Dark field microscopy is an optical microscopy illumination technique used to enhance the contrast in unstained samples. It works on the principle of illuminating the sample with light that will not be collected by the objective lens, so not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it. While an X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce images of very small objects. Unlike visible light microscopes, X-rays do not reflect or refract easily, and they are invisible to the human eye. Therefore the basic process of an X-ray microscope is to expose film or use a charge-coupled device or CCD detector to detect X-rays that pass through the specimen, rather than light which bounces off the specimen. It is a contrast imaging technology using the difference in absorption of soft x-ray in the water window region, wavelength region: 2.3 - 4.4 nm, photon energy region: 0.28 - 0.53 keV by the carbon atom, main element composing the living cell and the oxygen atom, main element for water. Early X-ray microscopes by Kirkpatrick and Baez used grazing-incidence reflective 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 1940’s. In the 1950’s 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. 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 or 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 level resolution but the relatively thick living cell cannot be observed as the sample has to be sliced thinly and then dried to get the image. However, it should be mentioned that cryo-electron microscopy allows the observation of biological specimens in their hydrated natural state. 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, there has been active research on using the soft x-rays emitted from the laser-produced plasma rather than synchrotron radiation light. 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.
April 12th, 2010 at 6:34 am
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[…]Darkfield and X-Ray Microscopes | Different Type of Microscope[…]
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