Over the last 15 years, light-sheet fluorescence microscopy has emerged to be a powerful imaging technique perfectly suited for live 3D imaging due to its gentle illumination scheme, high spatial resolution, and fast data acquisition. Imaging the development of entire living organisms at high resolution over long periods of time allows for a descriptive analysis of biological processes taking place, ranging from within the cell to full animal scale. However, observation alone is not always sufficient to directly probe some of these complex biological processes. Recently, there have been a plethora of tools developed that allow for manipulation, control and perturbation of biological processes to gain a deeper understanding of the underlying molecular mechanisms and signaling pathways. Laser-based manipulation has emerged at the forefront of these tools, allowing for a variety of different applications including laser dissection, laser ablation, laser cauterization, and optogenetics. Combined with rapid live-cell imaging, this powerful combination not only allows researchers to visualize but also to perturb living samples to learn more about the mechanisms under investigation.
The authors present an optical design that allows for rapid 3D imaging with a multi-view light-sheet (Luxendo MuVi SPIM) combined with IR femtosecond laser-based photomanipulation. Utilizing the model organisms Drosophila melanogaster and Zebrafish, the authors demonstrate the precise spatio-temporal targeting by preforming subcellular ablations of neurons, cells, tissue cauterization, and optogenetics while simultaneously preforming in toto live imaging at high temporal resolution. The combination of these two advanced techniques will allow researchers to address increasingly complex questions in a variety of model systems.