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

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.

Under-filling trapping objectives optimizes the use of the available laser power in optical tweezers.

For optical tweezers, especially when used in biological studies, optimizing the trapping efficiency reduces photo damage or enables the generation of larger trapping forces. One important, yet not-well understood, tuning parameter is how much the laser beam needs to be expanded before coupling it into the trapping objective. Here, we measured the trap stiffness for 0.5-2 µm-diameter microspheres for various beam expansions. We show that the highest overall trapping efficiency is achieved by slightly under-filling a high-numerical aperture objective when using microspheres with a diameter corresponding to about the trapping-laser wavelength in the medium. The optimal filling ratio for the lateral direction depended on the microsphere size, whereas for the axial direction it was nearly independent. Our findings are in agreement with Mie theory calculations and suggest that apart from the choice of the optimal microsphere size, slightly under-filling the objective is key for the optimal performance of an optical trap.