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is that “there was no equivalent to that in electron microscopy.” EMs create images by shooting a b

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5 W3915 D Women's W Propet Women's 10 Smooth Walker 10 White 5 US Vista Sneaker 8qw5x6w A new technique creates colorful stains that label proteins and cellular structures at higher resolution than ever before possible. 

Nov 3, 2016 Ben Andrew Henry

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Electron microscopes (EMs) generate the most detailed images of organic structures available, but the view they offer has always been in black and white. Now, for the first time, researchers have developed a method for capturing EM images in color. The technique, described today (November 3) in Cell Chemical Biology, involves staining cellular components with metal ions that emit unique signals under the scope, which can then be rendered as vivid colors. Previously, EMs could not distinguish between different types of molecules, so colors had to be painted on afterward. Removing this barrier may open a window onto fine-scale cellular organization that has never before been visible.

In their paper, researchers from the University of California, San Diego (UCSD), not only show that the method works, they also demonstrated three different applications that would not be feasible using standard EM sample preparation. They generate an image of two astrocytes sharing a synapse, track the movement of a peptide into a cell, and provide the first direct look at how a protein involved in memory formation functions.

The technique has been in the works for more than a decade. Several UCSD labs contributed to the study, including the lab of the late Roger Tsien. Port Royal Low Canvas Red Mens Vans Black Independent Chukka Brown Skateboarding qwfB8U, a biophysicist who studies EM at the National Institute of Biomedical Imaging and Bioengineering in Bethesda, Maryland, and was not involved in the research, observed that the methodological advance is analogous to Tsien’s Nobel Prize–winning research on green fluorescent protein (GFP). Fluorescent proteins allowed specific molecules to be tagged and illuminated in live cells under a light microscope.

The problem, Leapman said, is that “there was no equivalent to that in electron microscopy.”

EMs create images by shooting a beam of high-energy electrons straight through a sample and then analyzing the “shadow” that is cast. “When you look at an electron micrograph, what you’re looking at is just density,” Leapman explained, “there’s no chemical signature.” Specific proteins can be stained with gold particles delivered by antibodies, causing targeted locations to show extra dark on a micrograph, but the end result is still a low-contrast, black-and-white image.

Using gold labeling, “there’s no easy distinguishing between the specific label and the general cellular structure,” study coauthor Free RN 0 Men Nike D Khaki Blue Shoe 10 OCqfdw of UCSD told The Scientist. The antibodies that deliver gold particles to the cell are also introduced through holes punctured in the membrane, as a result of which “you sacrifice the ultrastructure [of the sample] to get the antibody in,” he added. Adams’s team worked out a way to resolve both problems.

The multicolor labeling process involves a succession of clever chemical reactions during sample preparation and some post-production image doctoring.

First, the researchers tag cell structures to be viewed in color with the same type of fluorescent labels that might be used in light microscopy. These small, commercially available molecules latch onto target molecules and penetrate tissue more effectively than larger antibodies. The researchers next bathe the sample in diaminobenzidine (DAB), a carrier molecule with a lanthanide ion as its cargo.

The process exploits a quirk in the chemistry of fluorescent labels. When certain fluorescent labels are hit with radiation of a specific wavelength, they generate an unstable, high-energy version of an oxygen molecule called a singlet. “That’s something biologists try to avoid” under normal circumstances, Adams said, “but we take advantage of it.”

In the presence of singlet oxygen, DAB turns into a sticky polymer that glues onto anything nearby. In this case, it effectively labels the target structures with lanthanide ions.

Currently, the researchers are able to employ two different lanthanide labels, Cerium (Ce) and Praseodymium (Pr). So they repeat this staining process for each element, irradiating the sample at different wavelengths of light to ensure each tag winds up where it should.

With Ce and Pr attached to their target proteins, researchers are able to “see” these two elements using a type of EM called electron energy-loss spectroscopy (EELS). EELS differentiates the two elements based on the way they deflect and absorb energy from an electron beam. The result is a set of three images: one showing the location of Ce, another the location of Pr, and a third, a normal transmission EM (TEM) micrograph.

The finished product is a multicolored  overlay of all three images. “When we first saw the images it was like, Wow! That’s better than we ever thought,” said Adams.

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