3D imaging ability of TLS-SPIM in resolving complex structures of multicellular specimens.(a-c) 3D volume renderings of a C. elegans embryo (OD95) expressing GFP::PLC∂PH (membrane) and H2B::mCherry (nucleus) at ~50 cell stage, ~200 cell stage and bean stage. (d-f) XY lateral slices through the longitudinal axis of the embryo in a, b and c. (g-i) YZ axial slices of the embryo in a-c through the positions marked in d-f. (j-o) XZ axial slices of the embryo in a-c through the positions marked in g-i. (p) 3D volume rendering of a ~15 h.p.f. zebrafish embryo tailbud expressing KikGR (nucleus). (q-r) A XY lateral slice and a XZ axial slice of the tailbud through the planes in p. Scale bars, 5 µm (d-o) and 20 µm (q-r).

3D imaging by TLS-SPIM with real-time light sheet optimization.(a) An ideal imaging method with both high spatial resolution and imaging speed acquires spatial and temporal information simultaneously. (b) A compromised solution acquires spatial information and temporal information separately by quick switching between a high spatial resolution, low imaging speed mode and a low spatial resolution, high imaging speed mode. (c,d) 3D volume renderings of two mesodermal cells in the tailbud of a ~15 hpf zebrafish embryo imaged in a low spatial resolution, high imaging speed mode and a high spatial resolution, low imaging speed mode. (e,f) Orthogonal views of the cells in c and d. (g) Ten continuous time points of the selected area in c imaged in the low spatial resolution, high imaging speed mode, showing the lamellipodia activities of the mesodermal cell. Scale bars, 5 µm (c-f), 2 µm (g).

3D imaging ability comparison of regular SPIM and TLS-SPIM on a nucleus-labeled zebrafish embryo. (a, b) XY and XZ orthogonal slices of a ~15 hpf zebrafish embryo tailbud imaged by TLS-SPIM (NAOD=0.2, NAID=0.05, single excitation beam, nine tiling positions, raw image). (c, d) XY and XZ orthogonal slices of the same embryo imaged by regular SPIM with a Gaussian light sheet (NAOD=0.07, single excitation beam, raw image). (e, f) Magnified views of the selected areas in a and c. (g, h) Magnified views of the selected area in b and d. Scale bars, 20 µm (a-d), 5 µm (e-h).

Acknowledgments
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