title: High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy.
abstract: Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil- and water-immersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution.
authors.affiliation: Applied Optics and Information Processing, Kirchhoff Institute of Physics, University of Heidelberg, INF 227, 69120, Heidelberg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Reymann
authors.forename: David
authors.initials: D
authors.lastname: Baddeley
authors.forename: Manuel
authors.initials: M
authors.lastname: Gunkel
authors.forename: Paul
authors.initials: P
authors.lastname: Lemmer
authors.forename: Werner
authors.initials: W
authors.lastname: Stadter
authors.forename: Thibaud
authors.initials: T
authors.lastname: Jegou
authors.forename: Karsten
authors.initials: K
authors.lastname: Rippe
authors.forename: Christoph
authors.initials: C
authors.lastname: Cremer
authors.forename: Udo
authors.initials: U
authors.lastname: Birk
mesh_terms: D002460:Cell Line; D022003:Cell Nucleus Structures / Q000648:ultrastructure*; D004867:Equipment Design; D006358:Hot Temperature; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008857:Microscopy, Interference / Q000295:instrumentation / Q000379:methods; D049329:Nanostructures / Q000648:ultrastructure; D055095:Optics and Photonics / Q000295:instrumentation