Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy
Lothar Schermelleh,1*
Peter M. Carlton,2*
Sebastian Haase,2,4
Lin Shao,2
Lukman Winoto,2
Peter Kner,2
Brian Burke,3
M. Cristina Cardoso,4
David A. Agard,2
Mats G. L. Gustafsson,5
Heinrich Leonhardt,1*
John W. Sedat2*
Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.
1 Center for Integrated Protein Science, Department of Biology, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.
2 Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.
3 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA.
4 Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
5 Department of Physiology and Program in Bioengineering, University of California, San Francisco, CA 94143, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: h.leonhardt{at}lmu.de (H.L.); sedat{at}msg.ucsf.edu (J.W.S.)
