Breakthrough in X-Ray Microscopy Opens New View Inside Living Cells

May 29, 1998

By Lynn Yarris,

What has been one of the better kept secrets on the Hill will soon be a secret no longer, and the view inside living cells will be better for it. Carolyn Larabell, a cell biologist and microscopist with the Life Sciences Division, working with Werner Myer-Illse, a physicist and x-ray microscopist with the Materials Sciences Division, plus other researchers from both divisions, has developed a technique for using x-ray microscopy to obtain unprecedented images of labeled proteins inside of whole cells in a hydrated state. Among many other things, this technique may help scientists resolve the long-standing debate over whether or not the cell nucleus has an organized internal structure.

With the combination of x-rays from the Advanced Light Source and a new protein-labeling technique, scientists can see the distribution of the nucleoli within the nucleus of a mammary epithelial cell

Using the x-ray microscopy beamline (XM-1) at the ALS, Larabell, Myer-Illse and their collaborators have produced images of intact hydrated cells that many scientists will have to see to believe. Compared to the 200 nanometer resolution in images obtained with the best visible light microscopes, the images produced with the soft (low energy) x-ray beams at XM-1 showed a resolution between 40 and 50 nanometers. Plus, these cells did not have to be stained in order to obtain high contrast images of cell structures.

"Soft x-ray microscopy could make a major contribution to the understanding of cell function and structure," Larabell says. "We have demonstrated a practical technique that gives cell biologists a whole new way of looking at their samples."

In perhaps no other scientific field does the adage "form follows function" hold more true than in biology, especially the biology of living cells, which is why our knowledge of cells starts with imaging. Throughout the 1990s, the best images of protein localizations in cells have probably come from confocal microscopy, a technique whereby blurring is avoided through the collection of thousands of pin-points of laser light. These pinpoints of collected light can be assembled into highly-focused images that are unmatched for contrast and clarity by conventional visible light micros-copy. Used in combination with fluorescent-labeling, in which fluorescently-tagged antibodies bind to specific proteins for identification, confocal microscopy has lit the way for many of the recent advances in cell biology. However, the information obtained will always be limited by the technique's relatively low spatial resolution. Electron microscopy provides outstanding resolution (down to one-tenth of a nanometer), but cells must be sectioned off into tissue-thin slices for imaging because even highly-energized electrons are poor penetrators. Also, the cell slices must be dehydrated, as electron microscopy can only be done in a vacuum.

"To section the cells, you have to first embed them in plastic, otherwise it's like trying to section a raw egg," says Larabell, who is highly experienced with both confocal and electron microscopy. "The chemicals used for dehydration and embedding in plastic can result in the loss of proteins, and the plastic can interfere with the ability of the antibodies to bind to the protein."

The need has been, she says, for an alternative form of microscopy that provides higher resolution information on thick hydrated cells without requiring elaborate specimen preparation.

As Larabell and her collaborators are showing, soft x-ray microscopy fits the bill.

A key to the success at XM-1 lies in how the cells are prepared for imaging. After being treated with a fixing agent to lock their proteins into position, the cells are washed in a special detergent that removes the lipids in their outer membrane. This yields a cell surface that is perforated with holes through which antibodies coated with a cluster of fluorescent and nano-sized gold particles can be introduced.

Inside the cell, the fluorogold-labeled antibodies attach themselves to specific proteins, including those within the nuclei. Because the nano-sized particles of gold are too small to be seen even with x-rays, prior to imaging at XM-1 the treated cells are enhanced with a coating of silver, similar to the use of silver emulsions to bring out images on photographic film.

Larabell's experiences with both visible light and electron microscopy were crucial to her developing this unique method of preparing cells for soft x-ray imaging. She also credits her success with being "flexible" about her protocols.

"Microscopists who work with (visible) light tend to use fast but harsh cell preparation techniques because at that resolution, they won't see their mistakes," she says. "With electron microscopy, every flaw is (literally) magnified so the preparation techniques are incredibly arduous. Having worked with both, I'm willing to change my protocols to find the best and most efficient."

Another key to the success at XM-1 has to do with the capabilities of the facility itself. XM-1 is a direct-imaging transmission x-ray microscope operating off an ALS bend-magnet beamline (6.1.2) under the direction of Meyer-Ilse of the Center for X-ray Optics, who was responsible for the facility's creation.

Members of Meyer-Ilse's team include CXRO's John Brown and Ajit Nair, who have been collaborators on the cell imaging work, along with Sophie Lelievre and Donna Hama-moto of Life Sciences Division.

The photon-energy range of XM-1 extends from 250 to 950 electron volts, a range that covers the so-called "water window"--the energy span over which water is transparent to x-rays, but carbon-containing materials are not. This means that high-contrast images of proteins and other interior cell structures can be obtained at XM-1 without the need for staining.

The combination of fluorogold-labeling and XM-1 has already been used to obtain detailed information on the distribution within cell nuclei of a "splicing factor" protein that plays an important role in the expression of certain genes.

This proof-of-principle experiment can be considered a preview of coming attractions.

Among the first areas to be explored will no doubt be the cell nucleus itself. The cell nucleus has been called one of the best known but least understood of all cell organelles, with some biologists arguing it has a distinct substructure, and others arguing that it is merely a membrane-bound sack full of DNA and other molecules. A major goal of cell biology is to finally resolve this dispute and get a better handle on nuclear organization.

Another major target for x-ray microscopy will be to investigate the interactions between cell interiors and a mass of protein support "scaffolding" outside the cell called the extracelluar matrix (ECM). LSD director Mina Bissell has demonstrated that breakdowns in the normal interaction between the interior of a cell and its ECM can lead to breast cancer.

X-ray microscopy could also be used to view other critical interactions, such as those between the cell's outer surface and its cytoplasm, between the cytoplasm and the nucleus, and even between the nucleus and the chromosomes.

Says Larabell, "We hope x-ray microscopy will help answer a lot of questions no other imaging technique can."

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