ARTICLE

Bridging the Resolution Gap: Imaging the Same Transcription Factories in Cryosections by Light and Electron Microscopy

Correspondence to: Peter R. Cook, Sir William Dunn Schl. of Pathology, U. of Oxford, South Park Road, Oxford OX1 3RE, UK.

The resolution of conventional light microscopy is limited to ~200 nm in the x- and y-axes and >500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100–200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of ~40 nm. (J Histochem Cytochem 47:471–480, 1999)

Key Words: BrUTP, CLSM, cryosection, EM, fluorescent–gold immunoprobe, immunofluorescence, Tokuyasu, transcription factories

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. B. Lawrence and C. M. Clemson Gene associations: true romance or chance meeting in a nuclear neighborhood? J. Cell Biol., September 22, 2008; 182(6): 1035 - 1038. [Abstract] [Full Text] [PDF]

				C. H. Eskiw, A. Rapp, D. R. F. Carter, and P. R. Cook RNA polymerase II activity is located on the surface of protein-rich transcription factories J. Cell Sci., June 15, 2008; 121(12): 1999 - 2007. [Abstract] [Full Text] [PDF]

				I. Faro-Trindade and P. R. Cook A Conserved Organization of Transcription during Embryonic Stem Cell Differentiation and in Cells with High C Value Mol. Biol. Cell, July 1, 2006; 17(7): 2910 - 2920. [Abstract] [Full Text] [PDF]

				M. Mori, G. Ishikawa, T. Takeshita, T. Goto, J. M. Robinson, and T. Takizawa Ultrahigh-resolution immunofluorescence microscopy using ultrathin cryosections: subcellular distribution of caveolin-1{alpha} and CD31 in human placental endothelial cells J. Electron Microsc. (Tokyo), April 1, 2006; 55(2): 107 - 112. [Abstract] [Full Text] [PDF]

				S. Q. Xie, S. Martin, P. V. Guillot, D. L. Bentley, and A. Pombo Splicing Speckles Are Not Reservoirs of RNA Polymerase II, but Contain an Inactive Form, Phosphorylated on Serine2 Residues of the C-Terminal Domain Mol. Biol. Cell, April 1, 2006; 17(4): 1723 - 1733. [Abstract] [Full Text] [PDF]

				S. Martin, A. V. Failla, U. Spori, C. Cremer, and A. Pombo Measuring the Size of Biological Nanostructures with Spatially Modulated Illumination Microscopy Mol. Biol. Cell, May 1, 2004; 15(5): 2449 - 2455. [Abstract] [Full Text] [PDF]

				T. Takizawa and J. M. Robinson Ultrathin Cryosections: An Important Tool for Immunofluorescence and Correlative Microscopy J. Histochem. Cytochem., June 1, 2003; 51(6): 707 - 714. [Abstract] [Full Text] [PDF]

				Y. Ren, M. J. Kruhlak, and D. P. Bazett-Jones Same Serial Section Correlative Light and Energy-filtered Transmission Electron Microscopy J. Histochem. Cytochem., May 1, 2003; 51(5): 605 - 612. [Abstract] [Full Text] [PDF]

				X. Mo and W. S. Dynan Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites Mol. Cell. Biol., November 15, 2002; 22(22): 8088 - 8099. [Abstract] [Full Text] [PDF]

				J. M. Robinson, T. Takizawa, A. Pombo, and P. R. Cook Correlative Fluorescence and Electron Microscopy on Ultrathin Cryosections: Bridging the Resolution Gap J. Histochem. Cytochem., July 1, 2001; 49(7): 803 - 808. [Abstract] [Full Text] [PDF]

				T. Takizawa and J. M. Robinson FluoroNanogold Is a Bifunctional Immunoprobe for Correlative Fluorescence and Electron Microscopy J. Histochem. Cytochem., April 1, 2000; 48(4): 481 - 486. [Abstract] [Full Text]

				K Sugaya, M Vigneron, and P. Cook Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein J. Cell Sci., January 8, 2000; 113(15): 2679 - 2683. [Abstract] [PDF]

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 1999