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Chromosome Research

, Volume 16, Issue 3, pp 367–382 | Cite as

High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy

  • Jürgen Reymann
  • David Baddeley
  • Manuel Gunkel
  • Paul Lemmer
  • Werner Stadter
  • Thibaud Jegou
  • Karsten Rippe
  • Christoph Cremer
  • Udo Birk
Article

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.

Key words

FPALM live cell imaging localization microscopy nanosizing PALM PALMIRA SALM SMI microscopy SPDM STORM Vertico-SMI microscope 

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Copyright information

© Springer 2008

Authors and Affiliations

  • Jürgen Reymann
    • 1
  • David Baddeley
    • 1
  • Manuel Gunkel
    • 1
  • Paul Lemmer
    • 1
  • Werner Stadter
    • 2
  • Thibaud Jegou
    • 3
  • Karsten Rippe
    • 3
  • Christoph Cremer
    • 1
    • 4
  • Udo Birk
    • 1
    • 5
  1. 1.Applied Optics and Information Processing, Kirchhoff Institute of PhysicsUniversity of HeidelbergHeidelbergGermany
  2. 2.University of Applied SciencesHamburgGermany
  3. 3.Research Group Genome Organization and Function, German Cancer Research Centre (DKFZ) & Bioquant CentreUniversity of HeidelbergHeidelbergGermany
  4. 4.Institute for Pharmacy and Molecular Biotechnology (IPMB)/Bioquant CentreUniversity of Heidelberg, Germany & Institute for Molecular Biophysics, The Jackson LaboratoryBar HarborUSA
  5. 5.In-vivo Optical Imaging GroupInstitute of Electronic Structure and Laser, FORTHHeraklionGreece

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