While significant progress has been made in optical microscopy to break the diffraction barrier, such techniques rely on fluorescent labeling technologies, which prohibit the quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image structures at a resolution of 3-5 nm, but this only works with thin or sectioned specimens.
Furthermore, although X-ray protein crystallography is currently the primary method used for determining the 3D structure of protein molecules, many biological specimens--such as whole cells, cellular organelles, some viruses and many important protein molecules--are difficult or impossible to crystallize, making their structures inaccessible. Overcoming these limitations requires the employment of different techniques.
Now, in an article published May 31, 2010, in the journal Proceedings of [U.S.] National Academy of Sciences (PNAS), scientists from the University of California, Los Angeles (UCLA; USA) and their collaborators demonstrate the use of a unique X-ray diffraction microscope that enabled them to reveal the internal structure of yeast spores. The team reports the quantitative 3D imaging of a whole, unstained cell at a resolution of 50 nm - 60 nm using X-ray diffraction microscopy, also known as lensless imaging.
Researchers identified the 3D morphology and structure of cellular organelles, including the cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, and nucleolus. The research may open a way to identifying the individual protein molecules inside whole cells using labeling technologies.
The lead authors on the study are Dr. Huaidong Jiang, a UCLA assistant researcher in physics and astronomy, and Dr. John Miao, a UCLA professor of physics and astronomy. The work is a culmination of a collaboration started three years ago with Dr. Fuyu Tamanoi, UCLA professor of microbiology, immunology, and molecular genetics. Dr. Miao and Dr. Tamanoi are both researchers at UCLA's California NanoSystems Institute. Other collaborators include teams at Riken Spring 8 (Hyogo, Japan) and the Institute of Physics, Academia Sinica (Taipei, Taiwan).
"This is the first time that people have been able to peek into the 3D internal structure of a biological specimen, without cutting it into sections, using X-ray diffraction microscopy, Dr. Miao noted. "By avoiding use of X-ray lenses, the resolution of X-ray diffraction microscopy is ultimately limited by radiation damage to biological specimens. Using cryogenic technologies, 3D imaging of whole biological cells at a resolution of 5 nm - 10 nm should be achievable. Our work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.”
Dr. Tamanoi prepared the yeast spore samples analyzed in this study. Spores are specialized cells that are formed when they are placed under nutrient-starved conditions. Cells use this survival strategy to deal with harsh conditions. "Biologists wanted to examine internal structures of the spore, but previous microscopic studies provided information on only the surface features. We are very excited to be able to view the spore in 3D,” Dr. Tamanoi said. "We can now look into the structure of other spores, such as Anthrax spores and many other fungal spores. It is also important to point out that yeast spores are of similar size to many intracellular organelles in human cells. These can be examined in the future.”
Since its first experimental demonstration by Dr. Miao and collaborators in 1999, coherent diffraction microscopy has been applied to imaging a wide range of materials science and biologic specimens, such as nanoparticles, nanocrystals, biomaterials, cells, cellular organelles, viruses, and carbon nanotubes using X-ray, electron, and laser facilities worldwide. Until now, however, the radiation-damage problem and the difficulty of acquiring high-quality 3D diffraction patterns from individual whole cells have prevented the successful high-resolution 3D imaging of biologic cells by X-ray diffraction.
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