title: Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination.
abstract: Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA. fiolkar@janelia.hhmi.org
authors.forename: Reto
authors.initials: R
authors.lastname: Fiolka
authors.forename: Lin
authors.initials: L
authors.lastname: Shao
authors.forename: E Hesper
authors.initials: EH
authors.lastname: Rego
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.forename: Mats G L
authors.initials: MG
authors.lastname: Gustafsson
mesh_terms: D000465:Algorithms; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*