Imaging crystalline protein grains in 3D using X-ray diffraction

1st April 2013

Preparing protein crystals has always been difficult because their growth mechanisms are not well understood. A better insight into these mechanisms is now possible, using a new imaging method based on coherent x-ray diffraction.

Scientists at the University of Illinois in the US have developed a lensless X-ray microscope that can create three-dimensional images of micron-size samples. The instrument can be used in metallurgical and semiconductor applications, and for studying the early growth stages of protein crystals.

Unlike conventional radiography, which looks at the variation in X-ray absorption (between bones and soft tissue, for example), the new imaging method is based on coherent X-ray diffraction. As a result, the technique can image microscopic crystals embedded in solids or liquids that are inaccessible to electron microscopy (Fig. 2).

"Coherent X-ray diffraction, coupled with our imaging capability, offers considerable potential for studying nanocrystalline materials, including proteins,“ said Ian Robinson, a professor of physics and a researcher at the Frederick Seitz Materials Research Laboratory on the UI campus. "The penetrating power of a beam of X-rays allows us to peer beneath the surface of the material and see features buried inside.“

In place of an objective lens, the X-ray microscope uses computer inversion of the diffraction pattern to produce an image. The computer algorithm is similar to that used in CAT scanning tomography for medical imaging.

To create an image, Robinson and his colleagues ­ graduate students Garth Williams, Mark Pfeifer and Sebastien Boutet with visiting scientist Ivan Vartanyants ­ illuminate a sample with a coherent beam of X-rays generated at the Advanced Photon Source, a synchrotron radiation facility located at Argonne National Laboratory near Chicago. The diffracted X-rays are collected by a charge-coupled device (CCD) detector and converted into signals that can be read by a computer (Fig. 1).

As the sample is rotated, a series of two-dimensional diffraction patterns is created, which can be reconstructed into a high-resolution, three-dimensional image. Such images can be extremely useful to structural biologists studying the growth of protein crystals.

"The preparation of protein crystals for X-ray crystallography can be a very challenging and time-consuming task,“ Robinson said. "Much of the effort that goes into solving the structure of a protein actually goes into finding the ideal growth conditions and obtaining the crystals,“ he added.

One of the bottlenecks in growing crystals, he said, is that the growth mechanisms, themselves, are not well understood. To facilitate the preparation of protein crystals, scientists must better understand the mechanisms involved in the nucleation process that occurs in the early stages of crystal growth.

"One way to accomplish that is to use the X-ray microscope to look at the nucleation stage of the protein aggregate as it transforms into a crystal. This could give valuable information on the structure of critical nuclei and provide new insight into the nucleation process.“

Image Station 1000

PerkinElmer Life Sciences, a leading provider of drug discovery, life science research and genetic disease screening solutions, is now the exclusive worldwide distributor of the new Kodak Image Station 1000.

Used for high-performance imaging of chemiluminescent, fluorescent and chromogenic labels, this system rivals films in its sensitivity.

Publication-quality images are generated in less time than with traditional autoradiography and with a significantly wider linear dynamic range (>4.5 orders of magnitude), enabling users to capture and produce precise intensity measurements of very bright and faint signals in a single image. The Image Station also gives users the ability to detect a wide variety of labels and sample types with a single instrument.

It is a thermoelectrically cooled CCD technology, which produces low noise and high sensitivity, allowing for long exposure times. CCD pixel density is 1024 x 1024 and 10x optical zoom produces high resolution (20 micron/pixel at full zoom) as well as 16- and 32-bit image acquisition, which the company says provide excellent linearity of response over a wide dynamic range for accurate intensity measurements. u





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