Nucleic Acids Research Advance Access published online on October 28, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp901
Methods Online |
Measurement of replication structures at the nanometer scale using super-resolution light microscopy
1Kirchhoff Institut für Physik, University of Heidelberg, 2Department of Biology, Technische Universität Darmstadt, Germany, 3Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia, 4Department of Biology II, Center for Integrated Protein Science, Ludwig Maximilians University Munich, Germany, 5MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, UK, 6Department of Biochemistry and Biophysics, University of California, San Francisco, USA, 7Max Delbrueck Center for Molecular Medicine, Berlin, 8Institute for Pharmacy and Molecular Biology, University of Heidelberg, Germany, 9Institute for Molecular Biophysics, The Jackson Laboratory/ME, USA
*To whom correspondence should be addressed. Tel: +49 6151 16 2377; Fax: +49 6151 16 2375; Email: cardoso{at}bio.tu-darmstadt.de
Correspondence may also be addressed to C. Cremer. Tel: +49 6221 549252; Fax: +49 6221 549112; Email: cremer{at}kip.uni-heidelberg.de
Received August 20, 2009. Revised October 2, 2009. Accepted October 7, 2009.
DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.
Present addresses: D. Baddeley, Department of Physiology, School of Medical Sciences, University of Auckland, Auckland, New Zealand.
S. Martin, Cancer Sciences Division, School of Medicine, University of Southampton, Southampton, UK.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.