High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics

Abstract

The ever-increasing speed and resolution of modern microscopes make the storage and post-processing of images challenging and prevent thorough statistical analyses in developmental biology. Here, instead of deploying massive storage and computing power, we exploit the spherical geometry of zebrafish embryos by computing a radial maximum intensity projection in real time with a 240-fold reduction in data rate. In our four-lens selective plane illumination microscope (SPIM) setup the development of multiple embryos is recorded in parallel and a map of all labelled cells is obtained for each embryo in <10 s. In these panoramic projections, cell segmentation and flow analysis reveal characteristic migration patterns and global tissue remodelling in the early endoderm. Merging data from many samples uncover stereotypic patterns that are fundamental to endoderm development in every embryo. We demonstrate that processing and compressing raw image data in real time is not only efficient but indispensable for image-based systems biology.

In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

“…Unprecedented views of how cells move individually and in concert in a developing embryo have been provided using light-sheet microscopy to track individual nuclei labeled with fluorescently-tagged histones (Huisken and Stainier, 2009;Keller et al, 2008). Selective plane illumination microscopy has provided a three-dimensional view of endodermal cell movement in older live embryos (Schmid et al, 2013). Multiphoton imaging has been used to investigate how neuronal circuits assemble during retinal development by tracking dynamic changes in cell structure and connectivity (Williams et al, 2013), and super-resolution microscopy has been used to image a single receptor molecule uncovering the role for receptor clustering in the antiviral immune response (Gabor et al, 2013).…”

“…An exquisite map of cell division and movement during these early developmental events has been achieved by tracking individually labeled nuclei using advanced microscopy (Keller et al, 2008;Schmid et al, 2013). By the end of 2 dpf, organogenesis is underway throughout the embryo and, by 5 dpf, most organs carry out specialized functions.…”

Augmented line-scan focal modulation microscopy for multi-dimensional imaging of zebrafish heart in vivo

“…Selective plane illumination microscopy (SPIM) or light sheet microscopy has emerged as a popular choice for in vivo fluorescence imaging of relatively transparent small animal models, including embryonic zebrafish and fruit fly [6][7][8][9][10][11][12][13]. Optical sectioning in SPIM is mainly achieved by illuminating the sample using a light beam propagating within the focal plane of a detection objective, although a confocal slit is employed in some recently reported setups to further enhance the contrast.…”

Light-sheet-based 2D light scattering cytometry for label-free characterization of senescent cells

“…Recently, light-sheet illumination has been developed to replace the point or full-field illumination in fluorescent microscopy, and performs optical sectioning of cells or tissues by exciting only the fluorescent molecules in the focal plane. Since Huisken and his colleagues applied the light-sheet technique for reconstruction and visualization of developmental embryo [26], light-sheet fluorescent microscopy has been shown with wide applications in biomedicine such as the long-time imaging in developmental biology [27], and the real-time tracking of molecule or cell dynamics in living tissues [28,29]. In flow cytometry, Chan et al have shown the sectioning of fluorescent ocean phytoplankton by adopting the light-sheet illumination [30].…”