Data gathering was done with XM-1, the Center for X-Ray Optics transmission x-ray microscope. (A dedicated biological x-ray microscope, dubbed "XM-2" will be in operation at the ALS by 2006.) First, light microscopy was used to position and examine rows of living yeast in their capillary container. Next, the living S. cerevisiae cells were rapidly frozen and placed in the x-ray microscope. The cells remained fully hydrated throughout image collection.

Because of the alignment of the yeast in their container, tomographic data sets could be collected from multiple cells by simply advancing the capillary into the field of view. Cells were rotated through 180 degrees and images collected every 4 degrees. A fully automated cryorotation stage has recently been developed that will enable collection of more images with 0.5- to 1.0-degree intervals, yielding better resolution data.

With light microscopy, a glass lens focuses visible light onto the sample. With x-ray microscopy, an x-ray zone plate is used to focus photons. The photons bombarding the sample have a 2.4-nm wavelength at 517 eV in a range known as the "water window." This means that the structures in the cells absorb approximately an order of magnitude more strongly than the surrounding water. The resulting natural contrast generates unprecedented views of the internal cellular architecture in a natural, albeit frozen, state. After tomographic reconstruction, computer algorithms can process data to reveal features of interest.

To get volume information, the researchers started with the surface of the yeast. They then took it away to reveal internal vesicles and organelles. To get density information, they color coded the structures by measuring x-ray absorption. In this way, the researchers were able to quantify information and differentiate cellular regions. Density information can also be superimposed on top of volume information to determine what densities are associated with what structures.

This organism was the first eukaryote sequenced. Eukaryotes, unlike bacteria, have nuclei. Some are single celled, like budding yeast, but with the nucleus also came the evolution of multicelled organisms, such as plants and animals. It was found that at least 31% of proteins encoded by yeast genes have mammalian equivalents, or homologs. Therefore, yeast are important model organisms for understanding cellular processes in higher eukaryotes. In fact, our connection to the budding yeast is even more ancient than originally believed. Nearly 50% of human genes implicated in heritable diseases have yeast homologs, making this distant relative a good predictor of human gene function.

High-magnification view of a budding yeast. (A) Projection image showing numerous superimposed organelles. (B–C) Computer-generated sections through the yeast reveal numerous organelles after tomographic reconstruction; the most dense (bright white circles) are filled with lipid.

X-ray tomography generates unique three-dimensional reconstructions of whole yeast without the need for chemical fixatives or contrast-enhancement reagents. Because the high-resolution data set is based on tomographically reconstructed local x-ray absorption, the information in these reconstructions is quantifiable, and because the cells were rapidly frozen from the living state and remain fully hydrated, the information retains biological fidelity.

Reconstructed data of the yeast shown above using different volume-analysis algorithms. (A) Opaque surface. (B) Transparent surface showing internal vesicles. (C) Volume-rendered thick-slice section: dense lipid droplets are white, least-dense vacuoles appear gray, structures of varying densities appear green, orange, and red. Click on image (C) to play a Quicktime movie showing the structural organization of the entire yeast.

Using this technology, researchers can now rapidly examine phenotypic consequences of genetic mutations and knockouts and observe changes not detectable with light microscopy. It is also possible to obtain quantifiable, three-dimensional information about the localization of molecules throughout an entire cell.

Research conducted by Carolyn Larabell (Berkeley Lab and University of California, San Francisco) and Mark Le Gros (Berkeley Lab).

Research funding: National Institute of General Medicine (GM 63948) and the U.S. Department of Energy, Office of Health and Environmental Research. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: C.A. Larabell and M.A. Le Gros, "X-ray Tomography Generates 3-D Reconstructions of the Yeast, Saccharomyces cerevisiae, at 60-nm Resolution," Molecular Biology of the Cell 15, 957 (2004).

ALSNews Vol. 245, September 29, 2004