A Matter of Time: Catching Organelles in Action in Vitro and in Vivo

The dynamics and fate of endosomal vesicles following endocytosis at the plasma membrane are central to the understanding of transport phenomena of various cargoes including signaling receptors in living cells. The complex dynamics of endosomes and their stochastic motility characteristics in the 3-dimensional milieu demands rapid volumetric imaging to decipher their organization within single cells on a coverslip (in vitro) as well as single cells within living organisms (in vivo). Light-sheet microscopy based on diffraction free beams such as the Bessel and Lattice light-sheet approaches are suitable to capture ensemble endosomal dynamics at whole cell volumes. Using Lattice light-sheet microscopy along with tailored image analysis routines, examples of ‘timekeeping’ in the endosomal network and will be described in the context of endosomal conversions. Further stochastic and rare events such as endosomal escape of lipid nanoparticles relevant to therapeutic delivery will be described in cell culture models. Lastly, can we image organelle within cells in their native environment of growing tissues? The use of Airy beams, that offer an increased flexibility between resolution, field-of-view, and insensitivity to optical obstacles in a light-sheet geometry for subcellular imaging in tissues will be discussed. Examples from our newly built microscope with data from model organisms such as Zebrafish and drosophila will be described where sub-cellular resolution with a step-change in temporal resolution has been achieved.