An X-ray microscope uses electromagnetic radiation in the 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 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 (an element of 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 keV) and are not re-focused.
Invention and development The history of X-ray microscopy can be traced back to the early 20th century. After the German physicist Röntgen 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 micrometers. In 1918, Einstein pointed out that the refractive index for X-rays in most mediums should be just slightly greater than 1, which means that 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.
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 the 1970s. In 1972, Horowitz and Howell built the first synchrotron-based X-ray microscope at the Cambridge Electron Accelerator. 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, the first TXMs using zone plates by Schmahl et al., and Stony Brook's experiments in STXM. 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. 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 new applications in soil science, geochemistry, polymer sciences, and magnetism. The hardware was also miniaturized, so that researchers could perform experiments in their own laboratories. Extremely high-intensity sources of 9.25 keV X-rays for X-ray phase-contrast microscopy, from a focal spot about 10 μm × 10 μm, may be obtained with a non-synchrotron X-ray source that uses a focused electron beam and a liquid-metal anode. This was demonstrated in 2003 and in 2017 was used to image mouse brain at a voxel size of about one cubic micrometer (see below). With the applications continuing to grow, X-ray microscopy has become a technique regularly 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, liquid-metal anode, and other laboratory light sources are being built around the world. X-ray optics and components are also being commercialized rapidly.
Instrumentation
X-ray optics
Synchrotron light sources
Advanced Light Source 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. 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.
Liquid-metal-anode X-ray source Extremely high-intensity sources of 9.25 keV X-rays (gallium K-alpha line) for X-ray phase-contrast microscopy, from a focal spot about 10 um x 10 um, may be obtained with an X-ray source which uses a liquid metal galinstan anode. This was demonstrated in 2003. The metal flows from a nozzle downward at a high speed and the high intensity electron source is focused upon it. The rapid flow of metal carries current, but the physical flow prevents a great deal of anode heating (due to forced-convective heat removal), and the high boiling point of galinstan inhibits vaporization of the anode. The technique has been used to image mouse brain in three dimensions at a voxel size of about one cubic micrometer.
Detection devices
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