Biomedical research with Ion Beam Techniques
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Ion beam techniques can be used both to modify the materials and to obtain structural and elemental information about the studied samples. In both cases there are many biological applications.

The knowledge of the elements, particularly trace elements, of biological samples can be very useful to resolve many medical problems. Ion Beam Analysis (IBA) techniques are a fast, effective and with high sensitivity (they can detect concentrations of about µg/g) tool to the detection of elemental concentrations.

The simplest studies with ion beam techniques in biological samples consist basically, in the determination of the bulk elemental concentration with an ion beam by PIXE (Particle Induced X-ray Emission). This technique can be applied to many tissues (teeth, uteri, brain, blood, liver…). The sample preparation normally is very simple and some mineralized tissues such as teeth do not need preparation, minimizing any problems of contamination or redistribution of elements. Using also a beam with a beam size of some millimeters RBS (Rutherford Backscattering Spectrometry) and ERDA (Elastic Recoil Detection Analysis) techniques have been used successfully to characterize biomaterials. However, most of the biomedical applications are carried out with nuclear microscopy techniques.

Nuclear microscopy is a MeV ion-based suite of IBA (Ion Beam Analysis) techniques that has the ability to image density variations in relatively thick tissues and map trace elements in the cell to the microgram per gram (dry weight) sensitivity (Fig.1). Nuclear microscopy consists of three complementary ion beam techniques (STIM, PIXE and RBS) that can be simultaneously applied. Although these techniques cannot yet be applied to living cells, its unique capability of mapping and measuring quantitative trace element concentrations at the cellular level in unstained cells, thereby minimizing any problems of contamination or redistribution of elements during sample preparation, make it a powerful tool for biomedical studies. Nuclear microscopy has been used successfully to the analysis of different biological tissues such as skin, bone, brain, pancreas, arteries, lung… The use of high energy ion beams focused to nanodimensions increases the possibility of STIM nano-imaging of single cells resolving easily the nucleus and several nucleolus (Fig. 2), and tissue [1]. The first results using nanoSTIM on whole cells have shown that this technique has high potential for nanoparticle counting, and structural imaging of nanoparticles within the cell (Fig. 3).

STIM can be used simultaneously with proton induced fluorescence (PIF) to study individual whole cells reaching resolutions of 200 nm, compared with 300–400 nm achieved by conventional optical fluorescence imaging. The combination of both techniques offers a powerful tool in the quest for elucidating cell function, particularly when the technical developments will allow in the near future to image down to sub-50 nm.

Tomography images can be also obtained making use of nuclear microscopy techniques. So far, 3D density images of single cells have been obtained. This technique offers information about inside the cells without any transversal section.

Finally, the great capacity of ion beam to modify materials has also two important application fields in biomedicine: the irradiation cell and the manufacture of different devices with biological applications. For both applications, beam size and control are decisive. The aim of irradiation cell experiments is to determine the effect of low radiation doses in living cells. The single culture cells are irradiated ion by ion in localized areas. Subsequently, the cell evolution is studied. It is well known that cellular behavior and function of cells are changed by geometric constraints and substrate chemical properties. Using a micro or nano-beam different for the cellular culture can be patterned. An ion beam can be used to manufacture 3D micro- and nano-channels in resists with very low rugosity in order to transport micro-fluid, to divide cells and to detect individual molecules using fluorescent microscopy. Biochip to support cells, DNA and proteins can be also manufactured.

The CMAM activity in biological field is firstly based on a whole equipment to prepare the biological which consist of: cryotome, freeze dryer, digestion pump and microwave oven, optical microscope, and diamond saw/polishing machine for mineralized tissue (teeth, bones…). The most important tool is the internal microbeam line placed at +30° where a micrometric or sub-micrometric beam will be available to work. This line is being optimized to analyze biological tissues and to study the interaction of nanoparticles of different sizes with mesenchymal stem cells (MSCs).

In the biosensors field interesting studies are being carried out at CMAM in order to modify materials, such as silicon, by heavy ions. The materials are amorphized and patterned. According with the patterning, preferential cellular orientations appear.

Fig. 1.  Mouse foot sole epidermis images. 1-Electronic microscopy image. 2- STIM map. 3- C map (RBS), 4- S map (PIXE), 5- P map (PIXE), 6- K map (PIXE). Stratum Corneum (SC), Stratum Lucidum (SL), Stratum Granulosum (SG), Stratum Spinosum (SS) and Dermis are identified without doubt. Image taken using a proton of 2 MeV with 1 µm spot size at CENBG (Centre d'Etudes Nucléaires de Bordeaux Gradignan).
Fig. 2.  Images of a N2A blastoma cell stained with SytoxR green nucleic acid stain: (a) confocal microscopy, (b) proton STIM, (c) alpha STIM, and (d) proton induced fluorescence. Proton images taken using 200 nm spot size, alpha STIM image taken using a 50 nm spot size alpha beam. [1]
Fig. 3.  STIM image of a whole cell (MRC-5 fetal lung fibroblast (ATCC No. CC-171)) imaged using 1.5 MeV alpha particles (STIM). The cell has been grown in an environment containing gold nanoparticles (more intense points). Spatial resolution 50nm (pending of publication).

[1] F. Watt, A.A. Bettiol, J.A. van Kan, M.D. Ynsa, Ren Minqin, R. Rajendran, Cui Huifang, Sheu Fwu-Shen, A.M. Jenner, Imaging of single cells and tissue using MeV ions, Nucl. Instrum. and Meth. B 267 (2009) 2113-2116.