title: Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens.
abstract: Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Gao
authors.forename: Lin
authors.initials: L
authors.lastname: Shao
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Higgins
authors.forename: John S
authors.initials: JS
authors.lastname: Poulton
authors.forename: Mark
authors.initials: M
authors.lastname: Peifer
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.forename: Xufeng
authors.initials: X
authors.lastname: Wu
authors.forename: Bob
authors.initials: B
authors.lastname: Goldstein
authors.forename: Eric
authors.initials: E
authors.lastname: Betzig
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology / Q000648:ultrastructure; D017173:Caenorhabditis elegans / Q000166:cytology / Q000254:growth & development; D002460:Cell Line; D045744:Cell Line, Tumor; D017484:Dermatitis, Phototoxic; D004023:Dictyostelium / Q000648:ultrastructure; D004331:Drosophila melanogaster / Q000166:cytology; D005347:Fibroblasts / Q000648:ultrastructure; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007621:Karyotyping / Q000379:methods; D007814:Larva / Q000166:cytology / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D008938:Mitosis; D061848:Optical Imaging / Q000379:methods*