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An X-ray microscopy image of a living 10-days-old canola plant.[1]

An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce magnified images of objects. Since X-rays penetrate most objects, there is no need to specially prepare them for X-ray microscopy observations.

Unlike visible light, X-rays do not reflect or refract easily, and they are invisible to the human eye. Therefore, an X-ray microscope exposes film or uses a charge-coupled device (CCD) detector to detect X-rays that pass through the specimen. It is a contrast imaging technology using the difference in absorption of soft X-rays in the water window region (wavelengths: 2.34-4.4 nm, energies: 280-530 eV) by the carbon atom (main element composing the living cell) and the oxygen atom (main element for water).

Microfocus X-ray also achieves high magnification by projection. A microfocus X-ray tube produces X-rays from an extremely small focal spot (5 µm down to 0.1 µm). The X-rays are in the more conventional X-ray range (20 to 300 kV), and they are not re-focused.


Invention and DevelopmentEdit

The history of X-ray microscopy can be traced back to the early 20th century. After the German physicist Rontgen discovered X-rays in 1895, scientists soon illuminated an object using an X-ray point source and captured the shadow images of the object with a resolution of several microns.[2] In 1918, Einstein pointed out that the refractive index for X rays in most mediums should be just slightly less than 1,[3] which means refractive optical parts would be difficult to use for X-ray applications.

Early X-ray microscopes by Paul Kirkpatrick and Albert Baez used grazing incidence reflective X-ray optics to focus the X-rays, which grazed X-rays off parabolic curved mirrors at a very high angle of incidence. An alternative method of focusing X-rays is to use a tiny Fresnel zone plate of concentric gold or nickel rings on a silicon dioxide substrate. Sir Lawrence Bragg produced some of the first usable X-ray images with his apparatus in the late 1940s.

Indirect drive laser inertial confinement fusion uses a "hohlraum" which is irradiated with laser beam cones from either side on its inner surface to bathe a fusion microcapsule inside with smooth high intensity X-rays. The highest energy X-rays which penetrate the hohlraum can be visualized using an X-ray microscope such as here, where X-radiation is represented in orange/red.

In the 1950s Sterling Newberry produced a shadow X-ray microscope which placed the specimen between the source and a target plate, this became the basis for the first commercial X-ray microscopes from the General Electric Company.

After a silent period in the 1960s, X-ray microscopy regained people's attention in the1970s. In 1972, Horowitzand Howell built the first synchrotron-based X-ray microscope at the Cambridge Electron Accelerator.[4] This microscope scanned samples using synchrotron radiation from a tiny pinhole and showed the abilities of both transmission and fluorescence microscopy. Other developments in this period include the first holographic demonstration by Sadao Aoki and Seishi Kikuta in Japan,[5] the first TXMs using zone plates by Schmahl et al.,[6] and Stony Brook’s experiments in STXM.[7][8]

The uses of synchrotron light sources brought new possibilities for X-ray microscopy in the 1980s. However, as new synchrotron source-based microscopes were built in many groups, people realized that it was difficult to perform such experiments due to insufficient technological capabilities at that time, such as poor coherent illuminations, poor quality x-ray optical elements, and user-unfriendly light sources.[9]

Entering the 1990s, new instruments and new light-sources greatly fueled the improvement of X-ray microscopy. Microscopy methods including tomography, cryo, and cryo-tomography were successfully demonstrated. With rapid development, X-ray microscopy found itsnew applications in soil science, geochemistry, polymer sciences, and magnetism. The hardware was also miniaturized so that researchers could perform experiments in their own laboratories.[9]

With the applications continued to grow since 2000, X-ray microscopy has become a routine, proven technique used in environmental and soil sciences, geo- and cosmo-chemistry, polymer sciences, biology, magnetism, material sciences. With this increasing demand for X-ray microscopy in these fields, microscopes based on synchrotron and laboratory light sources are being built around the world. X ray optics and components are also being commercialized rapidly.[9]


X-ray opticsEdit

Synchrotron Light SourcesEdit

Advanced Light SourceEdit

The Advanced Light Source (ALS) in Berkeley, California, is home to XM-1, a full-field soft X-ray microscope operated by the Center for X-ray Optics and dedicated to various applications in modern nanoscience, such as nanomagnetic materials, environmental and materials sciences and biology. XM-1 uses an X-ray lens to focus X-rays on a CCD, in a manner similar to an optical microscope. XM-1 held the world record in spatial resolution with Fresnel zone plates down to 15 nm and is able to combine high spatial resolution with a sub-100ps time resolution to study e.g. ultrafast spin dynamics. In July 2012, a group at DESY claimed a record spatial resolution of 10 nm, by using the hard X-ray scanning microscope at PETRA III.[10]

The ALS is also home to the world's first soft x-ray microscope designed for biological and biomedical research. This new instrument, XM-2 was designed and built by scientists from the National Center for X-ray Tomography. XM-2 is capable of producing 3-dimensional tomograms of cells.

Detection devicesEdit

Scanning TransmissionEdit

Sources of soft X-rays suitable for microscopy, such as synchrotron radiation sources, have fairly low brightness of the required wavelengths, so an alternative method of image formation is scanning transmission soft X-ray microscopy. Here the X-rays are focused to a point and the sample is mechanically scanned through the produced focal spot. At each point the transmitted X-rays are recorded with a detector such as a proportional counter or an avalanche photodiode. This type of Scanning Transmission X-ray Microscope (STXM) was first developed by researchers at Stony Brook University and was employed at the National Synchrotron Light Source at Brookhaven National Laboratory.


The resolution of X-ray microscopy lies between that of the optical microscope and the electron microscope. It has an advantage over conventional electron microscopy in that it can view biological samples in their natural state. Electron microscopy is widely used to obtain images with nanometer to sub-Angstrom level resolution but the relatively thick living cell cannot be observed as the sample has to be chemically fixed, dehydrated, embedded in resin, then sliced ultra thin. However, it should be mentioned that cryo-electron microscopy allows the observation of biological specimens in their hydrated natural state, albeit embedded in water ice. Until now, resolutions of 30 nanometer are possible using the Fresnel zone plate lens which forms the image using the soft x-rays emitted from a synchrotron. Recently, the use of soft x-rays emitted from laser-produced plasmas rather than synchrotron radiation is becoming more popular.


Additionally, X-rays cause fluorescence in most materials, and these emissions can be analyzed to determine the chemical elements of an imaged object. Another use is to generate diffraction patterns, a process used in X-ray crystallography. By analyzing the internal reflections of a diffraction pattern (usually with a computer program), the three-dimensional structure of a crystal can be determined down to the placement of individual atoms within its molecules. X-ray microscopes are sometimes used for these analyses because the samples are too small to be analyzed in any other way.

Biological ApplicationsEdit

One early applications of X-ray microscopy in biology was contact imaging, pioneered by Goby in 1913. In this technique, soft x-rays irradiate a specimen and expose the x-ray sensitive emulsions beneath it. Then, magnified tomographic images of the emulsions, which correspond to the x-ray opacity maps of the specimen, are recorded using a light microscope or an electron microscope. A unique advantage that X-ray contact imaging offered over electron microscopy was the ability to image wet biological materials. Thus, it was used to study the micro and nanoscale structures of plants, insects, and human cells. However, several factors, including emulsion distortions, poor illumination conditions, and low resolutions of ways to examine the emulsions, limit the resolution of contacting imaging. Electron damage of the emulsions and diffraction effects can also result in artifacts in the final images.[11]

X-ray microscopy has its unique advantages in terms of nanoscale resolution and high penetration ability, both of which are needed in biological studies. With the recent significant progress in instruments and focusing, the three classic forms of optics—diffractive,[12] reflective,[13][14] refractive[15] optics—have all successfully expanded into the X-ray range and have been used to investigate the structures and dynamics at cellular and sub-cellular scales. In 2005, Shapiro et al. reported cellular imaging of yeasts at a 30 nm resolution using coherent soft X-ray diffraction microscopy.[16] In 2008, X-ray imaging of an unstained virus was demonstrated.[17] A year later, X-ray diffraction was further applied to visualize the three-dimensional structure of an unstained human chromosome.[18] X-ray microscopy has thus shown its great ability to circumvent the diffractive limit of classic light microscopes; however, further enhancement of the resolution is limited by detector pixels, optical instruments, and source sizes.

A longstanding major concern of X-ray microscopy is radiation damage, as high energy X-rays produce strong radicals and trigger harmful reactions in wet specimens. As a result, biological samples are usually fixated or freeze-dried before being irradiated with high-power X-rays. Rapid cryo-treatments are also commonly used in order to preserve intact hydrated structures.[19]

A square beryllium foil mounted in a steel case to be used as a window between a vacuum chamber and an X-ray microscope. Beryllium, due to its low Z number is highly transparent to X-rays.

See alsoEdit


  1. ^ Karunakaran, Chithra; Lahlali, Rachid; Zhu, Ning; Webb, Adam M.; Schmidt, Marina; Fransishyn, Kyle; Belev, George; Wysokinski, Tomasz; Olson, Jeremy; Cooper, David M. L.; Hallin, Emil (2015). "Factors influencing real time internal structural visualization and dynamic process monitoring in plants using synchrotron-based phase contrast X-ray imaging". Scientific Reports. 5: 12119. Bibcode:2015NatSR...512119K. doi:10.1038/srep12119. PMC 4648396. PMID 26183486.
  2. ^ Malsch, Friedrich (1939-12-01). "Erzeugung stark vergrößerter Röntgen-Schattenbilder". Naturwissenschaften (in German). 27 (51): 854–855. doi:10.1007/BF01489432. ISSN 1432-1904.
  3. ^ Senn, E. (1989), "Grundsätzliche Überlegungen zur physikalischen Diagnostik und Therapie von Muskelschmerzen", Verhandlungen der Deutschen Gesellschaft für Innere Medizin, Springer Berlin Heidelberg, pp. 668–674, ISBN 9783540514374, retrieved 2019-05-05
  4. ^ Horowitz, P.; Howell, J. A. (1972-11-10). "A Scanning X-Ray Microscope Using Synchrotron Radiation". Science. 178 (4061): 608–611. doi:10.1126/science.178.4061.608. ISSN 0036-8075.
  5. ^ Aoki, Sadao; Kikuta, Seishi (1974). "X-Ray Holographic Microscopy". Japanese Journal of Applied Physics. 13 (9): 1385–1392. doi:10.1143/jjap.13.1385. ISSN 0021-4922.
  6. ^ Niemann, B.; Rudolph, D.; Schmahl, G. (1974). "Soft X-ray imaging zone plates with large zone numbers for microscopic and spectroscopic applications". Optics Communications. 12 (2): 160–163. doi:10.1016/0030-4018(74)90381-2. ISSN 0030-4018.
  7. ^ Rarback, H.; Cinotti, F.; Jacobsen, C.; Kenney, J. M.; Kirz, J.; Rosser, R. (1987). "Elemental analysis using differential absorption techniques". Biological Trace Element Research. 13 (1): 103–113. doi:10.1007/bf02796625. ISSN 0163-4984.
  8. ^ Rarback, H.; Shu, D.; Feng, Su Cheng; Ade, H.; Jacobsen, C.; Kirz, J.; McNulty, I.; Vladimirsky, Y.; Kern, D. (1988), "The Stony Brook/NSLS Scanning Microscope", Springer Series in Optical Sciences, Springer Berlin Heidelberg, pp. 194–200, ISBN 9783662144909, retrieved 2019-05-05
  9. ^ a b c Kirz, J; Jacobsen, C (2009-09-01). "The history and future of X-ray microscopy". Journal of Physics: Conference Series. 186: 012001. doi:10.1088/1742-6596/186/1/012001. ISSN 1742-6596.
  10. ^ Coherent X-Ray scanning microscopy at PETRA III reached 10 nm resolution (June 2012). Retrieved on 2015-12-14.
  11. ^ Cheng, Ping-chin. (1987). X-ray Microscopy : Instrumentation and Biological Applications. Jan, Gwo-jen. Berlin, Heidelberg: Springer Berlin Heidelberg. ISBN 9783642728815. OCLC 851741568.
  12. ^ Chao, Weilun; Harteneck, Bruce D.; Liddle, J. Alexander; Anderson, Erik H.; Attwood, David T. (2005). "Soft X-ray microscopy at a spatial resolution better than 15 nm". Nature. 435 (7046): 1210–1213. doi:10.1038/nature03719. ISSN 0028-0836.
  13. ^ Hignette, O.; Cloetens, P.; Rostaing, G.; Bernard, P.; Morawe, C. (June 2005). "Efficient sub 100nm focusing of hard x rays". Review of Scientific Instruments. 76 (6): 063709. doi:10.1063/1.1928191. ISSN 0034-6748.
  14. ^ Mimura, Hidekazu; Handa, Soichiro; Kimura, Takashi; Yumoto, Hirokatsu; Yamakawa, Daisuke; Yokoyama, Hikaru; Matsuyama, Satoshi; Inagaki, Kouji; Yamamura, Kazuya (2009-11-22). "Breaking the 10 nm barrier in hard-X-ray focusing". Nature Physics. 6 (2): 122–125. doi:10.1038/nphys1457. ISSN 1745-2473.
  15. ^ Schroer, C. G.; Kurapova, O.; Patommel, J.; Boye, P.; Feldkamp, J.; Lengeler, B.; Burghammer, M.; Riekel, C.; Vincze, L. (2005-09-19). "Hard x-ray nanoprobe based on refractive x-ray lenses". Applied Physics Letters. 87 (12): 124103. doi:10.1063/1.2053350. ISSN 0003-6951.
  16. ^ Shapiro, D.; Thibault, P.; Beetz, T.; Elser, V.; Howells, M.; Jacobsen, C.; Kirz, J.; Lima, E.; Miao, H. (2005-10-11). "Biological imaging by soft x-ray diffraction microscopy". Proceedings of the National Academy of Sciences. 102 (43): 15343–15346. doi:10.1073/pnas.0503305102. ISSN 0027-8424.
  17. ^ Song, Changyong; Jiang, Huaidong; Mancuso, Adrian; Amirbekian, Bagrat; Peng, Li; Sun, Ren; Shah, Sanket S.; Zhou, Z. Hong; Ishikawa, Tetsuya (2008-10-07). "Quantitative Imaging of Single, Unstained Viruses with Coherent X Rays". Physical Review Letters. 101 (15). doi:10.1103/physrevlett.101.158101. ISSN 0031-9007.
  18. ^ Nishino, Yoshinori; Takahashi, Yukio; Imamoto, Naoko; Ishikawa, Tetsuya; Maeshima, Kazuhiro (2009-01-05). "Three-Dimensional Visualization of a Human Chromosome Using Coherent X-Ray Diffraction". Physical Review Letters. 102 (1). doi:10.1103/physrevlett.102.018101. ISSN 0031-9007.
  19. ^ Super-resolution microscopy techniques in the neurosciences. Fornasiero, Eugenio F.,, Rizzoli, Silvio O.,. New York. ISBN 9781627039833. OCLC 878059219.CS1 maint: others (link)

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