Clear water, fresh air, pure earth – a longterm guaranteeing of the natural environment is a primary ecological goal. In places where natural resources have been exploited for decades or the delicate environmental balance disturbed due to accidents it is desirable to restore conditions to their original state. In the past few years biological methods have become more and more popular in this regard. One of the most well-known examples is of bacteria being able to break down the oil released during oil spills at sea. Various research projects are being supported to increase crop yields of soils that contain either impurities resulting from industry, or that for natural reasons (eg, very dry or saline soils) would be generally unsuitable for agriculture.
Stress caused plants by heavy metals
One widespread problem involves heavy metal levels in the soil. Reports of excessive concentrations found in farm fields, pastures or construction land are frequent. Plants growing in such soils suffer what is referred to as 'heavy metal stress' and thus do not thrive. There are, however, exceptions. The sea pink or sea thrift wildflower (armeria maritima) can tolerate high levels of copper; the hairy rock cress (cardaminopsis halleri) has no trouble dealing with astonishingly high zinc levels. So how do these plants manage it? That's what the 'heavy-metal-stress' research group at the Institute for Plant Biochemistry in Halle intends to find out – ie, what are the basic mechanisms responsible for these plants' remarkable tolerance?
Research aim and possible applications
The scientists in Halle are particularly interested in where and how these plants store heavy metals. Their research is on a fundamental level with specific application targets, however. For example, a more precise understanding of these mechanisms will help to increase crop yields of soils containing high levels of heavy metals. Another realistic possibility is that these plants could be used as soil purifiers. The heavy metals are extracted by practically 'growing them out' of the ground.
Ultra-thin sections in the electron microscope
For investigative purposes, the plant leaves are first frozen in propane and freeze substituted, embedded in synthetic resin and sliced into thin sections 50 nanometers in thickness. These ultra-thin sections are inserted into an energy-filtering transmission electron microscope (EFTEM). The energy filter means that not only electron-spectroscopic images (ESI) can be generated, but also energy-loss spectra of single points of the sample (EEL spectra, EELS). Only selected electrons of a specific energy loss contribute to an ESI image. The other electrons are filtered out by the energy filter.
ESI images of hairy rock cress
When imaging a thin section of hairy rock cress, eg, these researchers can select the electrons to image with whose energy loss is characteristic for a specific inner-shell transition of a zinc atom. The electrons which contribute to the ESI image are those which have caused this transition (ie, have 'flown through' a zinc atom), but also other electrons which in some other way coincidentally have lost the same energy. Without these 'other electrons', this ESI image would simply be an elemental map of zinc – thus displaying how the zinc atoms are distributed within the sample.
iTEM - the 'path' to obtaining an elemental map
Therefore, an ESI image is not the same as an elemental map. Not yet. In order to turn it into one, a multi-step process is necessary – eg, for displaying zinc distribution. This is what the researchers in Halle use the iTEM Solution EFTEM by Olympus Soft Imaging Solutions for. The iTEM Solution EFTEM makes conducting the various steps most convenient: the electron microscope can be remote controlled and a series of ESI images at various energy losses can be digitally acquired using a SIT camera. The automatic enhancement of image quality directly during acquisition is of critical importance here. Why? Because ESI images generally have a very small signal-to-noise ratio. One of the acquired images has an energy loss which is characteristic for zinc. This zinc ESI image, however, also includes (alongside contrast components originating from zinc atoms) a background which is zinc independent as well - this background is due to those 'other electrons' referred to above. Using the other ESI images, iTEM computes the corresponding background image that is then subtracted from the zinc-ESI image. The result will be the zinc distribution image desired.
Clarification of chemical bonding via EEL spectra
Obtaining an elemental map of silicon is done in the same way. The mixmap function of iTEM colors both of the images and then superimposes them onto a 'normal' electron-microscopical acquisition of the same image segment. The resulting image shows very clearly where concentrations of zinc (blue) and silicon (green) are located within the cell structure. Any spot where there is a blending of the two colors means that both elements are present – this is an indication that the zinc present is bonded as zinc silicate. Researchers can subsequently clarify whether or not this is actually the case by recording an EEL spectrum of such a spot. If the near-edge fine structure corresponds to the quantum-mechanically computed spectrum and to the spectrum of a standard substance, this is indeed the case. EEL spectra are generated and managed with the greatest convenience via iTEM.
Conclusion
The iTEM Solution EFTEM is an important tool for the scientists in Halle in their electron-microscope investigation of ultra-thin sections, thus making a contribution to illuminating what those mechanisms are that facilitate heavy-metal concentrations in plants. Furthermore, understanding these mechanisms will contribute to crop yield increases in impure soils as well as greatly increasing the likelihood that biological soil purification will soon become a reality.
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