METHOD, DEVICE AND MARKER SUBSTANCE KIT FOR MULTI-PARAMETRIC X-RAY FLUORESCENCE IMAGING

Abstract

A method for multi-parametric x-ray fluorescence imaging with maximized detection sensitivity and minimized radiation dose for a biological/living sample (10) containing a first marker substance comprises the steps of irradiation of the sample (10) with x-ray radiation (1), with x-ray fluorescence (2) of the first marker substance being excited, spatially resolved detection of the x-ray fluorescence (2) of the first marker substance, and determination of a distribution of the first marker substance in the sample (10) from the x-ray fluorescence (2) of the first marker substance, wherein the sample (10) contains at least one further marker substance which is excited to x-ray fluorescence (2) by the x-ray radiation (1), wherein fluorescence lines (3) of the first and the at least one further marker substances are different, at least one of the first and the at least one further marker substances is coupled with active ingredient molecules and/or ligand molecules provided for a specific interaction with the sample (10) or contained in cells, in order to be able to trace these, the detection comprises a spectrally resolved detection of the x-ray fluorescence (2) of the first and the at least one further marker substances, and additionally at least one distribution of the at least one further marker substance in the sample (10) is determined from the detected x-ray fluorescence (2) of the first and the at least one further marker substances. An imaging device (100) for multi-parametric x-ray fluorescence imaging and an optimized selection method for a marker substance kit for introducing marker substances into a sample (10) are also described.

Claims

1. A method for multi-parametric X-ray fluorescence imaging on a sample which comprises at least a part of a body of a biological organism and contains a first marker substance and at least one further marker substance, wherein at least one of the first and the at least one further marker substance is coupled to at least one of active substance molecules and ligand molecules which are provided for a specific interaction with the sample, comprising the steps of: irradiation of the sample with X-ray radiation, wherein X-ray fluorescence of the first marker substance and of the at least one further marker substance is excited by the X-ray radiation, and fluorescence lines of the X-ray fluorescence, of the first and the at least one further marker substance are different, spatially resolved detection of the X-ray fluorescence, comprising a spectrally resolved detection of the X-ray fluorescence of the first and the at least one further marker substance, and determination of a distribution of the first marker substance in the sample and in addition at least one distribution of the at least one further marker substance from the detected X-ray fluorescence, wherein the first and the at least one further marker substance exhibit fluorescence probabilities, attenuations of the X-ray fluorescence in the sample, and background noise levels on account of scattering in the sample which are equal or are similar to such an extent that the detection of the X-ray fluorescence at the same concentration of the marker substances results in comparable statistical significance levels, and the irradiation photon energy of the X-ray radiation is above the absorption edges of all the marker substances.

2. The method according to claim 1, wherein the statistical significance levels of the marker substances are maximized in that the irradiation photon energy of the X-ray radiation is selected so as to be of a distance above the highest absorption edge of the marker substances in the sample, such that the background noise levels of the marker substances are minimal and equal or approximately equal, and at the same time the fluorescence probabilities and through the sample are equal or approximately equal, and maximum.

3. The method according to claim 1, wherein the X-ray fluorescence of the first and the at least one further marker substance is excited and simultaneously detected using a common excitation beam of the X-ray radiation.

4. The method according to claim 1, wherein the X-ray fluorescence of the first and the at least one further marker substance is excited and simultaneously or sequentially detected using different excitation beams of the X-ray radiation which exhibit different energies.

5. The method according to claim 1, wherein the first and the at least one further marker substance in each case comprise nanoparticles and marker molecules.

6. The method according to claim 5, wherein the first or the at least one further marker substance in each case comprise nanoparticles, and the nanoparticles of the first and the at least one further marker substance comprises surfaces which are indistinguishable for the sample, wherein the nanoparticles comprise different elements on the inside.

7. The method according to claim 5, wherein the first marker substance comprises a first type of nanoparticles which primarily contain a first X-ray fluorescence element, and the at least one further marker substance comprises at least one further type of nanoparticles which in each case primarily contain at least one further X-ray fluorescence element.

8. The method according to claim 7, wherein each type of nanoparticles contains exclusively one of the first and the at least one further X-ray fluorescence element.

9. The method according to claim 5, wherein the first type of nanoparticles carries a first type of at least one of active substance molecules and ligand molecules which are provided for at least one of a chemical and a physical interaction with the sample, and the at least one further type of nanoparticles in each case carries different types of at least one of active substance molecules and ligand molecules which are provided for at least one of a chemical and a physical interaction with the sample, or does not carry any active substance molecules or any ligand molecules.

10. The method according to claim 9, wherein each type of nanoparticles carries exclusively one specific type of at least one of active substance molecules and ligand molecules.

11. The method according to claim 5, wherein at least one of the first and the at least one further type of nanoparticles has a core-shell structure comprising a particle core and a particle cover layer.

12. The method according to claim 11, wherein all the nanoparticles are of the core-shell structure.

13. The method according to claim 11, wherein the particle cover layer comprises a metal.

14. The method according to claim 11, wherein the particle cover layers of all the nanoparticles are produced from the same material, to which at least one of active substance molecules and ligand molecules can bind.

15. The method according to claim 5, wherein the nanoparticles in each case contain iridium, platinum, gold, bismuth, silver, iodine, palladium, cadmium or indium.

16. The method according to claim 5, wherein the nanoparticles in each case contain different X-ray contrast agent molecules.

17. The method according to claim 5, wherein each type of nanoparticles is of a different nanoparticle size.

18. The method according to claim 5, wherein the first marker substance comprises a first type of marker molecules which contain a first X-ray fluorescence element, and the at least one further marker substance comprises at least one further type of marker molecules which in each case contain at least one further X-ray fluorescence element.

19. The method according to claim 5, wherein the first marker substance comprises nanoparticles which contain a first X-ray fluorescence element, and the at least one further marker substance comprises marker molecules which in each case contain at least one further X-ray fluorescence element.

20. The method according to claim 18, wherein the marker molecules are bound to at least one of active substance and ligand molecules, the marker molecules in each case comprising one of the first and at least one further X-ray fluorescence element.

21. The method according to claim 18, wherein the X-ray fluorescence elements comprise silver, indium, palladium, cadmium, iodine or barium.

22. The method according to claim 1, wherein a time function of spatial distributions of the first marker substance and the at least one further marker substance in the sample is determined.

23. The method according to claim 1, wherein the active substances comprise biological cells, and the at least one of the first and the at least one further marker substance is coupled to the biological cells, and the determination of the distribution of the first marker substance and the at least one further marker substance comprises a detection of transport of the biological cells through the sample.

24. The method according to claim 1, wherein the first and the at least one further marker substance have in each case been introduced into the sample in different ways.

25. The method according to claim 1, wherein the first and the at least one further marker substance are formed such that they have the same effect for the sample, without coupled active substance molecules or ligand molecules.

26. An imaging apparatus which is configured for multi-parametric X-ray fluorescence imaging for investigating a sample by use of the method according to claim 1, wherein the sample comprises at least a part of a body of a biological organism and contains a first marker substance and at least one further marker substance, wherein at least one of the first and the at least one further marker substance is coupled to at least one of active substance molecules and ligand molecules which are provided for a specific interaction with the sample, comprising: an X-ray radiation source device which is arranged for irradiation of the sample with X-ray radiation, wherein X-ray fluorescence of the first marker substance and of the at least one further marker substance is excited, and fluorescence lines of the X-ray fluorescence of the first and the at least one further marker substance are different, a detector device which is configured for spatially and spectrally resolved detection of the X-ray fluorescence of the first marker substance and the at least one further marker substance, and an evaluation device which is configured for determination of a spatial distribution of the first marker substance and in addition a spatial distribution of the at least one further marker substance in the sample, from the detected X-ray fluorescence.

27. A marker substance kit which is configured for introducing marker substances into a sample for multi-parametric X-ray fluorescence imaging on the sample, comprising a first marker substance which emits X-ray fluorescence upon irradiation with X-ray radiation, and at least one further marker substance which emits X-ray fluorescence upon irradiation with X-ray radiation, wherein fluorescence lines of the first and the at least one further marker substance are different, and the first and the at least one further marker substance exhibit fluorescence probabilities, attenuations of the X-ray fluorescence in the sample, and background noise levels on account of scattering in the sample which are the equal or are similar to such an extent that the detection of the X-ray fluorescence at equal concentration of the marker substances results in comparable statistical significance levels, which can be maximized by use of a selection of the irradiation energy.

28. The method according to claim 19, wherein the marker molecules are bound to at least one of active substance and ligand molecules, the marker molecules in each case comprising one of the first and at least one further X-ray fluorescence element.

Description

[0091] Further details and advantages of the invention will be described in the following, with reference to the accompanying drawings, in which:

[0092] FIG. 1: is a schematic view of an imaging apparatus for X-ray fluorescence imaging according to an embodiment of the invention;

[0093] FIG. 2: is a flow diagram of a method for X-ray fluorescence imaging according to embodiments of the invention;

[0094] FIG. 3: shows examples of marker substances which can be used in the method according to the invention for X-ray fluorescence imaging;

[0095] FIG. 4: is a schematic view of a marker substance kit according to an embodiment of the invention;

[0096] FIG. 5: shows a measured X-ray spectrum for illustrating X-ray fluorescence emissions of two different X-ray fluorescence elements in biological cells;

[0097] FIG. 6: shows a measured X-ray spectrum for illustrating X-ray fluorescence emissions of four different X-ray fluorescence elements in a sample; and

[0098] FIG. 7: shows simulated X-ray spectra for illustrating X-ray fluorescence emissions of X-ray fluorescence elements in a sample for two different irradiation energies.

[0099] Features of preferred embodiments of the invention are described in the following, with reference, by way of example, to XFI on a human subject, with particular X-ray fluorescence elements. It is emphasized that the implementation of the invention in practice is not limited to the stated examples, but rather is possible, in a corresponding manner, using other samples, such as portions of a human subject, synthetic biological objects, animal subjects, or portions thereof. Embodiments of the invention are described in the following, in particular with reference to important features of the configuration of the marker substances, the execution of the XFI, and the structure of the imaging apparatus. Further features of the imaging apparatus can be implemented for example as described in [1]. [1] is incorporated by reference into the present disclosure, with respect to the structure and the function of the imaging apparatus. Details of the functionalization of nanoparticles, the coupling of nanoparticles with active substances, the selection of ligands, the coupling of marker molecules with active substances, the spectrally and spatially resolved detection of X-ray fluorescence, and the analysis of superimposed spectra composed of a plurality of fluorescence lines are not described, insofar as they are known per se from the prior art. The concentrations of the marker substances can be selected as is known per se from conventional XFI.

[0100] FIG. 1 schematically shows features of embodiments of an imaging apparatus 100 for X-ray fluorescence imaging for the study of a sample 10, such as a human subject, which is arranged on a sample holder 101, such as a bed. FIG. 2 schematically shows the steps of the method according to the invention using the imaging apparatus 100, comprising the supply of the marker substances (S1), the irradiation of the sample with X-ray radiation (S2), the detection of the X-ray fluorescence (S3), and the determination of marker substance distributions from the detected X-ray fluorescence (S4).

[0101] In addition, FIG. 2 shows a step S0, which comprises the selection of the marker substances and the irradiation photon energy. Is it sufficient for step S0 to be performed once, and separately from the implementation of the method according to the invention, for a considered sample or group of samples having the same scattering properties, for example for small animals of a particular species and size. Alternatively, step S0 can be provided as part of each execution of the method according to the invention.

[0102] The selection is made for example under the following considerations, on the basis of the fluorescence spectra shown by way of example in FIG. 7. In detail, FIG. 7 shows a direct comparison of two simulated spectra following excitation with monochromatic X-rays, firstly for an irradiation energy of for example 85 keV, and secondly for an irradiation energy of for example 53 keV. In the first case, it would be possible for example for gold nanoparticles to be excited (K edge at approximately 81 keV) which, however, have fluorescence lines in the region of the high peak around approximately 65 keV, said peak originating from just once-scattered photons. In contrast thereto, the 53 keV spectrum exhibits a pronounced background minimum at those fluorescence lines of moderately heavy elements which are in the range of between approximately 15 and 28 keV, because, for this, incoming photons usually have to scatter 5 times or more. Although the 85 keV spectrum also exhibits a minimum in the same energy range, this is higher, and the high irradiation energy would mean smaller yields for the fluorescence of moderately heavy elements.

[0103] Thus, for a marker kit of moderately heavy elements, the lower irradiation energy is more efficient, and for heavy elements of gold the higher energy is preferred. A variation of the detector position relative to the beam direction (in this case 150°) makes it possible for the minimum in the background range to be expanded slightly more, but the decisive parameter for the level of the minimum is the irradiation energy.

[0104] In the specific example of XFI on mice, for a first marker substance iodine can be selected as the X-ray fluorescence element, and 53 keV as the irradiation photon energy (see FIG. 7). The irradiation photon energy of 53 keV is significantly above the iodine edge of 33 keV. Incoming photons, which, after multiple scattering, fall into the energy range of the X-ray fluorescence of iodine (approx. 29 keV), would thus have to perform a plurality of successive Compton scatterings, in particular approximately 5 Compton scatterings. With each further Compton scattering after the preceding scattering, the overall probability reduces, such that the background is minimized. At the same time, upon excitation at 53 keV, iodine has a sufficiently high fluorescence probability. In this example, an element that is closely adjacent to iodine in the PTE, such as indium, is provided as a further marker substance, which is excited at the same irradiation photon energy of 53 keV.

[0105] The selection of the marker substances and the irradiation photon energy is performed for example by simulation of the scattering behavior of the sample and/or test, according to the above-mentioned optimization, such that the irradiation photon energy of the X-ray radiation is at a distance from a highest absorption edge of all the marker substances in the sample, at which, at the same time, the background noise levels of the marker substances are minimal and the effective cross sections are similar.

[0106] In a further example of XFI on larger samples, for example gold and an element closely adjacent to gold in the PTE, such as platinum, would be used as the X-ray fluorescence element, and an irradiation photon energy of the X-ray radiation of for example 85 keV would be used.

[0107] Step S1 comprises for example oral delivery and/or an injection of the marker substances and can, if an intervention into a biological body is provided, be considered to be no part of the invention.

[0108] The imaging apparatus 100 comprises an X-ray radiation source device 110, which is configured for irradiating the sample 10 with X-ray radiation 1 (step S2), and emits for example a photon energy of 50 keV or 100 keV. The X-ray radiation source device 110 is preferably a compact laser-based Thomson source (X-ray radiation source which generates X-ray radiation based on the Thomson scattering of laser light at relativistic electrons), as is described for example in [8], but can also comprise a synchrotron source or in general an X-ray source, e.g. an X-ray tube, which generates X-ray radiation having a sufficiently low divergence and high intensity, in particular in the energy range above the K edge of the X-ray fluorescence elements of the marker substances, and should be as monochromatic as possible, in order for the above-described optimization of the irradiation energy to be improved.

[0109] The X-ray radiation 1 may be generated in the shape of a parallel radiation beam having a diameter which covers the entire cross section, to be investigated, of the sample 10. In this case, all the regions of the sample are irradiated simultaneously, and the marker substances present therein are excited to X-ray fluorescence 2. Alternatively, the X-ray radiation 1 can be generated as a pencil beam, in particular having a smaller diameter than the cross section of the sample, transversely to the beam direction, and can be moved (scanned) relative to the sample 10 by means of X-ray optics (not shown). In this case, the regions of the sample are irradiated (“scanned”) in succession and the marker substances present therein are excited to X-ray fluorescence 2. Since the scanning movement of a pencil beam over the sample 10 can take place within a scanning duration which is negligible compared with typical transport times of marker substances in the sample 10, a snapshot of the X-ray fluorescence 2 is also acquired effectively in the case of scanning of the X-ray radiation 1.

[0110] The imaging apparatus 100 further comprises a detector device 120 which is arranged for spectrally and spatially resolved detection of the X-ray fluorescence 2 of marker substances in the sample 10 (step S3). The detector device 120 comprises a plurality of detector elements (not shown) which in each case acquire an X-ray spectrum of the X-ray fluorescence 2. The corresponding solid angle range, which covers a predetermined geometric portion in the sample 10, can be restricted by collimators of the individual detector elements or on groups of detector elements. The detector device 120 is constructed for example as is described in [1]. A collimator may be arranged between the detector device and the sample, which collimator can reduce scattered radiation, as described in [7].

[0111] In a manner deviating from FIG. 1, the detector device 120 may be provided with just one detector element, which is movable relative to the sample 10 and is arranged for spectrally resolved detection of the X-ray fluorescence 2 of marker substances in the sample 10. The sample 10 can be sampled (scanned) by means of the movable detector element, in order to acquire a spatial distribution of the marker substances, if a collimator cuts out only certain regions of the sample in the solid angle range of the detector element. According to a further alternative, a single detector element can be arranged so as to be fixed in position relative to the sample 10, and arranged for spectrally resolved detection of the X-ray fluorescence 2 of marker substances in a particular portion of the sample 10. In this case, too, the X-ray fluorescence 2 is acquired in a manner spatially restricted to a defined part of the sample 10, e.g. an organ, if a collimator is used.

[0112] Alternatively, the marker substances can be located without collimators, by scanning of the X-ray beam 1, e.g. as is described in [7].

[0113] The sample 10 contains at least two marker substances each having different X-ray fluorescence elements, which are excited to X-ray fluorescence 2, by the X-ray radiation 1. The detector device 120 delivers output signals in the form of X-ray spectra, which are in each case associated with pre-determined portions (geometric positions) in the sample 10 and which contain a superimposition of the fluorescence lines 3 of the X-ray fluorescence elements (see schematic graph of a spectrum in FIG. 1, and example measurement in FIGS. 5 and 6).

[0114] The imaging apparatus 100 furthermore comprises an evaluation device 130 for receiving the output signals (spatially resolved X-ray spectra) of the detector device 120, and for determining spatial distributions 4 of the marker substances in the sample 10 from the detected X-ray fluorescence 2 (step S4). The evaluation device 130 comprises for example a computer device which is coupled to the detector device 120. The evaluation device 130 is configured for executing a computer program, by means of which the intensities of the fluorescence lines at the geometric positions in the sample 10, and from these the sought distributions 4 of the marker substances, are determined from the output signal of the detector device 120, preferably taking into account a previously determined background spectrum.

[0115] The distributions 4 of the marker substances (see schematic illustration in FIG. 1) are output for example as an image (map) or tabular values. Upon acquisition of a temporal distribution of the marker substances, it is possible for a sequence of moving images, e.g. a video sequence, to be output, which represents the movement of the marker substances in the sample 10 and/or an accumulation of at least ones of the marker substances in a portion of the sample 10, such as an organ.

[0116] The computer device can optionally additionally be provided as a controller of the imaging apparatus 100, in particular for controlling and/or monitoring the X-ray radiation source device 110 and/or the detector device 120.

[0117] The sample 10 contains a first and at least one further marker substance, which differ in terms of their fluorescence lines 3 and will be described in the following, by way of example, with reference to FIG. 3. FIGS. 3A to 3C show a first type of marker substances in the form of nanoparticles 11, 12, while FIGS. 3D to 3E show a second type of marker substances in the form of marker molecules 14, 15. The nanoparticles 11, 12 may be spherical in shape (as is shown by way of example), or may be of a different shape, e.g. an angular shape having a plurality of side faces, and/or a rod shape.

[0118] According to FIG. 3A, a nanoparticle 11 may be produced from a single X-ray fluorescence element, in particular may consist entirely of the X-ray fluorescence element, such as gold or platinum, and have a diameter of for example 10 nm. According to FIG. 3B, a nanoparticle 12 may have a core-shell structure comprising a particle core 13 and a particle cover layer 14. Just like the nanoparticles 11 according to FIG. 3A, the particle core 13 may be produced from a single X-ray fluorescence element, in particular may consist entirely of the X-ray fluorescence element, such as platinum. The particle cover layer 14 consists of a different material from the particle core 13, e.g. of gold or a polymer (see [5]). The particle cover layer 14 has a thickness of e.g. 2 nm. According to FIG. 3C, a nanoparticle 12 having a core-shell structure and/or a particle cover layer can be functionalized, i.e. provided on the surface thereof with ligand and/or active substance molecules. The ligand and/or active substance molecules are illustrated schematically in FIG. 3C by means of triangles, and can in particular comprise entire biological cells.

[0119] Each marker substance comprises a plurality of nanoparticles 11, 12, the substance amount of which is selected depending on the desired concentration in the sample and the desired sensitivity in the measurement of the X-ray spectra using the detector device 120. The selection of the X-ray fluorescence elements of the nanoparticles, and optionally the functionalization of the nanoparticles, are implemented taking into account the following considerations.

[0120] In the Periodic Table of the Elements (PTE), elements that are located close to one another have physically very similar properties with respect to the production probability and the attenuation of X-ray fluorescence. At a given energy of the X-ray photons radiated in, the first variable depends only on the element. Accordingly, the X-ray fluorescence elements of the nanoparticles are selected such that they are directly adjacent in the PTE or are so close together that the X-ray fluorescence of all the X-ray fluorescence elements can be measured at a comparable sensitivity. X-ray fluorescence elements in different nanoparticles comprise for example at least two of iridium, platinum, gold and bismuth, because the four heavy elements are close to one another in the PTE (Ir, Pt and gold are indeed directly adjacent). Thus, their behavior is very similar, and all four can be used simultaneously for XFI. A further expedient variant would be moderately heavy X-ray fluorescence elements from zirconium to cerium. In contrast, it would be unfavorable to form nanoparticles of two different marker substances for example such that some nanoparticles consist of gold, on the inside, and other nanoparticles consist of an iodine compound. The two elements gold and iodine are so far apart from one another in the PTE that they can emit X-ray fluorescence simultaneously only if the irradiation energy is above what is known as the gold edge—if the energy were below said edge, no gold X-ray fluorescence would be excited. However, since the iodine edge is far distant from this, the probability of an iodine fluorescence also being generated reduces significantly. In addition, there is also the problem of the background in XFI—the (multiple) Compton scattering can lead to a strong background in the X-ray spectrum in the signal region of the actual fluorescence lines (see [1] and [7]), and would be significantly higher for gold than for iodine, such that overall the two elements could not be measured at a similar level of sensitivity.

[0121] It is advantageous if the sample, in particular the body of the subject, cannot distinguish the gold and platinum nanoparticles, because both are of the same size, have the same surface (e.g. an identical polymer particle cover layer), and are of equal or very similar masses. If, however, for example only the platinum nanoparticle, comprising a gold particle cover layer thereon, is functionalized, but the gold nanoparticle remains non-functionalized, and both are introduced into the sample, measured differences in the local concentrations can be traced back to the action of the ligands, because the two nanoparticle types are otherwise indistinguishable by the body. Only if the ligands bond purposely, in the body, will the local concentration at the binding site of the platinum nanoparticles be higher than that of the non-functionalized gold nanoparticles, which thus serve as the reference concentration. These local concentration differences can advantageously be measured by means of the XFI according to the invention. For this purpose, it is particularly advantageous for the measuring sensitivities of both internal X-ray fluorescence elements of the nanoparticles to be sufficiently high, and preferably equal or very similar (possible differences with respect to the evaluation of the detector output signals are negligible).

[0122] A further variant of the use of nanoparticles is possible such that these do not contain any heavy elements on the inside, but rather lighter molecules comprising X-ray fluorescence elements, for example the two contrast agents mentioned below for computerized tomography (CT), and bear a polymer shell as the particle cover layer.

[0123] FIGS. 3E and 3D relate to variants of the invention in which active substances, such as medicament molecules, are directly connected to marker molecules 15, 16, such as smaller complexes comprising X-ray fluorescence elements, such as a triiodobenzene ring or a ring of barium atoms. Triiodobenzene and barium are advantageously available CT contrast agents, wherein the two elements, iodine and barium, are arranged close together in the PTE. The multi-parametric XFI comprising marker molecules thus uses X-ray fluorescence element complexes comprising different X-ray fluorescence elements, to which for example different medicament molecules can be bound. The marker molecules preferably have a chemically equal or very similar effect on the sample, such that they do not influence the kinetics of the medicaments in the sample, or only in a manner that is insignificant for the measurement.

[0124] Regarding the use of different CT contrast agents as marker substances, it is noted that the conventional CT methods would not allow for multi-parametric measurement, because the measuring difference in the different absorptions of the contrast agents would be far too small for these to be measurable simultaneously. An important advantage of the invention compared with CT is that the XFI is a spectroscopic method where every element produces its own characteristic lines, and is not purely an absorption method.

[0125] FIG. 4 schematically shows, by way of example, a marker substance kit 200 for introducing marker substances into a sample for X-ray fluorescence imaging according to the invention. The marker substance kit 200 comprises a container 210, such as a flexible pouch, which is filled with a marker substance suspension 220. The marker substance suspension 220 comprises a physiological fluid, such as a physiological salt solution, in which nanoparticles 11, 12 comprising different X-ray fluorescence elements are arranged. The design of the nanoparticles 11, 12, the volume of the container 210, and the concentration of the nanoparticles 11, 12 in the marker substance suspension 220 are selected depending on the specific XFI application. In order to use the marker substance kit 200, the container 210 is connected to a blood vessel of a subject, via a line and an injection needle, and the marker substance suspension 220 comprising the nanoparticles 11, 12, is conducted into the blood vessel.

[0126] Alternatively, the marker substance kit 200 according to FIG. 4 can be provided for oral ingestion. According to a further alternative, a marker substance kit can be provided in dry form, e.g. in the form of a tablet, comprising the nanoparticles 11, 12 and a physiological binder.

[0127] With test measurements by the inventors, biological cells were provided with both gold and platinum nanoparticles in predetermined concentrations, and arranged in a reagent vessel (Eppendorf vessel) having a diameter of 6 mm. The reagent vessel was then pushed into a piece of animal flesh which was of a similar size to a mouse. The sample, comprising the flesh having the inserted reagent vessel, was irradiated with monochromatic X-ray radiation by the German Electron Synchrotron (DESY) (DESY Hamburg). In the case of further test measurements, four different fluorescent elements were arranged in a reagent vessel and irradiated with X-ray radiation from DESY synchrotron. The test measurements, which were directed to the distinguishable nature and quantitative evaluability of the measured X-ray fluorescence, yielded the results shown in FIGS. 5 and 6. The corresponding measurements with spatial resolution can for example also be performed using the technique described in [1].

[0128] According to FIG. 5, the gold and platinum fluorescence lines, detected simultaneously at a high degree of sensitivity, can be clearly distinguished. The evaluation of the superimposed fluorescence lines, comprising a numerical deconvolution for determining the individual intensities of the fluorescence lines, resulted, taking account of predetermined reference or calibration data, in the concentrations of the gold and platinum nanoparticles.

[0129] FIG. 6 shows a measured X-ray spectrum in the case of the reagent vessel, in which the four elements iridium, platinum, gold and bismuth were in solution, in predetermined concentrations. It is possible to clearly identify the four elements in the spectrum. The respective concentrations, which corresponded very well with those used, could be determined from all the fluorescence lines present.

[0130] FIGS. 5 and 6 in each case also show a background spectrum, which was measured when the reagent vessels contained only water. The measurement of the background spectrum illustrates that knowledge of the background is important for the quantitative evaluation of the individual fluorescence lines in the spectrum, in order to be able to conclude the number of corresponding fluorescence photons. The background can be measured specifically upon each use, or can be determined using reference or calibration data. In particular, the background can be selected so as to be approximately the same for all marker elements, and so as to be equally minimal for all, as far as possible.

[0131] The background curve can also be taken into account when selecting the X-ray fluorescence elements. If the absorption of the fluorescence photons of an element is too strong, such that it can barely be distinguished from the background in the spectrum at the location of the fluorescence energy, this element would be unusable.

[0132] The features of the invention disclosed in the above description, the drawings, and the claims, can be of significance both individually and in combination or sub-combination for implementing the invention in the various embodiments thereof.