Structural Biochemistry/Imaging cellular architecture with X-rays

An interesting look at a paper by Carolyn A Larabell and Keith A Nugent
Much knowledge pertaining to cell architecture and its redevelopment during normal and disease filled processes has been derived from imaging. For centuries, the use of light microscopy techniques has reached its peak and has yielded a tremendous amount of information concerning cell and molecular dynamics. There exist two main types of microscopies, of which include fluorescence microscopy and transmission light microscopy. Though these two techniques have significantly advanced our understanding of cellular processes, they tend to provide limited details. Due to the low penetrating capabilities of electrons, it is not possible to examine the eukaryotic cell as a whole. Thus, most cells are sectioned into 60-500 nm slices. This tedious process requires initial fixation, dehydration, and plastic embedment as well as application of heavy metal stains to generate contrast. As can be seen, extensive work is required to obtain three-dimensional structural information of a whole cell, which is why most studies tend to be limited to small sections of cells.

All this leads to the emergence of X-ray imaging technologies, a crucial new tool for cellular imaging. X-rays are versatile, for they can penetrate thick cells and tissues, eliminating the need to section the specimen further. There are in fact three main types of X-ray imaging techniques, of which include soft X-ray microscopy, soft X-ray tomography, and coherent diffraction imaging. X-ray microscopy has the ability to penetrate thick cells deeply and initiate immediate structural development and outlook. Soft x-ray tomography can generate quantitative three-dimensional images of cells in the near-native state (at a better than 50 nm isotropic resolution). The most unique technique, which goes by the name of coherent diffraction imaging (the name speaks for itself), offers the possibility of high resolution x-ray imaging of cell using computation and high speed computers rather than an X-ray optic to phase the image. Let us take a closer look at the specifics of each X-ray imaging technique. Soft X-ray microscopy involves the use of particular zone plate lenses to allow the release of X-rays towards the cell of interest. These microscopes are operated using photons with energies in the ‘water window’, which is the region of the spectrum that lies between the K shell absorption edges of carbon and oxygen. However, lens-based imaging can also with energies outside the water window, where images can be generated using phase contrast techniques. To date, the most gripping images have been obtained using bright field, absorption contrast at a water window wavelength operation. To further specify the operations of each plate optic, it can be said that a condenser zone plate optic focuses X-rays on the cell itself and an objective zone late optic focuses the transmitted light onto the detector.

Soft x-ray tomography is in a sense similar to light and electron microscopes in that it can also generate two-dimensional representations of a three dimensional specimen. However, with the ability to collect projection images of the cell at angles around a rotation, it is possible to mathematically compute a three-dimensional reconstruction of the specimen of interest. It has been noted that all biological materials are damaged when exposed to intense light. This intensity can either come from ultra violet illumination in a fluorescence microscope or photons from an X-ray microscope. However, by cryosplicing, optical beams can be focused at a much lower temperature, thus avoiding the possibility of radiation cell damage altogether. As a matter of fact, when cells are imaged at liquid nitrogen temperatures, more than a thousand soft X-ray projection images can be retrieved without any sort of radiation damage. As stated before, it is crucial that specialized cryogenic tomography stages occur during this process of X-ray imaging. A key fact illustrating soft X-ray tomography’s versatility is that is can be applied to virtually any imaging investigation in cell biology, from imaging simple bacteria, to yeast and even algae. The first recorded evidence of X-ray tomographic reconstruction was algae. The distinctive ability to obtain high resolution images of intact eukaryotic cells is something that cannot be repeated by any sort of light or electron microscopy. In addition, x-ray tomography can be used to examine even the most complex cells, such as white blood cells and malaria-infested red blood cells.

The last, but certainly not least, technique of discussion is the method of coherent diffractive imaging. It is simply a method that offers the possibility of removing the need for a lens, thus avoiding the limitations imposed by the state-of-the-art in fabrication technology. As basic arrangement of a CDI experiment can be seen as a coherent beam which is used to illuminate the sample and its far-field diffraction pattern is measured. The beam will typically interact quite weakly with the sample, and the undiffracted component of the beam is held to be dominant and will damage the detector unless a beam-stop is initiated. The measurement of the beam at very low diffraction angles closely follows this previous process. Instead of a lens, a zone plate is used instead in coherent diffraction imaging, as it is used to create a focus and the sample is placed in the beam diverging from it. Sure enough, soft X-ray microscopy and soft X-ray tomography seem to be more in use at this time, but coherent diffraction imaging has great promise in the future. The procedural techniques must be sharpened further for it to play a bigger role in three-dimensional imaging. After all, CDI offers access to the quantitative amplitude and phase information that are not easily available through other forms of X-ray imaging. One thing is for sure: X-ray imaging techniques will seek development in the years to come.

Hard X-ray Fluorescence Tomography
Hard X-ray Fluorescence Tomography was a method originally created to detect the presence of very small amounts of transition metals in biological tissue, like copper, zinc, magnesium, etc. Metal ions like zinc and magnesium, for example, act as cofactors in many catalytic enzymatic reactions, and are hence very important for the cell. Using hard X-rays, a large number of transition metals can be detected and mapped all at once, since transition elements automatically fluoresce when hit by hard X-rays. Although this method is very powerful, there are and have been a number of technical problems with the usage of this method.

Hard X-ray tomography is a technique that uses a very large number of two-dimensional pictures of slices of a tissue in order to assemble a three-dimensional picture of the given sample of tissue. There are three main broad categories of tomography – full-field, projection tomography and confocal tomography. Full-field projection tomography is the process of utilizing the entire field of the specimen to carry out the tomography. However, this is still a young, developing technology with many problems. Projection tomography on the other hand, is moderately successful, and certain errors have to be accounted for in the final image produced. Confocal tomography is the most interesting of the three – it gives scientists the ability to analyze a very small portion of a specimen, although it is difficult to find the particular target using this method.

There are a few challenges being faced by scientists using this method to analyze biological tissues. The first is that the hard X-rays are very strong, and hence very harful to the tissue being studied. This results in only hard tissues like seeds being able to be studies, since other tissues die off so easily under exposure. Samples currently analyzable are freeze-dried or chemically fixed to endure the radiation. Scientists also face a number of time constraints due to speed limitations in dealing with the technical difficulties of using this technique.

Reference
Imaging cellular architecture with X-rays. Nugent and Carolyn A Larabell. Hard X-ray fluorescence tomography —an emerging tool for structural visualization. Martin D de Jonge and Stefan Vogt.