As indicated previously, there are a number of biological investigations where the use of microfocus technology is becoming crucial to maximising the information that can be obtained on the structure and behaviour of biological tissues. The following study is representative of the work carried out on beamlines ID22 and ID18F.

In vivo Fluorescence Microtomography of Plants

Plant physiologists and microbiologists are trying to link the macroscopic appearance to the molecular biology of a plant, especially with a view to understanding the influence of single genes on a plants large scale structure. The current research of the plant physiology group at the IBI, Forschungszentrum Jülich, Germany, includes the study of ion channels and their influence on the long range transport of ions through the plant [1]. To that end, valuable information is obtained from a determination of the ion concentrations inside the plant.

In an experiment at beamline ID22/ID18F the concentration of physiologically relevant ions inside various plants has been determined by fluorescence microtomography. This method, currently under further development in collaboration with the University of Technology in Aachen, Germany, allows non-destructive determination of the element specific inner structure of a sample with a resolution in the micrometer range [2]. It is particularly suited to imaging the elemental distributions inside biological bulk samples at a cellular level with a minimum of sample preparation. It does not require vacuum, thus allowing wet samples to be investigated. Since the fluorescence is excited with monochromatic X-rays rather than charged particles (such as electrons in EDXA), the signal to background ratio is high, and even small elemental concentrations can be detected.

As a scanning technique, fluorescence microtomography uses a microbeam of high intensity produced using a compound refractive lens for hard X-rays [3]. The monochromatic microbeam (19.5 keV) had a lateral size of 6 µm by 1.6 µm (horizontal by vertical FWHM) with a flux of 1.1x1010 photons/s. The sample is scanned through the microbeam. Inside the sample, the atoms along the path of the microbeam are excited and emit characteristic fluorescence radiation that is recorded by an energy dispersive detector (Figure 5). After a line scan, the sample is rotated by a small angle and the scanning is repeated. The whole procedure is continued until the sample has completed a full rotation. From this data, the distribution image for each element is reconstructed by computer tomographic techniques.

Figure 6a shows the fluorescence tomograph of a root of the mahogany plant (Swietenia macrophylla) without the surrounding soil. With a resolution of 7 µm the cellular structures of the plant are clearly resolved. The distribution of chlorine, potassium, iron, and rubidium is shown. While iron is not absorbed from the soil into the root and sticks to the roots surface, rubidium is incorporated by the plant in a similar way to potassium. However, it is about three orders of magnitude less concentrated than potassium. The reconstruction illustrates the high sensitivity of the method. In combination with other tomography techniques, such as phase contrast microtomography, the elemental distribution can be directly related to the structure of the sample. Figure 6b shows the phase contrast tomograph of the mahogany root.

Due to the small amount of sample preparation and its non-destructive character, fluorescence microtomography may become an important tool in many disciplines, including bio-medicine, environmental and earth science, condensed matter physics, and chemistry.

References
[1] W.H. Schröder, G.L. Fain, Nature, 309, 268-270 (1984).
[2] A. Simionovici, M. Chukalina, M. Drakopoulos, I. Snigireva, A. Snigirev, C. Schroer, B. Lengeler, K. Janssen, F. Adams, in Developments in X-ray Tomography II, Ulrich Bonse, Editor, Proceedings of SPIE, 3772, 304-310 (1999).
[3] B. Lengeler, C. Schroer, J. Tümmler, B. Benner, M. Richwin, A. Snigirev, I. Snigireva, M. Drakopoulos, Journal of Synchrotron Radiation, 6(6), 1153-1167 (1999).

Authors
W. Schroder (a), C. Schroer (b), B. Lengeler (b), J. Tummler (b), F. Gunsler (b), A. Simionovici (c), A. Snigirev (c).

(a) IBI, Forschungszentrum, Jülich (Germany)
(b) RTWH, Aachen (Germany)
(c) ESRF