zebrafish. These are the optimal size for imaging, and transparent mutants are readily available resulting in less scattering of optical radiation. They are also genetically tractable and many transgenic lines with fluorophore labels have been created. This thesis reports on the use of optical projection tomography (OPT) and fluorescence lifetime imaging (FLIM) to detect enzyme activity in whole live zebrafish. FRET biosensors for Caspase 1 and 3 activity were generated and the Caspase 3 FRET biosensor was validated in mammalian cell culture. Transgenic zebrafish expressing the biosensors ubiquitously under control of the ubiquitin (Ubi), macrophage (mpeg) and neutrophil (LysC) promoters were generated and used to validate the biosensors within live zebrafish. Caspase 3 is activated as part of the apoptotic pathway, in response to gamma irradiation. After exposure to 25 Gy irradiation, Caspase 3 activity is evident in the head region of 24 hpf zebrafish ubiquitously expressing the Caspase 3 biosensor, through both confocal and OPT FLIM imaging. FLIM OPT imaging allows visualisation of activity via production of 3D lifetime maps. Programs were generated to enable qualification and quantification of the data. Preliminary validation of the novel Caspase 1 biosensor indicates the biosensor is functional and it is possible to use FLIM to detect Caspase 1 activity in live zebrafish, in response to immune activation via tail transection. Overall, these findings demonstrate that FLIM OPT is a useful tool in mesoscopic imaging, able to identify enzyme activity in a whole live zebrafish, in 3D.