A Multi-modal Imaging Platform Using Light-Sheet and Photostable Probes for Quantitative Cell and Tissue-Scale Biology

Imaging dynamic molecular-scale events with high spatial and temporal resolution is central to discovering fundamental principles underlying molecular and cellular organization relevant to health and disease. Achieving this goal requires combined advances in imaging technology and fluorescent probe development applicable to a variety of biological questions and easily adaptable in multiple laboratory settings. Light-sheet microscopy is an attractive solution for live imaging due to its speed and low phototoxicity, achieved by restricting illumination to a sheet of light placed right at the focal plane perpendicular to the optical axis. We propose a simple yet powerful light-sheet microscope design, combining virtual light-sheet illumination via a fast-scanning laser beam with slit confocal detection. This enables high-resolution, volumetric, and multicolor imaging of diverse fluorescence properties, such as fluctuations, lifetime and polarization, which reveal dynamic molecular behaviours in live cells and tissues. In parallel, to meet the need for bright and photostable probes compatible with these advanced imaging modalities, we will develop a suite of multicolour, genetically-encoded probes based on mStayGold and other recently described fluorescent proteins. In Phase 1 of this project, feasibility, proof-of-principle, and ease of use of the prototype light-sheet system with three detection modalities using existing and new fluorescent proteins will be established. In Phase 2, we explore a breadth of biological questions through investigation of model membranes, cultured cells (yeast, mammalian), and animal models (nematode, fly, fish). Our ultimate goal is to develop transformative imaging technology including optics and probes for widespread use by quantitative cell biologists everywhere