Contemporary fluorescence microscopy connects our understanding of molecular events from biochemistry and structural biology with their activities in living cells. Lattice light sheet microscopy (LLSM) has made it possible for us to track phenomena such as endocytic vesicle assembly or lipid kinase recruitment across an entire cell with high resolution, in both space and time, and with nearly single-molecule sensitivity. Development during the past year of lattice light sheet microscopy with adaptive optics (AO-LLSM) has overcome the optical limitations that have so far restricted most studies to individual cells in culture, allowing us to achieve comparable resolution and sensitivity in the complex optical environment of an intact, living, multicellular organism. It promises to bridge the gap between cells and organisms, through high sensitivity, volumetric imaging, with diffraction-limited resolution, of living tissues and developing embryos. We propose a research program in three overlapping stages: implementation of AO-LLSM (in collaboration with its developer), development of the new kinds of visualization and analysis software required by the scale and complexity of the datasets, and use of AO-LLSM to solve a problem in vertebrate development. To meet the computational challenges of analyzing the 4D data sets (from low signal-to-noise, the often non- punctate characteristics of the objects being studied, the temporally varying spatial complexity of the data, and the size of the data sets), we will develop new approaches using deep learning and related algorithms, with consultation from experts. As a paradigm application, we will study the consequences of Notch signaling and the related membrane-traffic and protein translocation events for cell differentiation in zebrafish early neurogenesis. AO-LLSM will for the first time allow us to relate molecular signaling events occurring on a timescale of seconds at cell interfaces to the ultimate fate of daughter cells many hours later. We therefore expect that in the course of resolving some long- standing issues in cell fate determination, we will develop microscopy approaches and computer visualization tools that are widely applicable to a range of model systems and biological questions.
We will implement and apply a novel, live-cell imaging strategy (Lattice Light-Sheet Microscopy with Adaptive Optics: AO-LLSM) that spans, with diffraction-limited resolution, a range from molecules to tissues and from seconds to hours. We will use this new technology to study cell differentiation in the embryonic zebrafish brain, concentrating on how Notch-mediated signaling exerts long-range control over neuronal development. Obtaining accurate and comprehensive models of the underlying biology will require that we devise new and generalizable ways to display and analyze complex data sets, while overcoming the computational challenges posed by the low SNR of the imaging regime, the time-varying spatial complexity of the data, and the size of the data sets.
