Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.
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.
Schermelleh
Lothar
L
Center for Integrated Protein Science, Department of Biology, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.
Carlton
Peter M
PM
Haase
Sebastian
S
Shao
Lin
L
Winoto
Lukman
L
Kner
Peter
P
Burke
Brian
B
Cardoso
M Cristina
MC
Agard
David A
DA
Gustafsson
Mats G L
MG
Leonhardt
Heinrich
H
Sedat
John W
JW
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure*; D002843:Chromatin / Q000648:ultrastructure*; D005456:Fluorescent Dyes; D006570:Heterochromatin / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D007211:Indoles; D007399:Interphase; D034882:Lamins / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D032446:Myoblasts; D009685:Nuclear Envelope / Q000648:ultrastructure*; D034881:Nuclear Lamina / Q000648:ultrastructure; D022022:Nuclear Pore / Q000648:ultrastructure; D055095:Optics and Photonics
D002843:Chromatin; D005456:Fluorescent Dyes; D006570:Heterochromatin; D007211:Indoles; D034882:Lamins; C007293:DAPI