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X-ray diffraction gets the inside scoop on whole yeast cells

Erin Podolak

A new microscopy technique can capture high-resolution 3-D images of the internal structures of whole, unstained yeast cells.

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Researchers from the University of California, Los Angeles (UCLA) have developed an X-ray diffraction microscopy technique capable of capturing 3-D images of internal structures in whole, unstained cells. At a resolution of 50–60 nm, these pictures represent the first high-resolution images that do not require fluorescent tagging or staining to identify specific organelles.

UCLA Researchers have developed an X-ray diffraction microscopy technique capable of capturing 3-D volume renderings of a yeast spore, showing nucleus (orange), ER (green), vacuole (white), mitochondria (blue), and granules (light blue). Source: UCLA/California NanoSystems Institute.

“X-ray diffraction microscopy can, in principle, be used to perform high-resolution 3-D imaging of whole biological cells that are too thick for electron microscopy,” John Miao, a UCLA professor of physics and astronomy and a researcher at UCLA’s California NanoSystems Institute, told BioTechniques. While electron microscopy can image structures at a resolution of 3–5 nm, the technique requires thin or sectioned specimens. Since X-rays are capable of deeper penetration into a sample, researchers have looked for ways of using X-ray microscopy to image the interiors of cells.

The highest resolution currently achievable using X-ray diffraction microscopy on biological samples is 10–20 nm. However, this resolution can only be achieved using a stained or tagged sample, not a whole unstained cell, which is favorable for analysis since it enables researchers to study cells without manipulation.

Led by Miao, the researchers created a new X-ray diffraction microscope that does not rely on lenses and captures a series of 2-D diffraction patterns by tilting a sample. To reconstruct a 3-D image from this series of diffraction patterns, the authors had to design a more robust image reconstruction algorithm using a tomographic method.

The UCLA team—who worked with colleagues from Riken SPring 8 (Japan) and the Institute of Physics at Academia Sinica (Taiwan) to develop the microscope—used the new technique to identify the 3-D morphology and structure of organelles in yeast spores including the cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, and nucleolus at a resolution of 50–60 nm. Although the potential of radiation damage to specimens limits higher resolution using X-ray diffraction microscopy, the authors say that cryogenic technologies could be used to mitigate such damage and improve resolution to 5–10 nm, matching electron microscopy resolution.

“Our work paves a way for quantitative 3-D imaging of a wide range of biological specimens at nanometer-scale resolutions," said Miao. The researchers plan to continue work on their instrument to further enhance the resolution, which they say, with additional modification, has the potential to resolve individual protein molecules inside whole cells.

The paper, “Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy,” was published online ahead of print June 4 in the Proceedings of the National Academy of Science.