Whole-animal functional and developmental imaging with isotropic spatial resolution.
Imaging fast cellular dynamics across large specimens requires high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To meet these requirements, we developed isotropic multiview (IsoView) light-sheet microscopy, which rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. Combining these four views by means of high-throughput multiview deconvolution yields images with high resolution in all three dimensions. We demonstrate whole-animal functional imaging of Drosophila larvae at a spatial resolution of 1.1-2.5 μm and temporal resolution of 2 Hz for several hours. We also present spatially isotropic whole-brain functional imaging in Danio rerio larvae and spatially isotropic multicolor imaging of fast cellular dynamics across gastrulating Drosophila embryos. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
Raghav K
RK
Chhetri
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
Fernando
F
Amat
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
Yinan
Y
Wan
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
Burkhard
B
Höckendorf
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
William C
http://orcid.org/0000-0003-4541-738X
WC
Lemon
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
Philipp J
PJ
Keller
D000818:Animals; D001921:Brain / Q000196:embryology / Q000648:ultrastructure*; D004330:Drosophila / Q000196:embryology; D004625:Embryo, Nonmammalian / Q000502:physiology / Q000648:ultrastructure*; D047108:Embryonic Development; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007814:Larva; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D051598:Whole Body Imaging / Q000295:instrumentation / Q000379:methods*; D015027:Zebrafish / Q000196:embryology