Abstract
Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos.
Highlights
Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging
Every light sheet balances the properties of light sheet thickness, the illumination light confinement, and the light-sheet size differently, which results in different 3D spatial resolution, optical sectioning capability and field of view (FOV), respectively[13,14] (Supplementary Fig. 1)
Either a Gaussian light sheet, a Bessel light sheet or a Lattice light sheet can be used in the microscope, and it takes less than a millisecond to either tile or change the light sheet by applying different binary phase maps to the spatial light modulators (SLM) (Supplementary Figs 5–10, Supplementary Movie 1,2)
Summary
Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. In order to understand cell behaviours in a multicellular process, it is necessary to study all involved cells at subcellular level to acquire the dynamic information of their intracellular activities, by which the underlying connections between cellular behaviours and intracellular activities of the involved cells can be revealed For this reason, fluorescence imaging techniques that allow low-invasive 3D imaging of multicellular specimens with high spatial and temporal resolution are required. Multiview SPIM cannot bypass the fundamental tradeoff of SPIM set up by the light sheet Both high-speed subcellular dynamics and the optical aberration and light scattering introduced by the sample and the agarose gel usually used to mount the sample make it more difficult to fuse different views accurately with high spatial resolution. Imaging multicellular specimens with subcellular spatial resolution remains a problem with multiview SPIM
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