RICERCA ITALIANA     

Research program

ADVANCED TOOLS OF MOLECULAR IMAGING AND GENETIC ENGINEERING TO STUDY DISTRIBUTION AND DYNAMICS OF CELL MAGNESIUM: NOVEL APPROACHES TO LINK MAGNESIUM HOMEOSTASIS AND CELL FUNCTIONS.

University Co-ordinator

Consiglio Nazionale delle Ricerche - ()

Research Unit Leader

Stefano Lagomarsino

Description

The main objective of the activity at the O.U. at CNR is the development of x-ray microfluorescence and microimaging methodologies and the analysis, with such techniques, of selected samples, in order to give useful information on the Mg intracellular distribution, complementary to those obtainable with the other molecular imaging techniques proposed in this project. It is worth to note that the application of x-ray micro-imaging and micro-fluorescence techniques to the problem of element distribution in cells, as can be inferred by bibliography, is quite recent. Furthermore, the examples cited in the literature refer generally to heavier elements, as Fe and Cu, which are more easily characterized with respect to Mg. Indeed the low energy of the absorption edge of Mg (1300 eV) gives non negligible problems: low fluorescence yield (ratio between fluorescence photons and Auger electrons for one photoabsorption event), high absorption of the radiation by air, substrate or windows of vacuum chambers. We must therefore design very carefully the experiments. On the other hand these methodologies have a very great potentiality not only for the studies on Mg distribution; indeed this kind of analysis can be extended to other light elements, essential to the cellular functions, as Na and K, whose spatial distribution and intracellular dynamics are still unexplored. We therefore expect at least two main results: from one hand the set-up of experimental and data analysis protocols for the general problem of localization, quantification and oxidation state of elements in cells, and on the other hand a significant contribution to the specific problem of Mg localization. This will be faced in this project with contributions from several, quite different experimental techniques, whose merging will allow reciprocal validation, and which hopefully will give a comprehensive understanding of cellular magnesium homeostasis.

The techniques which will be used in this O.U. will be:

1. X-ray micro-fluorescence. The incident beam will be focused with suitable x-ray optics (in general Fresnel Zone Plates) down to sub-micrometer dimensions (of the order of few hundreds of nm), then the sample will be scanned in front of the beam, and for each sample position both the transmitted intensity, measured with an area detector, and the fluorescence spectrum, measured with an energy dispersive solid state detector, will be recorded. The transmitted intensity will give the image of the cell, with the contrast due to spatial density variations, whilst the analysis of the fluorescence spectrum will give the spatial distribution of the excited elements, that for an incident radiation of 2 KeV will be essentially Na and Mg. It will be then possible also to study the co-localization of these two elements.
2. Differential absorption. The photoelectric absorption coefficient for x-rays, for any element, reduces for increasing incident photon energy E, being inversely proportional to the cube of E. However, at the absorption edges the absorption coefficient increases suddenly, with a variation which can be even higher than ten-fold. For Mg, whose absorption edge is at 1300 eV, the absorption coefficient is 447 cm2/g for an energy of 1295 eV, and 5914 cm2/g for an energy of 1305 eV. In this narrow energy interval the absorption coefficient of the other cell constituents is practically constant. Digitally subtracting the two images, taken for ex. at the two energies mentioned above, it will be obtained a contrast due only to the presence of Mg, from which the spatial distribution of Mg in the different cellular compartments will result. Preliminary evaluations confirm that the contrast due to Mg will be appreciable. This kind of method will be implemented with two different experimental techniques: imaging with the aid of x-ray lenses, and lens-less imaging
a) Imaging with the aid of x-ray lenses: in this case it will be used a full field x-ray microscope where, in analogy with optical microscope, the image of the sample will be projected on the detector plane (in general an X-ray CCD camera provided with a scintillator screen and suitable optics), with 100-fold, or more, magnification. The obtainable spatial resolution is of the order of some tens of nm.
b) Lens-less imaging: it is possible to obtain high resolution images even with a lens-less set-up, using contact microscopy and a suitable high spatial resolution area detector. This last can be a Lithium Fluoride (LiF) film (see for ex. R.M. Montereali, in Handbook of thin film materials, Vol 3, ch. 7 Ed. H.S. Nalwa (2002) and S. Almaviva et al., Appl. Phys. Lett., 89, 054102 (2006)). In this case the X radiation creates, in the LiF structure, crystalline point defects, named Color Centers (CC), which luminesce when excited by light at about 450 nm. The image is then read by a confocal fluorescence microscope, with a spatial resolution of the order of 250-300 nm. Because the production of CC is proportional to the intensity of radiation impinging on the LiF film, it is possible to record the spatial variations of the intensity transmitted by the sample (in our case the cell). Also in this case, as in the imaging with lenses, two images at two different energies will be recorded, one just before and one just after the Mg absorption edge, at two different positions of the detector in order not to superpose the images. These will be then read with the confocal microscope, and subtracted one from the other, in order to put in evidence the presence of Mg.

The differential absorption, in both the experimental techniques, allows also in principle to distinguish between different valence states of the element under examination. In fact the binding energy of the level 1s, that is responsible for the absorption edge at 1300 eV, is influenced by the chemical state of the element. Then the absorption edge of Mg, for different oxidation states, can vary of some eV. Recording images for different incident energies it should be possible to distinguish between ionized and bound Mg. We said “it should” because of the poor knowledge on how Mg is chemically bound to the different cellular components, and therefore on the effective absorption edge variations, and on the real spatial distribution and separation between free and bound Mg, which could be too small to allow its detection. Anyway we hope that this kind of experiment can shed also some light on these aspects.

All the experimental techniques above described will be implemented, using different synchrotron radiation facilities; in particular we will carry out experiments at the X-ray microscopy beamline ID21 of the European Synchrotron Radiation Facility (ESRF) in Grenoble, at the beamline TWINMIC at Elettra in Trieste, and at the UV and soft x-ray beamline at DAFNE in Frascati.
At the ESRF microfluorescence measurements will be carried out, using the Scanning Transmission X-ray Microscope (STXM), and a Ge solid state detector for x-ray fluorescence detection. The incident energy will be 2 KeV, the spatial resolution 0.3x0.6 microns;.
At the TWINMIC beamline in Elettra x-ray full field microscopy measurements are planned, using Fresnel Zone Plates for a spatial resolution of the order of 60 nm. Several images will be recorded close in energy to the Mg absorption edge at 1300 eV, for differential absorption measurements and with the hope to put in evidence different spatial distributions for free and bound Mg.
At the UV and soft x-rays beam line of DAFNE we will instead carry out lens-less absorption measurements using LiF films as high spatial resolution detector. Also in this case, images at different energies around the Mg edge at 1300 eV will be taken.

In all cases, it will be extremely important to set up protocols to deposit and to fix the cells onto suitable substrates. Indeed the standard slides cannot be used because they are not transparent at such low x-ray energy. In the literature we found indications that for this kind of measurements, electron microscopy grids covered with formvar films are used, or Si substrates with very thin Si3N4 windows. Furthermore it will be not possible to examine with these techniques living cells, because of the radiation damage which could alter the cell conformation and functionality. It will be therefore necessary to set up protocols to fix the cells in order to carry out the planned experiments. Also in this case it is possible to find in the literature examples of suitable procedures, but these must be tested and optimized for the specific cases of this project.
Another important aspect to consider is the need to compare the results obtained using the x-ray methodologies, with those achieved using the fluorescent molecular probes. To this purpose it will be necessary to use sample holders suitable for both the x-ray and the confocal microscopes, and to have on them precise reference points, which allow the identification of the single cells studied with both techniques. This will be made possible by suitable markers made with microfabrication techniques available at the O.U of CNR.

Concerning the temporal sequence, we will adopt the following workplan:

- In the first six months of the project we will set up, in collaboration with the O.U. of Rome and Milan, the protocols to fix the cells, and the preparation of substrates and sample holders, as explained before. In particular we will take care, using the microfabrication facility at CNR, of the deposition of markers, to identify the single cells.
- In the following twelve months the experiments at the described synchrotron radiation facilities will be carried out, using the HC11 cells prepared by the O.U. in Rome. Basal and stimulated cells, as described by the O.U. in Rome, will be studied, both marked and non marked with the fluorescent probes. The results will be then compared with those obtained with confocal microscopy and spectrofluorimetry respectively in Rome and Bologna.
- In the last six months we will mainly study the cellular lines prepared by the O.U. in Milan, genetically modified for the expression of Mg channels.

As a whole, the activities carried out by the different O.U. characterized by complementary competences, will allow to set up, for the first time in Italy, a coherent experimental protocol system of molecular imaging at very high spatial resolution (of the order of 100 nm) for the assessment of the intracellular spatial distribution of elements essential to cell functions, bringing a forefront contribution in methodologies and basic knowledge.