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Vol. 15, Issue 5, 2449-2455, May 2004
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* MRC, Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom;
Applied Optics and Information Processing, Kirchoff Institute for Physics, University of Heidelberg, 69120 Heidelberg, Germany
Submitted January 16, 2004;
Revised February 23, 2004;
Accepted February 24, 2004
Monitoring Editor: Joseph Gall
Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads (
40–100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.
Abbreviations used: CLSM, confocal laser scanning microscopy; GFP, green fluorescent protein; mAb, monoclonal antibody; pol, RNA polymerase; SMI, spatially modulated illumination.
Online version of this article contains supporting material. Online version is available at www.molbiolcell.org.
These authors contributed equally to this study.
Present address: Physics of Condensed Matter, Institute of Physics, University of Potsdam, Am Neuen Palais 10, 14469 Potsdam, Germany.
|| Corresponding author. E-mail address: ana.pombo{at}csc.mrc.ac.uk.
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