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Electron microscopy: New type of genetic tag illuminates life in never-before-seen detail

Date:
April 6, 2011
Source:
Public Library of Science
Summary:
By modifying a protein from a plant that is much favored by science, researchers have created a new type of genetic tag visible under an electron microscope, illuminating life in never-before-seen detail.

Confocal fluorescence images of miniSOG-targeted endoplasmic reticulum (A), Rab5a (B), zyxin (C), tubulin (D), -actin (E), -actinin (F), mitochondria (G), and histone 2B (H) in HeLa cells; scale bars, 10 m.
Credit: Xiaokun Shu, Varda Lev-Ram, Thomas J. Deerinck, Yingchuan Qi, Ericka B. Ramko, Michael W. Davidson, Yishi Jin, Mark H. Ellisman, Roger Y. Tsien. A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms. PLoS Biology, 2011; 9 (4): e1001041 DOI: 10.1371/journal.pbio.1001041

By modifying a protein from a plant that is much favored by science, researchers at the University of California, San Diego School of Medicine and colleagues have created a new type of genetic tag visible under an electron microscope, illuminating life in never-before-seen detail.

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Led by Nobel laureate Roger Tsien, PhD, Howard Hughes Medical Institute investigator and UCSD professor of pharmacology, chemistry and biochemistry, a team of scientists radically re-engineered a light-absorbing protein from the flowering cress plant Arabidopsis thaliana. When exposed to blue light, the altered protein produces abundant singlet oxygen, a form of molecular oxygen that can be made visible by electron microscopy (EM).

The findings are published in the online, open access journal PLoS Biology.

Tsien was co-winner of the 2008 Nobel Prize in chemistry for his role in helping develop and expand the use of green fluorescent protein (GFP), a protein from jellyfish that is now widely employed in light microscopy to peer inside living cells or whole animals and observe molecules interacting in real-time. Tsien said the development of the small, highly engineered Arabidopsis protein, dubbed "miniSOG," may elevate the abilities of electron microscopy in the same way that GFP and its relatives have made modern light microscopy in biological research much more powerful and useful.

"The big advantage of EM is that it has much higher spatial resolution than light microscopy. You can get up to a hundred-fold higher useful magnification from EM than from light microscopy," said Tsien. The result has been extraordinarily detailed, three-dimensional images of microscopic objects at resolutions measuring in the tens of nanometers, tiny enough to meticulously render the internal anatomy of individual cells. But current EM technologies do not distinguish or highlight individual proteins in these images. Although individual proteins can be tagged with GFP or other fluorescent proteins to aid localization by light microscopy, there has been no equivalent technology for the higher-resolution images provided by EM.

To create this ability, the scientists began with a protein from Arabidopsis that absorbs incoming blue light. It's normal function is to trigger biochemical signals that inform the plant how much sunlight it is receiving. "We rationally engineered the protein based on its atomic model so that it changes incoming blue light into a little bit of green fluorescence and a lot of singlet oxygen," said the paper's first author, Xiaokun Shu, now an assistant professor at UC San Francisco. Established methods were then used to convert singlet oxygen production into a tissue stain that the electron microscope can "see." The scientists tested the modified protein's utility as an EM marker by first using it to confirm the locations of several well-understood proteins in mammalian cells, nematodes and rodents, and then used miniSOG to successfully tag two neuronal proteins in mice whose locations had not been known.

Tsien is optimistic that miniSOG will grant new powers to electron microscopy, permitting scientists to pursue answers to questions previously impossible to ask. MiniSOG will especially be useful to scientists who investigate cellular and subcellular structures including neuronal circuits at nanometer resolution in multicellular organisms since previous methods have great difficulty in achieving both efficient labeling and good preservation of the structures under study. While EM can provide much higher useful magnification than light microscopy, EM will not replace light microscopy. "When we use miniSOG, we see the tagged proteins plus the landmarks that we are used to navigating by," said Tsien. "On the other hand, EM has the disadvantage that it gives a snapshot of cells before we killed them (to make the image), whereas light microscopy can show the dynamics in live cells. Each technique has different complementary strengths and weaknesses."

Co-authors of the paper include: Varda Lev-Ram, UCSD Department of Pharmacology; Thomas J. Deerinck, National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, UCSD; Yingchuan Qi and Yishi Jin, Howard Hughes Medical Institute, UCSD and UCSD Division of Biological Science; Ericka B. Ramko and Michael W. Davidson, National High Magnetic Field Laboratory and Department of Biological Science, Florida State University; and Mark H. Ellisman, National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, UCSD and UCSD Department of Neurosciences


Story Source:

The above story is based on materials provided by Public Library of Science. Note: Materials may be edited for content and length.


Journal Reference:

  1. Xiaokun Shu, Varda Lev-Ram, Thomas J. Deerinck, Yingchuan Qi, Ericka B. Ramko, Michael W. Davidson, Yishi Jin, Mark H. Ellisman, Roger Y. Tsien. A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms. PLoS Biology, 2011; 9 (4): e1001041 DOI: 10.1371/journal.pbio.1001041

Cite This Page:

Public Library of Science. "Electron microscopy: New type of genetic tag illuminates life in never-before-seen detail." ScienceDaily. ScienceDaily, 6 April 2011. <www.sciencedaily.com/releases/2011/04/110405174849.htm>.
Public Library of Science. (2011, April 6). Electron microscopy: New type of genetic tag illuminates life in never-before-seen detail. ScienceDaily. Retrieved November 25, 2014 from www.sciencedaily.com/releases/2011/04/110405174849.htm
Public Library of Science. "Electron microscopy: New type of genetic tag illuminates life in never-before-seen detail." ScienceDaily. www.sciencedaily.com/releases/2011/04/110405174849.htm (accessed November 25, 2014).

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