During its first hours of development, the zebrafish embryo presents a large microtubule array in the yolk region, essential for its development. Despite of its size and dynamic behavior, this network has been studied only in limited field of views or in fixed samples. We designed and implemented different strategies in Light Sheet Fluorescence microscopy for imaging the entire yolk microtubule (MT) network in vivo. These have allowed us to develop a novel image analysis from which we clearly observe a cyclical re-arrangement of the entire MT network in synchrony with blastoderm mitotic waves. These dynamics also affect a previously unreported microtubule array deep within the yolk, here described. These findings provide a new vision of the zebrafish yolk microtubules arrangement, and offers novel insights in the interaction between mitotic events and microtubules reorganization.

Light-sheet fluorescence microscopy (LSFM) has become an important tool for biological and biomedical research. Although several illumination and detection strategies have been developed, the sample mounting still represents a cumbersome procedure as this is highly dependent on the type of sample and often this might be time consuming. This prevents the use of LSFM in other promising applications in which a fast and straightforward sample-mounting procedure and imaging are essential. These include the high-throughput research fields, e.g. in drug screenings and toxicology studies. Here we present a new imaging paradigm for LSFM, which exploits modularity to offer multimodal imaging and straightforward sample mounting strategy, enhancing the flexibility and throughput of the system. We describe its implementation in which the sample can be imaged either as in any classical configuration, as it flows through the light-sheet using a fluidic approach, or a combination of both. We also evaluate its ability to image a variety of samples, from zebrafish embryos and larvae to 3D complex cell cultures.

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.