The Experimental Setup
To perform a X-ray diffraction experiment, we need an x-ray source. In many cases a laboratory X-ray source such as a rotating anode generator producing a X-ray beam of a characteristic wavelength is used. Intense, tunable X-ray radiation produced by a Synchrotron provides additional advantages. The primary X-ray beam is monochromated by either crystal monochromators, focusing mirrors, or complex multilayer optics. After the beam passes through a helium flushed collimator it passes through the crystal mounted on a pin on a goniometer head. The head is mounted to a goniostat which allows to position the crystal in different orientations in the beam. The diffracted X-rays are recorded using imaging plates, Multiwire detectors (now obsolete), CCD detectors (most common) and the new superfast pixel array detectors (PADs).
Old ADSC Multi-wire Detector System ADSC Quantum 315 CCD System
In an IP X-ray detector, a layer of small crystalline grains consisting of a doped phosphor and organic binders is sandwiched between a support layer and protective layer on a flexible backing. Irradiation excites the crystals in their luminescence center to a metastable state. Image information formed by this excitation is stable for many hours but decays within days. For readout, the phosphor is photo-stimulated to luminescence by exposure to a visible laser, the light pulses recorded, and the plate finally erased by further exposure to a high intensity halogen lamp. IPs have high quantum efficiency, a wide dynamic range, good linearity of response, a high spatial resolution, a large active area size, a high counting rate capability, and are the least expensive detectors. Their drawback in synchrotron use is the slow readout time, which is generally not an issue for the much less intense home-lab X-ray sources.
CCDs are comprised of a 2-dimensional semiconductor array which directly delivers a digital image of the diffraction pattern. Just as for IPs, the X-ray photons fall onto a phosphorescent screen, but the screen is bonded to an optical taper leading to a photon-sensitive CCD chip. The photons generated by the X-rays absorbed in the phosphorescent screen generate free electrons in the silicon of the CCD in proportion to the number of photons. Depending on the specific design of the detector, fast readout electronics generate a raw electronic image of the diffraction pattern, which is further processed by the data collection computer. CCD detectors exhibit high sensitivity, low noise, and excellent linearity of response. However, they can be saturated by very intense X-rays, and multiple passes with suitable exposure times may be necessary to capture data from crystals that diffract strongly. Exposure times for single data frames can be as fast as seconds on synchrotrons, and a whole data set can sometimes be collected within minutes.
Flash cooling of protein crystals to cryogenic temperatures (~100 K) offers many advantages, the most significant of which is the elimination of radiation damage. in the most common single axis rotation method, the crystal rotated in small increments around a single axis and exposed to X-rays in each orientations. A part of the X-rays passing through the crystal is scattered in different directions into a detector. The detector collects an image of the diffraction spots. A large number of these images recorded from the different crystal orientations are processed (integrated, scaled and merged) into a final list of indexed reflection intensities.
You can read the first pages 1, 2, 3 of Chapter 8 (Data Collection) of my book Biomolecular Crystallography or buy the book from Amazon.
To learn more about cryo-cooling of crystals click on the picture
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This World Wide Web site conceived and maintained by Bernhard Rupp. Last revised Dezember 27, 2009 01:40