# Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/X-ray microscope

An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce images of very small objects.
Unlike visible light, 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 (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-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 Paul Kirkpatrick and Albert 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.
The Advanced Light Source (ALS) in Berkeley CA is home to XM-1 (http://www.cxro.lbl.gov/BL612/), 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 still holds the world record in spatial resolution with Fresnel zone plates down to 15nm and is able to combine high spatial resolution with a sub-100ps time resolution to study e.g. ultrafast spin dynamics.
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 (http://ncxt.lbl.gov). XM-2 is capable of producing 3-Dimensional tomograms of cells.
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 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, more researchers have begun to use the soft x-rays emitted from laser-produced plasma rather than synchrotron radiation.
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.

## Imaging Cell Structure with X-rays

Currently, there are several ways in which cell architecture is observed and imaged: fluorescence microscopy, transmission electron microscopy, and X-ray microscopy. Fluorescence microscopy only detects fluorescent molecules in a specimen, leaving much of the cellular structure invisible. Transmission electron microscopy is limited by the low penetrating power of electrons, making it impossible to examine an intact eukaryotic cell due to its size, and requiring the cell to be sectioned into slices.
The researchers, Larabell and Nugent (2010), propose X-ray microscopy as being the future for studying cellular architecture. X-rays eliminate the need for sectioning the specimen since X-rays can penetrate cells and tissues. Due to the unique properties of photons, soft X-ray wavelengths also make it possible to image cells without the application of heavy metal stains, which are used in transmission electron microscopy. Soft X-ray microscopes use photons with energies in the ‘water window’ (the region of the spectrum between the K shell absorption of carbon and oxygen). Thus, carbon-containing organic material and nitrogen-containing organic material absorb the X-rays at much higher magnitude than water.
Advances in X-ray imaging technologies have established X-ray microscopy as a significant tool for cellular imaging and research. Soft X-Ray tomography (SXT) can produce quantitative 3D images of cells. Soft X-ray microscopes produce 2D representations of a 3D specimen. By collecting images at incremental angles (1-2 degree increments through 180 degree rotation) about an axis, a 3D tomographic reconstruction of the specimen can be mathematically computed. During this process, the specimen undergoes high exposure to radiation from the X-rays that causes structural damage. To circumvent this, the specimen is imaged at liquid nitrogen temperature, which allows more than a thousand X-ray projection images to be taken without noticeable signs of radiation damage.
A limitation to current X-ray imaging is the reliance on optical elements to produce an image of the sample. Lens technology will most likely hit a barrier due to the difficulty of producing better structures. Larabell and Nugent (2010) discuss the possibility of eliminating the need for lenses through coherent diffractive imaging (CDI). The principle behind CDI is that any sample can be uniquely defined by its diffraction pattern. An advantage of CDI is that it gives access to the quantitative amplitude and phase information which can make it easier to explore different imaging wavelengths. Currently, CDI does not produce images as clear as the images produced through lenses, but may indeed be the future of X-ray microscopy imaging.
REFERENCES:
Larabell & Nugent, Imaging cellular architecture with X-rays, 2010, Department of Anatomy, University of California at San Francisco, Elsevier Ltd.