Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy

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Item Type:	Article
Title:	Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy
Creators Name:	Pombo, A., Hollinshead, M. and Cook, P.R.
Abstract:	The resolution of conventional light microscopy is limited to approximately 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 approximately 40 nm. (J Histochem Cytochem 47:471-480, 1999)
Keywords:	BrUTP, CLSM, Cryosection, EM, Fluorescent-Gold Immunoprobe, Immunofluorescence, Tokuyasu, Transcription Factories
Source:	Journal of Histochemistry and Cytochemistry
ISSN:	0022-1554
Publisher:	Histochemical Society
Volume:	47
Number:	4
Page Range:	471-480
Date:	April 1999
Official Publication:	https://doi.org/10.1177/002215549904700405
PubMed:	View item in PubMed

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