Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.

The sympathetic nervous system is critical in maintaining the homeostasis of renal functions. However, its three-dimensional (3D) structures in the kidney have not been elucidated due to limitation of conventional imaging methods. CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) is a newly developed tissue-clearing technique, which enables whole-organ 3D imaging without thin-sectioning. Comprehensive 3D imaging by CUBIC found that sympathetic nerves are primarily distributed around arteries in the mouse kidney. Notably, the sympathetic innervation density was significantly decreased 10 days after ischemia-reperfusion injury (voluminal ratio of innervation area to kidney) by about 70%. Moreover, norepinephrine levels in kidney tissue (output of sympathetic nerves) were significantly reduced in injured kidneys by 77%, confirming sympathetic denervation after ischemia-reperfusion injury. Time-course imaging indicated that innervation partially recovered although overall denervation persisted 28 days after injury, indicating a continuous sympathetic nervous abnormality during the progression of chronic kidney disease. Thus, CUBIC-kidney, the 3D imaging analysis, can be a strong imaging tool, providing comprehensive, macroscopic perspectives for kidney research.

Heart function and hemodynamic analysis for zebrafish embryos.

The Zebrafish has emerged to become a powerful vertebrate animal model for cardiovascular research in recent years. Its advantages include easy genetic manipulation, transparency, small size, low cost, and the ability to survive without active circulation at early stages of development. Sequencing the whole genome and identifying ortholog genes with human genome made it possible to induce clinically relevant cardiovascular defects via genetic approaches. Heart function and disturbed hemodynamics need to be assessed in a reliable manner for these disease models in order to reveal the mechanobiology of induced defects. This effort requires precise determination of blood flow patterns as well as hemodynamic stress (i.e., wall shear stress and pressure) levels within the developing heart. While traditional approach involves time-lapse brightfield microscopy to track cell and tissue movements, in more recent studies fast light-sheet fluorescent microscopes are utilized for that purpose. Integration of more complicated techniques like particle image velocimetry and computational fluid dynamics modeling for hemodynamic analysis holds a great promise to the advancement of the Zebrafish studies. Here, we discuss the latest developments in heart function and hemodynamic analysis for Zebrafish embryos and conclude with our future perspective on dynamic analysis of the Zebrafish cardiovascular system. Developmental Dynamics 246:868-880, 2017. © 2017 Wiley Periodicals, Inc.

Complete Visualization of T Follicular Helper Cells in Germinal Centers by Light Sheet Fluorescence Microscopy.

Long-lasting immunity depends on generation of antibody forming cells in germinal centers (GCs). Conventional methods such as immunohistology and intravital live imaging have been used extensively to investigate the location of cellular assemblies within tissues as well as their dynamic motility and cellular interactions. Two photon laser scanning microscopy (TPLSM) intravital imaging allows scanning of large areas within tissues and reveals multiple immune cell niches. Nonetheless, this type of imaging is limited by the depth of penetration and cannot capture effectively all of the GC niches within lymphoid organs. Here we describe a method to visualize antigen-specific T and B cells in multiple microanatomical locations and niches at the level of a whole organ. This large-scale imaging approach can greatly increase our understanding of the spatial distribution of immune cells and help obtain detailed 3D maps of their locations and quantities.

In Vivo Light Sheet Fluorescence Microscopy of Calcium Oscillations in Arabidopsis thaliana.

Calcium imaging in plants requires a high-resolution microscope, able to perform volumetric acquisition in a few seconds, inducing as low photobleaching and phototoxicity as possible to the sample. Light sheet fluorescence microscopy offers these capabilities, with the further chance to mount the sample in vertical position, mimicking the plant's growth and physiological conditions.A protocol for plant preparation and mounting in a light sheet microscope is presented. First, the growth of Arabidopsis thaliana in a sample holder compatible with light sheet microscopy is described. Then, the requirements for sample alignment and image acquisition are detailed. Finally, the image processing steps to analyze calcium oscillations are discussed, with particular emphasis on ratiometric calcium imaging in Arabidopsis root hairs.