Method and apparatus for multiparametric x-ray fluorescence imaging - Patents.com

Abstract

A multiparametric X-ray fluorescence imaging method with maximum detection sensitivity and minimum radiation dose on a biological / living sample (10) containing a first marker substance includes the steps of irradiating the sample with X-rays (1), which excite X-ray fluorescence (2) of the first marker substance, spatially resolved detection of the X-ray fluorescence of the first marker substance, and determining the distribution of the first marker substance in the sample from the X-ray fluorescence of the first marker substance. The sample includes at least one other marker substance that is excited to X-ray fluorescence by X-rays. The fluorescence radiation (3) of the first marker substance and the at least one other marker substance is different. To enable tracking of this, at least one of the first and at least one other marker substances binds to active ingredient molecules and / or ligand molecules for specific interactions with the sample or in cells. Detection includes spectrally resolved detection of the X-ray fluorescence of the first and at least one other marker substance. The distribution of at least one of the at least one other marker substance in the sample is determined from the detected X-ray fluorescence of the first and at least one other marker substance. The imager 100 for multiparametric X-ray fluorescence imaging and the optimal selection of a marker substance kit for introducing marker substances into a sample are also described.

Description

[Technical field]

[0001] The present invention relates to a method for multiparametric X-ray fluorescence imaging on a sample, in which a marker substance distribution in the sample is detected. The present invention further relates to an imaging device configured to perform X-ray fluorescence imaging on a sample under investigation, and to a marker substance kit configured for introduction into the sample for X-ray fluorescence imaging on the sample. The present invention can be used for X-ray fluorescence imaging of a sample, in particular a biological or non-biological sample. [Background technology]

[0002] Reference is made herein to the following prior art which represents background to the present invention: [1] DE 10 2017 003 517 [2] U.S. Patent Application Publication No. 2012 / 0307962 [3] U.S. Patent Application Publication No. 2016 / 0252471 [4]T. Pellegrino et al. "Nano Lett." Vol.4, No.4, 2004, p.703-707 [5]HD Fiedler et al. "Anal Chem." 2013, 85(21):10142-8 [6]R. Zhang et al. "Am. J. Nucl. Med. Mol. Imaging" 2018, 8(3):169-188 [7]F. Gruner et al. "Sci. Rep." Vol.8, 2018, p.16561 [8]K. Khrennikov et al. "Phys. Rev. Lett." Vol.114, p.195003 (2015) [9]M. Hossain et al. "Applied Physics Letters" Vol.97, 2010, p.263704-1-263704-3

[10] DP Cormode et al. "Contrast Media & Molecular Imaging" 2014, No.9, p.37- 52

[11] Y. Li et al. "Contrast Media & Molecular Imaging" Vol.2018, 2018, Article ID 8174820, p.1-7 (with Supplementary material)

[0003] In pharmacology, it is interesting to measure the local and / or time-dependent distribution of physiologically acting substances (here also referred to as active substances or active substance molecules or pharmacologically active substances), in particular biomarkers, antibodies, antibody fragments, living cells and / or pharmaceuticals (drug molecules), in the body of a patient or test animal (pharmacokinetic measurements). Pharmacokinetic measurements aim in particular to capture the local concentration of the applied active substance in the body in a time-dependent form. The reason is that the effect of the active substance directly depends on the concentration (and the time period) at which it binds at the therapeutic action site, for example by binding to receptors on the cell surface. Conventional pharmacokinetic methods have only limited informative value. For example, if a blood sample is taken after administration of a drug to measure its concentration in the blood, the local distribution of the drug in the body is not recorded and in particular no information is given as to whether, when and in what concentration the drug has reached the desired site of action. When several active substances are applied (herein commonly referred to as multimedication), these can only be recorded individually if laborious and complex process steps are applied.

[0004] A further important application would be to measure the distribution of different immune cell types, for example to record the effect of drugs for the treatment of Crohn's disease and / or ulcerative colitis, as well as immune system mediated inflammatory diseases, where different immune cell types are usually involved, creating an unsolved problem of measuring the dynamics of different immune cell types separately, simultaneously and at the same location in the body.

[0005] Positron Emission Tomography (PET) is commonly known as a method that can record, for example, the distribution of drugs in the body. PET is based on the binding of a radioactive tracer molecule to a drug molecule. The tracer molecule emits a positron in the investigated body, which annihilates with an electron, resulting in the generation of two X-ray photons with a characteristic energy of 511 keV. The two X-ray photons are detected and by multiple simultaneous measurements the position of the emission and therefore the position of the drug can be measured or determined. However, PET has a series of drawbacks that result from the radiation exposure of the body, the complex instrumental technology and the limited informative value (Aussagekraft: informative, conclusion) in cases of multimedication.

[0006] In the case of multimedication, PET provides only limited information because only a single drug distribution can be recorded in one measurement (simultaneous pharmacokinetics of several active substances cannot be obtained). Even if several drugs are administered simultaneously, they cannot be measured separately. Since only photons with an energy of 511 keV are generated in all annihilation events, the combination of different tracer molecules with different drug molecules cannot be distinguished, and therefore the specific emission position of the drug cannot be detected. Although sequential measurements are possible with PET, where the first drug is injected and detected, and then the second drug is injected and detected, this is not practical because the diagnostic time window is very narrow due to the fast decay of the tracer molecules. Therefore, the application of PET in multimedication is practically excluded.

[0007] A more commonly known functional imaging technique is Single Photon Emission Computed Tomography (SPECT), which likewise cannot be used to detect the active agent distribution in cases of multimedication. In the case of SPECT, different tracer molecules could be used that would be distinguishable by their difference in emission energy, but the tracer molecules can only be bound to the biomolecules in very complex forms (by radiochemistry). Furthermore, the tracer molecules available for SPECT have very different emission energies and half-lives and therefore can be difficult to detect together. Also, the efficiency of the detector depends heavily on the energy of the incoming photons, and depending on the half-life the number of photons emitted respectively decreases to very different degrees. Finally, the available tracer molecules only offer a limited diagnostic time window, which would usually be too short to study the temporal distribution of the active substance. A SPECT-based solution would be considerably more complicated. This is because binding of SPECT tracer molecules to drug molecules is difficult, especially for different tracer molecules intended to be used simultaneously, and measurement of activity immediately prior to injection, which is essential for quantitative assessment, would also be difficult in this case.

[0008] The combination of PET and SPECT would not be able to overcome the limitations of PET, because if two different tracer molecules were used simultaneously, it would be possible to track two different agents with both methods, but this would only be possible in their diagnostic time window, which is rather limited by their respective half-lives. Moreover, so far, no combination or combined device is known that allows both PET and SPECT simultaneously, since the operation of the two methods is different.

[0009] X-ray fluorescence imaging (XFI) (Roentgenfluoreszenz-Bildgebung (RFB)) is another method for detecting the distribution of drugs in the body (see, for example, references [1]-[3], [9] and

[11] ). Diagnostic X-ray fluorescence imaging (diagnostische Roentgenfluoreszenz-Bildgebung) is based on the application of marker substances, e.g. nanoparticles, to the body of the subject and their spatially resolved detection by induced fluorescence in the X-ray wavelength range. If ligands are bound to the nanoparticles (functionalized nanoparticles) and the ligands are linked to or contain active substances, e.g. antibodies or antibody fragments, biological cells, biomarkers or drugs, spatially resolved measurements of the X-ray fluorescence provide information about the distribution of the active substances in the body. In XFI studies, gold nanoparticles are usually used because they are easy to synthesize and their functionalization is based on well-studied coupling chemistry. Other nanoparticles have been described in [4], [5], [9],

[10] (in the context of computer tomography) and in

[11] . The detection of different toxic metals already present in a living organism prior to the measurement by XFI based on their respective X-ray fluorescence in the organism is known from [6].

[0010] However, it is known that even non-functionalized nanoparticles accumulate at different concentrations in different organs of biological organisms in studies using XFI or invasive methods. Furthermore, nanoparticles have a highly asymmetric mass ratio compared to typical drug molecules, and therefore drug transport in the body may be dominated by nanoparticle transport. These properties of nanoparticles limit the informative value of conventional XFI methods. That is, when the concentration of drug-functionalized gold nanoparticles in a certain organ is measured by XFI, it cannot be concluded that it was determined by the drug based only on the measured concentration value. The gold nanoparticles could have reached the same concentration without the bound drug.

[0011] Therefore, in the literature [7], it is proposed to measure nanoparticles with ligands in a first organism (biological organism) and nanoparticles without ligands in a second organism (in this case a mouse) for comparison. However, this method requires high demands on the laboratory animals and ignores individual differences between individual organisms. Moreover, it is not practical for medical treatment of patients. It would therefore be interesting to carry out a comparative measurement in one organism at the same time. This comparative measurement would furthermore make it possible to detect non-specific physiological background in the target area. For example, if blood vessels and non-bound nanoparticles in the vessels are located there, this "background image" can be subtracted from the XFI image. The difference image then displays only the specifically bound nanoparticles. This comparative measurement cannot be performed using conventional techniques, and this comparative measurement would furthermore make it possible to detect non-specific physiological background in the target area. For example, if blood vessels and non-bound nanoparticles in the vessels are located there, this "background image" can be subtracted from the XFI image. The difference image then displays only the specifically bound nanoparticles.

[0012] Another drawback of regular XFI is that it is not possible to simultaneously track several different drugs or biomarkers in the case of multimedication. This also applies to the simultaneous imaging of the dynamics of different immune cells. However, this kind of measurement would be important in a series of medical studies, for example for the visualization of inflammatory processes in the body or for tumor diagnosis with several different antibodies. For example, in the case of an inflammatory process, it is intended to record different immune cell types, which arrive at the site of inflammation at different times and thus affect the progression of the inflammation. Thus, in the case of Crohn's disease, it would be interesting to simultaneously track four different immune cell types. However, it is not possible to distinguish between the different cell types by regular XFI. Pharmacokinetic measurements in the case of multimedication would be interesting to study interactions between drugs or to compare the effects of drugs. Generally, new drugs often fail after being marketed. The reason is that patients have to take several drugs at the same time, which block each other's binding sites in the body. However, this goal has not yet been achieved because current techniques do not allow for the same sensitivity in determining all immune cell types to be simultaneously measured.

[0013] Another important issue for conventional XFI is the notorious dependence of nanoparticle dynamics on their size: practical and effective methods to track the dynamics of multiple nanoparticle types of different sizes together with the same sensitivity and to directly compare them with each other are not yet available.

[0014] For the determination of pharmacokinetics in cases of multimedication, sequential measurements can also be performed with XFI, but this would require unacceptably long measurement times. Different drugs could be studied sequentially only after each one has been degraded and excreted. Therefore, with regular XFI it is not possible to measure multiparametric pharmacokinetics under practical routine conditions.

[0015] [9] describes multiplexed biomarker detection of different nanoparticle types using X-ray fluorescence. The authors of [9] confirmed or found that different nanoparticle types can be distinguished based on their different spectral fluorescence properties, allowing spatially resolved measurements of different nanoparticle types at different positions. The technique according to [9] was studied on the basis of a model system in which nanoparticles on a thin aluminum substrate are irradiated with high excitation intensity X-ray radiation. The high excitation intensity allows the generation of X-ray fluorescence with sufficiently high fluorescence intensity, which can therefore be detected with high sensitivity. By using the aluminum substrate, the interference scattered radiation (background radiation) due to multiple scattering can be largely avoided.

[0016] However, the technique according to [9] cannot be applied to biomedical imaging, because for radiation protection reasons, the intensity of the excitation X-ray radiation required is rather low, and because in living tissues rather strong scattered radiation is generated, because subjects (e.g. mice) have a rather large amount of tissue in which a lot of scattering occurs. To overcome this problem, [9] proposes to use monochromatic X-ray radiation for its excitation, but this turned out to be insufficient in practice to achieve a sufficient sensitivity of X-ray fluorescence detection, especially for subjects that are much larger than the substrates used in [9]. The reason is that due to their large volume, the scattered radiation results in a large background in the detection spectrum, and at the minimum radiation dose the weak XFI signal can no longer be detected. Also, the specific choice of marker kits is not described in [2].

[0017] Reference [2] describes a method of computed tomography XFI, which in principle allows XFI to be performed with compositions of several different nanoparticle types. However, in this case, the spectrally distinct emissions or emissions of the different nanoparticle types in the evaluation of the detected X-ray fluorescence are not used for imaging on living organisms.

[0018] A nanoparticle library for X-ray fluorescence tomography is described in

[11] . In the supplementary material of

[11] , the authors show that different nanoparticles of different elements cannot be detected with the same level of sensitivity, because the intensity of the scattered radiation varies over several orders of magnitude. Overcoming this problem by adapting the nanoparticle concentration in XFI on biological samples, for example by increasing the concentration of nanoparticles with high background by a factor of 1000, is subject to strict physiological constraints. Therefore,

[11] proposes as a selection criterion for the combination of nanoparticles that the K-edge (absorption edge) of the emitting element of the nanoparticles is as close as possible to the (average) energy of the X-ray source. However, this does not allow the background to be minimized. Therefore, in

[11] , the background in the signal region of the studied marker elements is very different (variable) and not globally minimized. Therefore, a highly sensitive XFI is not possible. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] DE 10 2017 003 517 [Patent Document 2] US Patent Application Publication No. 2012 / 0307962 [Patent Document 3] US Patent Application Publication No. 2016 / 0252471 [Non-patent literature]

[0020] [Non-Patent Document 1] T. Pellegrino et al. "Nano Lett." Vol.4, No.4, 2004, p.703-707 [Non-Patent Document 2] HD Fiedler et al. "Anal Chem." 2013, 85(21):10142-8

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Disclosure of the Invention

[0021] The object of the present invention is to realize an improved method for X-ray fluorescence imaging on a sample, whereby the drawbacks of the usual technique are avoided. Another object of the present invention is to realize an improved imaging device for X-ray fluorescence imaging for investigating a sample, whereby the drawbacks of the usual technique are avoided. Another object of the present invention is to realize an improved marker substance kit adapted to be introduced into a sample for X-ray fluorescence imaging on a sample. The present invention is intended in particular to realize a highly sensitive XFI with increased informative value, to allow the measurement of the spatial and / or temporal distribution of one or more active substances in the body of the organism to be investigated and / or to present new applications of XFI, in particular under real application conditions. In this case, in vivo imaging, which for example includes the detection of one or more active substances, in particular biomarkers, antibodies, antibody fragments, living cells, such as immune cells, and / or drugs, is of particular interest. XFI is intended in particular to enable research or testing in cases of multimedication with reduced method complexity and / or to avoid respective limitations on short diagnostic time windows.

[0022] Summary of the Invention These objects are achieved by a method for X-ray fluorescence imaging, a fluorescence X-ray imaging device and a marker substance kit, respectively, with the features of the independent claims. Preferred embodiments and applications of the invention are set out in the dependent claims.

[0023] According to a first general aspect of the invention, the above mentioned object is achieved by a method for multi-parametric X-ray fluorescence imaging of a sample, comprising the steps of: applying X-ray radiation, preferably monochromatic, to the sample under investigation; (Monochrome)A sample is irradiated with a broadband or at least narrowband X-ray radiation, exciting the X-ray fluorescence of the first marker substance. The sample is generally a shape-retaining object, preferably a body (or a body part) of a biological organism, particularly preferably a body (or a body part) of a human or animal subject. Alternatively, other non-natural objects, such as synthetic organisms, or artificial organs, such as implants or skin models, or technical objects, can also be investigated. A spatially resolved detection of the X-ray fluorescence of the first marker substance is carried out, in particular by scanning the sample. From the detected X-ray fluorescence of the first marker substance, the distribution of the first marker substance in the sample can be determined.

[0024] According to the invention, the sample contains, in addition to the first marker substance, at least one further marker substance which is excited to X-ray fluorescence by irradiation with X-rays. If the at least one further marker substance is located in the same irradiation area (beam volume of irradiation positions, scanning position) as the first marker substance, the excitation of X-ray fluorescence is preferably performed simultaneously. The marker substances are molecular or particulate substances, each containing at least one X-ray fluorescent element as a pure element or in a compound. The marker substances are foreign to the body, are intentionally supplied to the sample before performing the XFI method, and after performing the XFI method, after a substance-specific residence time, they leave the sample again and are separated, for example by a transport process. The active substance molecules and / or ligand molecules, which show a specific biological and / or chemical interaction with the sample and are transported and / or bound in a specific portion of the sample, for example by a transport process, for example metabolic or blood transport, are bound to at least one of the first and at least one further marker substance. According to a preferred variant, in particular in applications in which it is intended to monitor cells by the method according to the invention, when the active substance comprises (consists of) a living cell, for example a living cell for immune cell therapy, the bound marker substance is preferably contained within the cell or bound to its surface. The first and each further marker substance are characterized by a different X-ray fluorescence. In the measurement spectrum, the fluorescence lines of the X-ray fluorescent elements of the first and each further marker substance are different, in particular they have respective maxima at different energies and / or different spectral widths. According to the invention, the detection comprises a spectrally and spatially resolved capture of the X-ray fluorescence (X-ray fluorescence radiation) of the first marker material and each further marker material, preferably in a single measurement procedure, for example by scanning with an X-ray beam. In addition to the distribution of the first marker material, at least one distribution of at least one further marker material in the sample is determined from the detected X-ray fluorescence of the first marker material and each further marker material.

[0025] As a result, the multi-parametric method provides multiple unique distributions of the first marker substance and each of the other marker substances in the sample. The determined distributions typically do not constitute diagnostic information in themselves. The determined distributions according to the method of the present invention can be used to provide additional medical diagnoses regarding the sample.

[0026] The first marker substance and the at least one further marker substance preferably exhibit a fluorescence probability, an attenuation of the X-ray fluorescence in the sample, and a background noise level in the sample that are equal or close to each other, such that detection of the X-ray fluorescence at the same concentration of these marker substances results in a comparable statistical significance level. Furthermore, the emitted photon energy of the exciting X-rays is preferably above the absorption edge of all marker substances. The term "concentration" refers to the mass of the marker substance per (beam) area in the sample, i.e., the "area density" or "mass fraction" (Massenbelegung). The "fluorescence probability" refers to the effective cross-section (Wirkungsquerschnitt) of the individual atoms of the marker substance. The number of fluorescence photons generated by a marker substance, which gives rise to a detectable signal after the fluorescence has penetrated the sample, is determined by the product of the areal density and the effective cross-section for the fluorescence of the relevant marker substance. Advantageously, if the effective cross-sectional areas and permeabilities of the two marker materials are equal or similar, and the areal densities are equal or similar, then equal or similar detection signals of the marker materials can be obtained.

[0027] The marker substances preferably comprise nanoparticles, which in particular preferably comprise an external surface that is indistinguishable from the sample, preferably similar or isomorphous, and can only be distinguished by target functionalization, and whose interior consists of different elements, each with its characteristic "fluorescence fingerprint". These nanoparticles may be introduced exogenously into the sample, as in the case of conventional nanoparticle-based XFI (RFB).

[0028] In particular, targeted selection of multiple X-ray fluorescent elements of multiple marker materials is advantageous for multiparametric XFI. This selection preferably meets the following criteria: (a) The emitted photon energy is higher (above) all absorption edges. (b) The X-ray fluorescent elements exhibit fluorescence probabilities that are similar to each other. (c) The attenuation of the fluorescence lines of each of these X-ray fluorescent elements is also similar. (d) The fluorescence lines of those X-ray fluorescent elements should have background noise levels that are close to each other due to scattering (single and / or multiple scattering) in the sample and, together with (b) and (c), that the statistical significance of each of the detected fluorescence lines of those elements has levels that are close or similar to each other if the areal densities (concentrations) of those elements in the X-ray beam volume are the same.

[0029] For example, the ratio of the number of recorded fluorescence photons to the root (square root) of the number of background photons, or a variable that quantitatively represents this ratio, can be used as a measure of the level of statistical significance, i.e. in particular a measure of the sensitivity of X-ray fluorescence detection. The root of the number of background photons is a measure of the statistical background noise. The number of fluorescence photons and that of the background (number of photons) can be determined using available numerical methods by mathematical fit functions of the measured X-ray spectrum in the region of the fluorescence lines.

[0030] Criteria (b)-(d) are particularly important discoveries of the inventors and are particularly advantageous in implementing embodiments of the present invention. For example, if two X-ray fluorescent elements are selected in the periodic law so far apart that their fluorescence probabilities (i.e., effective cross sections), attenuation in the sample, and background noise levels are too different from each other, the two elements will be detected with significantly different sensitivities even if the areal densities of both elements in the X-ray beam volume are the same. This may result in only one type of the two marker material types being effectively detectable, and as a result, multi-parametric XFI (RFB) will not be achievable.

[0031] The effective cross sections of the atoms of the marker material are known from standard measurements and published tables, and the attenuation of the X-ray fluorescence in the sample can be determined by reference measurements and / or simulations. The background is formed by both the scattering of the incoming photons in the sample and by the detector effects. The nature of the background of the X-ray fluorescent elements can be determined by numerical simulations or measurements (see e.g. Ref. [7]), which allows an optimal selection of the X-ray fluorescent elements according to criteria (b) to (d).

[0032] The introduction of marker substances into the sample is not considered part of the present invention insofar as it requires invasive intervention with the biological material, such as injection.

[0033] According to a preferred embodiment of the invention, the statistical significance level (Signifikanz-Niveaus) of each marker material is maximized by selecting the emitted photon energy of the X-ray radiation at a distance (energy) above the highest absorption edge of the marker material in the sample, such that the background noise levels of each marker material are equal or approximately equal to each other at a minimum, and at the same time the effective cross sections and the transmission rates of the X-ray fluorescence through the sample are equal or approximately equal to each other at a maximum. It is preferable that the aforementioned variables are approximately equal to each other, if the respective differences in the background noise levels or the effective cross sections and the transmission rates have a negligible effect on the detection of the X-ray fluorescence.

[0034] The selection of the emitted photon energy of the X-ray radiation and the selection of each marker material are preferably performed by optimization, the emitted photon energy being selected to be as large (high) as possible compared to the highest absorption edge of each marker material in the sample, and selected so that the incident photon is scattered as much as possible until it enters the energy range of the X-ray fluorescence line and undergoes a corresponding energy loss (minimization of background noise due to the required multiple scattering, which becomes less likely the more scattering is required for the total energy loss from the emitted energy to the lower fluorescence energy range). At the same time, the emitted photon energy should not be selected so high that the effective cross section is excessively reduced.

[0035] The present invention realizes for the first time multiparametric X-ray fluorescence imaging on samples suitable for practical use. In [9], as mentioned above, two different nanoparticles have already been used, but these are only used in vitro on very thin substrates. In contrast, the present invention allows reliable XFI on real objects, such as subjects at least of the mouse size, due to the selection of marker substances not described in [9]. The selection of marker substances according to the invention is not described in [2] or

[11] either, and therefore the marker substances described, for example, in

[11] have very different imaging sensitivities, which excludes effective multiparametric XFI.

[0036] Unlike the above-mentioned method described in reference [7], the present invention allows comparative measurements on a single sample, since the simultaneous comparative measurements have the same imaging sensitivity as the real measurements, so that unambiguous results are obtained.

[0037] According to a second general aspect of the invention, the above mentioned object is achieved by an imaging device configured for performing multi-parametric X-ray fluorescence imaging for investigating a sample, the imaging device comprising an X-ray radiation source device, a detector device and an evaluation device. The X-ray source device is configured for irradiating the sample with X-ray radiation, whereby X-ray fluorescence of the first marker material and at least one further marker material is excited in the sample. The detector device comprises at least one spectrally resolved X-ray detector, preferably a plurality of spectrally resolved X-ray detectors, and is configured for spectrally resolved detection of the X-ray fluorescence of the first marker material and the at least one further marker material in the sample, the X-ray fluorescence of the first marker material and the at least one further marker material respectively having different fluorescence lines. The evaluation device is configured for determining (determining) respective spatial distributions of the first marker material and the at least one further marker material in the sample from the detected X-ray fluorescence radiation. The imaging device is preferably configured for performing a method according to the first general aspect of the invention or an embodiment thereof.

[0038] According to a third general aspect of the invention, the above-mentioned object is achieved by a marker substance kit configured for introduction into a sample for multi-parametric X-ray fluorescence imaging on the sample, the marker substance kit comprising at least two marker substances emitting X-ray fluorescence upon irradiation with X-ray radiation, the fluorescence radiation of the marker substances being different from each other. The marker substance kit is preferably intended for use in a method according to the first general aspect of the invention or an embodiment thereof, in particular for application to a sample. The marker substance kit can be realized in liquid or solid form. The composition of the marker substance kit (substance, concentration, grain size or particle size) and the preferred photon energy of the X-ray radiation irradiated therein can be determined by test or reference measurements and / or numerical simulations.

[0039] Spatially resolved detection of X-ray fluorescence of a first marker material and at least one further marker material in a sample includes detection of X-ray fluorescence of those marker materials only (exclusively) in at least one spatially limited region in the sample, such as a region of at least one organ and / or at least one other part of the organism, and / or spatially resolved detection of X-ray fluorescence of those marker materials throughout the sample.

[0040] The distribution of the first marker substance and at least one further marker substance each includes the assignment of a quantitative value, such as, for example, the concentration (or areal density), the absolute amount of the substance and / or the relative occurrence frequency of the different marker substances in the plurality of marker substances, in at least one spatially limited area and / or to position coordinates in the body. The distribution of the at least two marker substances results, for example, from their transport in (through) the sample by diffusion and / or by a carrier fluid, for example blood, and from biological / chemical interactions with the sample. Their quantitative values ​​can be determined directly from the amplitude of the X-ray fluorescence radiation of the marker substances, since the amplitude of the X-ray fluorescence radiation (emission, emission) of the marker substances is a measure for the number of detected X-ray fluorescent elements. The detected amplitude of the X-ray fluorescence radiation further depends on the transmission of the emitted fluorescence photons in the sample, which is known per se. It is possible to determine the spatial and / or temporal distribution of each marker substance.

[0041] The spatial distribution of each marker substance in the sample involves assigning a quantitative value of the marker substance to at least one spatially limited region and / or location coordinate within the body at a particular time.

[0042] The temporal distribution of each marker substance in the sample comprises a time-dependence of the assignment of a quantitative value of the marker substance to at least one spatially limited region and / or to a position coordinate within the body, respectively.

[0043] Thus, a distribution may include, for example, the average concentration value (and / or its function of time) in a particular organ, and / or a mapping of mass values ​​to particular position coordinates, such as, for example, a particular scan line or a particular scan plane or a particular scan volume.

[0044] According to the invention, XFI is advantageously extended so that the specific distribution of several different marker substances, e.g. several molecular marker elements or several nanoparticles each carrying several different active substance molecules (active substances) such as drugs or antibodies, or several whole living cells with or without them (active substance molecules) can be measured in vivo simultaneously with the same sensitivity and with a minimum radiation dose, which was not possible in principle with conventional XFI (RFB) or PET. In comparison with conventional XFI methods using a single marker substance, the preferably simultaneous detection of the distribution of several different marker substances ("multiparametric X-ray fluorescence imaging") provides additional information about the sample under investigation, e.g. pharmacokinetic information and / or diagnostically assessable information. Thus, for example, multiparameter pharmacokinetic measurements and multiparameter tumor diagnosis become possible as new applications of XFI. The present invention makes it possible for the first time to determine (determine, measure) the effect on the kinetics of nanoparticles that can be used, for example, as marker substances, since at least one comparative control group containing non-functionalized nanoparticles is also measured simultaneously.

[0045] For example, it will be possible for the first time to determine in vivo whether one drug is present in sufficient concentration at a particular location in a sample and whether another drug that interacts with it (e.g., has an inhibitory effect) is also present in significant concentration at the same location.

[0046] Another application is to simultaneously measure the pharmacokinetics of drugs, for example a new drug and an approved drug or an alternative or generic drug.

[0047] According to the present invention, it is possible to investigate not only the effectiveness of drugs, but also their interactions in the case of multimedication, i.e. when several drugs are administered simultaneously. By binding to different marker substances and detecting their distribution, the distribution of different drugs can be determined or measured. Thus, it is possible to detect exactly where or when different drugs with specific concentrations in a sample may interact with each other and thus interfere with each other's respective efficacy (effect). This is of considerable advantage in drug development.

[0048] Methods for applying multiparametric tumor diagnostics may be implemented, for example, in such a way that different marker substances are bound to different antibodies, respectively, in order to distinguish the subtypes of the tumor studied. This is of great advantage for the subsequent treatment, since the optimal treatment depends on the tumor subtype. Especially in cases where biopsy is not possible (e.g. tumors of the respiratory center of the brain), knowing the subtype is a decisive advantage for the subsequent treatment.

[0049] An important feature of the present invention is the application of XFI with different marker substances, which are here referred to as a first marker substance and another marker substance. The different marker substances are essentially different from each other, for example in their interiors, by different fluorescent elements (X-ray fluorescent elements), resulting in different fluorescent radiation. Another important feature is that in the non-functionalized state, i.e. without the binding of active substances, the different marker substances can be the same with regard to their interaction with the sample, whereby they have the same biological, chemical, physiological and / or physical effect, i.e. are the same with respect to the sample. In the non-functionalized state, each marker substance is indistinguishable from its surroundings (environment), especially biologically and / or chemically.

[0050] Furthermore, the fluorescent lines of each marker material are different from each other to such an extent that a detector having a finite spectral resolution (energy resolution) can distinguish each X-ray fluorescent element in the measured X-ray spectrum of the X-ray fluorescence emission of each marker material. The X-ray fluorescent elements of each marker material are preferably selected such that the K and L-α / β lines of these elements have a spectral spacing that allows the detector to distinguish these lines in the measured X-ray spectrum. Furthermore, each X-ray fluorescent element of the marker materials preferably has equal or similar fluorescence probability (effective cross section) and transmission through the sample, as well as comparable minimum background noise levels.

[0051] These characteristics are met for each of the elements preferably used, as listed below by way of example, where typically the Kα lines of two adjacent elements in the periodic table overlap, whereas for the other two elements they do not overlap and therefore appear separately in the spectrum. Since all four lines have similar energies and are therefore similarly absorbed in the sample, all four lines can be measured with the same or comparable precision.

[0052] By spectrally resolved detection of the X-ray fluorescence, an X-ray spectrum is obtained in which the fluorescence lines of the marker substances are added together. The individual quantitative contributions of the fluorescence lines can be determined from the X-ray spectrum by numerical deconvolution and / or by comparison with a predefined reference scale. The desired quantitative values ​​of the marker substances (e.g. concentrations, absolute amounts of substances and / or relative occurrences of different marker substances) are obtained from the respective quantitative contributions of the fluorescence lines. Furthermore, even if all types of marker substances are present in the X-ray beam volume, the X-ray fluorescence of all X-ray fluorescent elements can be clearly distinguished from one another in the X-ray spectrum, so that their relative occurrences can be obtained or determined from the X-ray spectrum.

[0053] In practice, optimization can be performed for the particular sample under consideration by numerical simulation of the background noise and / or test measurements with X-ray radiation of different emitted photon energies.

[0054] According to a further preferred embodiment of the invention, the X-ray fluorescence radiation of the first marker substance and of the at least one further marker substance are excited with a common excitation beam (or interrogation beam) of X-ray radiation and detected simultaneously, thereby advantageously minimizing radiation exposure of the sample and shortening the duration of the treatment.

[0055] According to an alternative embodiment of the invention, the X-ray fluorescence radiation of the first marker material and at least one further marker material are excited and detected, respectively, simultaneously or sequentially, with different excitation beams of X-ray radiation having different energies (emission photon energies). In this embodiment, the X-ray radiation source device is configured to generate multiple excitation beams of X-ray radiation, for example by multiple sources directed at the sample being operated simultaneously or sequentially (for example in direct succession) with different energies. Advantages can be obtained by adapting the energy of each excitation beam to the absorption of the X-ray fluorescent element of each marker material respectively. Simultaneous excitation and detection is advantageous in terms of minimizing the duration of the process or method. Sequential excitation and detection means that different marker materials are excited and the associated fluorescence is detected stepwise, preferably in direct succession. In this variant, there is the advantage of directly detecting each marker material from its respective X-ray spectrum recorded in succession.

[0056] Another advantage of the present invention is that different types of marker substances including nanoparticles (or target particles) and marker molecules are available. The first marker substance and each of the other marker substances each contain a plurality of nanoparticles and / or a plurality of marker molecules. Nanoparticles are particles with typical dimensions ranging from 2 nm to 100 nm or more, the respective surfaces of which are suitable or purposely prepared for the binding of ligands and / or active substance molecules. Marker molecules are single molecules or molecular aggregates containing an X-ray fluorescent element and suitable for the binding of active substance molecules. It is possible that all the parts of a marker substance with a particular X-ray fluorescent element consist only of nanoparticles or only of marker molecules, or that a marker substance consists of nanoparticles and marker molecules, both containing the same or different X-ray fluorescent elements.

[0057] The marker substances in the form of nanoparticles are particularly advantageous for binding the active substances. The ligands to which the active substance molecules can be bound or which themselves are used as active substances are bound on the surface of the nanoparticles. The active substance molecules are located on the surface of the nanoparticles. Optionally, the active substance molecules can also be located inside the particles. In this case, the nanoparticles have the advantage that they form an active substance carrier, as in conventional drug carrier technology. Thus, to realize different marker substances (so that it is possible to study where in the body each ligand docks), it is possible to use nanoparticles in which different ligand molecules are bound on the surface and at the same time the same or other active substance molecules are located inside. However, in most applications, the ligand molecules on the particle surface simultaneously form the active substance.

[0058] Each marker substance, which is made up of distinct nanoparticles, may, for example, include a first plurality of nanoparticles (or group or type of nanoparticles) that are not functionalized and at least one other plurality of nanoparticles, each of which has bound thereto at least one predetermined drug under investigation. Multiparametric XFI using the aforementioned marker substances provides the local concentrations of non-functionalized nanoparticles and functionalized nanoparticles in one measurement, either simultaneously or sequentially, the difference between the two concentrations being attributable to the drug to which the functionalized nanoparticles are bound, since the two types of nanoparticles preferably differ from each other only in the active substance or ligand molecules for the sample. In another variant, a third type of nanoparticles can be functionalized with a second drug. This variant of multiparametric XFI allows the basic distribution of non-functionalized nanoparticles to be subtracted from the measured concentration of functionalized nanoparticles and also allows the two different drugs to be differentiated in the process. It is also possible to realize the corresponding method (procedure) using a marker substance containing a marker molecule instead of nanoparticles.

[0059] The nanoparticles are particles consisting of only one X-ray fluorescent element (optionally in a chemical compound) or of a composition of one X-ray fluorescent element (optionally in a chemical compound) with at least one other element. Thus, according to a variant of the invention, each type of nanoparticle can contain only one of the X-ray fluorescent elements, optionally in a composition with a non-fluorescent element. According to an alternative variant of the invention, the first marker substance may comprise nanoparticles of a first type containing predominantly a first X-ray fluorescent element, and the at least one further marker substance may comprise at least one further type of nanoparticles each containing predominantly at least one further X-ray fluorescent element. Thus, the nanoparticles can each contain at least two X-ray fluorescent elements, of which one X-ray fluorescent element is respectively decisive (dominant) for the fluorescence radiation to be detected. This can be advantageous in the design of the nanoparticles, for example in the case of a core-shell structure as described below.

[0060] According to another advantageous embodiment of the invention, a first type of nanoparticles can carry a first type of active agent molecule that has a chemical and / or physical interaction with the sample, while another type of nanoparticles carries a different type of active agent molecule that can have a different chemical and / or physical interaction with the sample from the first type, or no active agent molecule at all. It is particularly preferred that each type of nanoparticle carries one specific type of active agent molecule. This advantageously increases the informative value of XFI. Thus, advantageously, nanoparticles offer high flexibility in adapting to specific XFI research questions.

[0061] According to another preferred embodiment of the invention, at least one type of nanoparticle may have a core-shell structure comprising a particle core and a particle coating layer (hybrid nanoparticles). The core-shell structure advantageously allows the separation of two functions of the nanoparticles, firstly the function related to X-ray fluorescence emission and secondly the function related to interaction with the environment. Thus, the particle core can be made of X-ray fluorescent elements with the desired fluorescence line of the respective type of nanoparticle, while the particle coating layer is made of a material different from the core and forms a surface for binding ligands and / or active substance molecules to generate a predetermined biological and / or chemical interaction with the sample. The particle coating layer may be made of fluorescent or non-fluorescent elements.

[0062] According to a preferred variant of the invention, the particle coating layer is formed of a metal, in particular gold, or a non-metallic material, in particular a polymer or a liposomal or micelle material. Since the coupling chemistry of active substances with nanoparticles has been studied so far in particular with metal nanoparticles, in particular with gold nanoparticles, it is preferred that the nanoparticles with a core-shell structure have an X-ray fluorescent element in the particle core, with an atomic number as large as that of gold (so that the X-ray fluorescence is in a relatively high energy range in order to measure these photons with high sensitivity outside the sample), which is preferably coated with a metal layer, in particular a gold layer, in order to bind the active substance (ligand). The thickness of the particle coating layer is preferably, for example, less than or equal to ¼ of the particle diameter. The volume fraction of gold compared to the other X-ray fluorescent elements is thus negligible and the XFI signal appears as if only the X-ray fluorescent element of the particle core is present. Another possibility is to produce nanoparticles from various X-ray fluorescent elements and use polymers instead of metals in the particle coating layer, as described for example in reference [5]. Corresponding ligands, i.e., for example drugs or antibodies, can then be attached to the above-mentioned polymer particle coating layer. The polymer layer nanoparticles can also be combined with suitable intrinsic X-ray fluorescent elements to achieve multiparametric XFI, where all nanoparticles used have similar sensitivities (ratio of signal intensity vs. associated background statistical noise) depending on the specific application conditions (e.g., size and amount of drug to be studied).

[0063] It is particularly preferred that all nanoparticles of the different marker substances have a core-shell structure, the particle cores of the different marker substances being formed from different X-ray fluorescent elements and the particle coating layers of all marker substances being formed from the same element to which at least one of the active substance molecules and the ligand molecules can be bound. Optionally, the particles of one marker substance of the multiple marker substances can be entirely formed from one element and the particle coating layers of the remaining other marker substances can be formed from the same element.

[0064] According to a preferred design, the nanoparticles are externally indistinguishable while they contain different elements inside, preferably with the same size. Nanoparticles with a core-shell structure preferably have an external particle coating layer, preferably the same, made of the same material. They are therefore indistinguishable in the sample, in particular in the body of the biological organism being investigated, unless they are functionalized in different ways. The above-mentioned nanoparticles contain different elements inside, the X-ray fluorescence energies of which differ from one another. It is therefore possible to distinguish different nanoparticle types from one another in the measured XFI spectrum and to determine their respective concentrations at the same time, while they are indistinguishable in the sample, except for the functionalization.

[0065] According to a particularly preferred embodiment of the invention, the nanoparticles contain iridium or platinum or gold or bismuth, respectively, as X-ray fluorescent element, in particular in the particle core. As an advantage, these elements can be detected with equal sensitivity, since their X-ray fluorescence energies are similar to each other but distinguishable. As a result, it is also possible to simultaneously track up to four different drugs, (immune) cell types and / or subtype-specific (specific) antibodies, for example in a biological organism. According to an alternative variant, the nanoparticles may each contain different X-ray contrast agent molecules. As an advantage, X-ray contrast agent molecules such as iodine or barium or gadolinium are widely available in practice and well studied with regard to their absorption properties (behavior, properties). Iodine and barium are particularly preferred for imaging on small animals. Nanoparticles made of silver, palladium indium, cadmium or iodine are also advantageous for imaging on small animals.

[0066] According to a further preferred embodiment of the invention, a further degree of freedom is obtained in multiparametric XFI if the different nanoparticles, i.e. different marker substances with different X-ray fluorescent elements, are of different nanoparticle sizes. As a result, it is possible to advantageously further increase the informative value of XFI and / or to investigate the properties (behavior) of the nanoparticles in the sample. For example, it is known in practice that nanoparticles of different sizes have different dynamics. For example, a distribution of nanoparticles of up to four different sizes could be detected by X-ray fluorescence. In this case, typical sizes are selected to be in the diameter intervals 2 nm to 5 nm, 6 nm to 10 nm, 11 nm to 20 nm, and 21 nm to 50 nm. The different sizes of nanoparticles are preferably of the same surface type, and only the size and the corresponding X-ray fluorescent element depending on the size differ from each other (change).

[0067] Alternatively or additionally, nanoparticles of different marker substances may differ with respect to their delivery into the sample, for example, multiparametric XFI can be used to investigate the effect of different routes of administration, e.g., oral and intravenous administration of nanoparticles, on the distribution of marker substances within the sample.

[0068] Marker substances in the form of marker molecules are particularly advantageous for transport in (through) a sample. Marker molecules are much smaller than nanoparticles and therefore transport in the sample is more similar to molecular mass transport in samples, particularly in biological organisms. The first marker substance preferably comprises a first type of marker molecule containing a first X-ray fluorescent element, and each of the other marker substances preferably comprises a different type of marker molecule, each of which contains at least one other X-ray fluorescent element.

[0069] According to another advantageous embodiment of the invention, if the first marker substance comprises nanoparticles containing a first X-ray fluorescent element and at least one further marker substance comprises marker molecules each containing at least one further X-ray fluorescent element, advantages are achieved with respect to new applications of XFI, for example it is possible to combine several, for example four, marker substances with different nanoparticles with one or more other marker substances formed of marker molecules in the form of contrast agent molecules.

[0070] For example, it is possible to bind three different types of immune cells with three different nanoparticles and to investigate a drug to which a fluorescent marker molecule is attached. The marking of the different immune cell types with different nanoparticles is carried out, for example, by previously collecting a number of cells and then introducing them into a sample with nanoparticles attached thereto, or by using nanoparticles functionalized to respectively bind in vivo only to certain different immune cell types. The nanoparticles for the immune cells should be indistinguishable for the cells and the sample, while the drug is distinguished from the immune cells. In other words, in this case the drug does not necessarily have to be attached to the nanoparticles. This application can be particularly advantageous in the study of immune system diseases, such as Crohn's disease. The distribution of the different immune cell types and the distribution of the drug could be measured simultaneously, so that the effectiveness of the drug can be investigated in vivo. In this case the drug can change the frequency of immune cell types at the site of inflammation, with the effect that, for example, immune cell types that alleviate inflammation appear more frequently.

[0071] Another advantageous application of the present invention, in which both nanoparticles and marker molecules are used as different marker substances, is the so-called drug carrier. For example, two different marker substances can be used in different nanoparticles, one group of nanoparticles being non-functionalized and the other group of nanoparticles being functionalized with a given ligand intended to bind to a target structure in the sample. In this case, both nanoparticles may act as drug carriers, i.e. carry inside them the actual drug, which in turn has a molecular marker attached. By multiparametric XFI it is possible to determine the location and optionally the timing (time) at which the nanoparticles as drug carriers release their load. For example, if the distribution of the drug does not correspond to that of the nanoparticles, an early release would be identifiable. By comparing the distribution of non-functionalized nanoparticles with that of nanoparticles carrying a ligand, it is shown how the ligand specifically finds its target location. If both distributions were the same, the drug carrier would be determined to have reached the target location by chance (randomly) rather than being targeted, as would actually be achieved by the ligand.

[0072] The marker molecules are preferably bound to the active substance molecules, each of which comprises a first X-ray fluorescent element and at least one further X-ray fluorescent element. It is particularly preferred that the X-ray fluorescent elements comprise medium-weight elements such as zirconium to cerium, which can significantly reduce the background noise level, or heavy elements such as iridium, platinum, gold and bismuth. These two groups of elements have the advantage that their fluorescent properties are similar to each other and have already been well studied, so that they can be easily bound to drug or ligand molecules.

[0073] According to the invention, the spatial and / or temporal distribution of the marker substance can be determined (obtained, measured). In this case, in a particularly advantageous preferred embodiment of the invention, the measurement is resolved spatially and temporally and the time function of the spatial distribution of the first marker substance and at least one further marker substance in the sample is determined (obtained). This embodiment has a particularly high informative value (Aussagekraft: usefulness, informativeness, significance, meaning) with respect to the transport of the marker substance in the sample from its introduction into the sample to its binding in the sample.

[0074] According to another feature that is preferably implemented, the emitted photon energy of the X-ray radiation for exciting the marker substances and the X-ray fluorescence properties of the marker substances are preferably selected such that the emitted photon energy of the X-ray radiation is above the absorption edge of the X-ray fluorescent elements of all the marker substances and such that the fluorescence probability that the X-ray fluorescent elements of all the marker substances have, the decay of the X-ray fluorescence in the sample and the signal-to-background noise level in the sample are equal or close to each other to such an extent that detection of the X-ray fluorescence at the same concentration results in a comparable signal intensity. This feature allows the relative occurrence frequency of different marker substances to be more easily determined directly from the detected X-ray spectrum. For example, one substance of the marker substances may have a much lower concentration than the other marker substances, for example because the ligands on these nanoparticles are less likely to bind to the target structures in the sample. This behavior can be observed directly from the detected X-ray spectrum when detecting the X-ray fluorescence with comparable sensitivity. If all the marker materials have the same or similar areal density as each other, then their signals will have comparable strength.

[0075] According to a preferred application of the invention, the sample to be investigated is a human or animal subject or a part of its body. The marker substances are introduced beforehand into the subject, for example orally or by other administration or injection. The first marker substance and at least one further marker substance may each be introduced into the sample by different methods or routes. A preparatory step involving the introduction of a marker substance by injection into the body of the subject does not form part of the invention.

[0076] According to another advantageous application of the invention, the active substance comprises (consists of) biological cells, at least one of the first marker substance and the at least one further marker substance is bound to the biological cells, and the determination (measurement) of the distribution of the first marker substance and the at least one further marker substance comprises the detection of the transport of the biological cells in (through) the sample, so that, advantageously, it is possible, for example, to determine the transport of different immune cells in the sample.

[0077] Further details and advantages of the invention are explained below with reference to the drawings. [Brief description of the drawings]

[0078] [Figure 1] FIG. 1 is a schematic diagram of an imaging apparatus for X-ray fluorescence imaging according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a flow diagram of a method for X-ray fluorescence imaging according to an embodiment of the present invention. [Diagram 3] FIG. 3 shows examples of marker substances that can be used in the method for X-ray fluorescence imaging according to the invention. [Figure 4] FIG. 4 is a schematic diagram of a marker material kit according to an embodiment of the present invention. [Diagram 5] FIG. 5 shows the measured X-ray spectra showing the X-ray fluorescence emissions of two different X-ray fluorescent elements in living cells. [Figure 6] FIG. 6 shows the measured X-ray spectrum to show the X-ray fluorescence radiation of four different X-ray fluorescent elements in the sample. [Figure 7] FIG. 7 shows a simulated X-ray spectrum to show the X-ray fluorescence radiation of an X-ray fluorescent element in a sample for two different radiation (single beam) energies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] The features of preferred embodiments of the invention are described below with reference to XFI on a human subject using a specific X-ray fluorescent element as an example. It should be emphasized that the practical realization of the invention is not limited to the described examples, but is possible in a corresponding manner with other samples, for example parts of a human subject, synthetic organisms (living objects), animal subjects or parts thereof. The embodiments of the invention are described below with particular reference to the important features of the composition of the multiple marker substances, the execution of XFI and the structure of the imaging device. Other features of the imaging device can be implemented, for example, as described in document [1], which is incorporated by reference into the present disclosure with respect to the structure and function of the imaging device. Details of the functionalization of the nanoparticles, the binding of the nanoparticles to the active substances, the selection of the ligands, the binding of the marker molecules to the active substances, the spectrally and spatially resolved detection of the X-ray fluorescence and the analysis of the superimposed spectrum consisting of multiple fluorescent lines will not be described in so far as they are known per se in the prior art. The concentration of each marker substance can be selected in a manner known per se for conventional XFI.

[0080] Figure 1 shows diagrammatically the features of an embodiment of an imaging device 100 for X-ray fluorescence imaging for the investigation or inspection of a sample 10, e.g. a human subject, arranged on a sample holder 101, e.g. a bed. Figure 2 shows diagrammatically the steps of a method according to the invention using the imaging device 100, including the provision of a plurality of marker substances (S1), irradiating the sample with X-rays (S2), detecting the X-ray fluorescence (S3) and determining the marker substance distribution from the detected X-ray fluorescence (S4).

[0081] 2 further shows a step S0, which includes the selection of a number of marker materials and emitted photon energies. It is sufficient that step S0 is performed only once for a sample or group of samples under consideration having the same scattering properties, for example for small animals of a certain species and size, separate from the implementation of the method according to the invention. Alternatively, step S0 may be provided as part of each implementation of the method according to the invention.

[0082] The selection is made, for example, on the basis of the fluorescence spectrum illustrated in FIG. 7, for example, under the following considerations: In particular, FIG. 7 shows in direct comparison two simulated spectra after excitation with monochromatic (monochrome) X-rays, first with a radiation energy of, for example, 85 keV, and second with a radiation energy of, for example, 53 keV. In the first case, for example, gold nanoparticles can be excited (K-edge (absorption edge) at about 81 keV), but they have several fluorescence lines in the range of a high peak approximately around 65 keV, which originates from photons scattered only once. In contrast, the 53 keV spectrum shows a significant background minimum in the fluorescence lines of medium-weight elements in the range of about 15 to 28 keV, because in this case the incoming photons will usually be scattered more than five times. The 85 keV spectrum also shows a minimum in the same energy range, but it is higher, and the higher radiation energy would mean a lower fluorescence yield for medium-weight elements.

[0083] Therefore, for marker kits of medium weight elements lower radiation energies are more efficient, and for the heavy element gold higher energies are preferred. A change in the detector position relative to the beam direction (150° in this case) allows a slightly larger extension of the minimum in the background range, but the critical parameter for the level of the minimum is the radiation energy.

[0084] In the specific example of XFI on a mouse, iodine can be selected as the X-ray fluorescent element and 53 keV as the emitted photon energy (see FIG. 7) for the first marker material. The emitted photon energy of 53 keV is much higher than the edge of iodine at 33 keV. Thus, the incoming light that falls into the energy range of iodine's X-ray fluorescence (about 29 keV) after multiple scattering will undergo multiple successive Compton scatterings, in particular about 5 Compton scatterings. Each additional Compton scattering after the previous scattering reduces the overall probability so that the background is minimized. At the same time, when excited at 53 keV, iodine has a sufficiently high fluorescence probability. In this example, an element close to iodine in the PTE, such as indium, is prepared or provided as another marker material, which is excited with the same emitted (single beam) photon energy of 53 keV.

[0085] The choice of marker material and emitted (single beam) photon energy is determined by, for example, determining whether the emitted photon energy of X-ray radiation is at a certain distance from the highest absorption edge of all marker materials in the sample. (distant location) This is carried out by simulating the scattering properties (behaviour) of the sample and / or test according to the above mentioned optimization, where the background noise level of the marker material is minimal and the effective cross section is close.

[0086] In another example of performing XFI on a larger sample, gold and one element proximate to gold in the PTE, e.g. platinum, would be used as the x-ray fluorescent elements and an emitted photon energy of x-ray radiation, e.g. 85 keV, would be used.

[0087] Step S1, which for example involves oral administration and / or injection of a marker substance, cannot be considered as part of the present invention if an intervention into the body of a living organism is performed.

[0088] The imaging device 100 comprises an X-ray radiation source device 110 configured to irradiate the sample 10 with X-ray radiation 1 (step S2), e.g. emitting photon energy of 50 keV or 100 keV. The X-ray radiation source device 110 is preferably a compact laser-based Thomson-Quelle X-ray source (an X-ray radiation source generating X-ray radiation based on Thomson scattering of laser light with relativistic electrons), e.g. as described in document [8], but can also comprise a synchrotron source or an X-ray source in general, e.g. an X-ray tube, generating X-ray radiation with sufficiently low divergence and high intensity, especially in the energy range higher than the K-edge of the X-ray fluorescent element of the marker material, and should be as monochromatic as possible in order to improve the optimization of the radiation energy mentioned above.

[0089] The X-ray radiation 1 can be generated in the form of a parallel radiation beam with a diameter covering the entire investigated cross-section of the sample 10. In this case, all respective areas of the sample are irradiated simultaneously and the respective marker substances present therein are excited to generate X-ray fluorescence 2. Alternatively, the X-ray radiation 1 can be generated as a pencil beam transversely to the beam direction, in particular with a diameter smaller than the cross-section of the sample, and moved (scanned) relative to the sample 10 by X-ray optics (not shown). In this case, the respective areas of the sample are irradiated sequentially ("scanned") and the respective marker substances present therein are excited to generate X-ray fluorescence 2. Since the scanning movement of the pencil beam over the sample 10 can be performed within a scanning duration that is negligible compared to the typical transport times of the marker substances in the sample 10, a snapshot of the X-ray fluorescence 2 is also effectively captured in the case of a scanning of the X-ray radiation 1.

[0090] The imaging device 100 further comprises a detector arrangement 120 arranged for a spectrally and spatially resolved detection of the X-ray fluorescence 2 of the marker material in the sample 10 (step S3). The detector arrangement 120 comprises a number of detector elements (not shown) each capturing an X-ray spectrum of the X-ray fluorescence 2. The corresponding solid angle range covering a given geometrical portion in the sample 10 can be limited by a collimator of the individual detector elements or of groups of detector elements. The detector arrangement 120 is constructed, for example, as described in [1]. A collimator may be arranged between the detector arrangement and the sample, which collimator is capable of reducing scattered radiation, as described in [7].

[0091] Unlike in FIG. 1, the detector arrangement 120 may be provided with only one detector element, which is movable relative to the sample 10 and is arranged for a spectrally resolved detection of the X-ray fluorescence 2 of each marker substance in the sample 10. If a collimator cuts out only a region of the sample within the solid angle of the detector element, the sample 10 can be sampled (scanned) by the movable detector element in order to capture the spatial distribution of the marker substances. According to another option, a single detector element can be arranged in a fixed position relative to the sample 10 and arranged for a spectrally resolved detection of the X-ray fluorescence 2 of several marker substances in a specific part of the sample 10. In this case too, if a collimator is used, the X-ray fluorescence 2 is captured in a spatially limited manner in a defined part of the sample 10, for example of an organ.

[0092] Alternatively, the marker material can be located by scanning the X-ray beam 1 without using a collimator, as described, for example, in [7].

[0093] The sample 10 contains at least two marker substances each having a different X-ray fluorescent element which is excited by the X-ray radiation 1 and produces X-ray fluorescence 2. The detector device 120 provides output signals in the form of X-ray spectra, each associated with a respective predefined portion (geometrical position) in the sample 10 and comprising a superposition (superposition) of fluorescence lines 3 of the X-ray fluorescent elements (see schematic curve (graph) of the spectrum in FIG. 1 and measurement examples in FIGS. 5 and 6).

[0094] The imaging arrangement 100 further comprises an evaluation arrangement 130 for receiving the output signal of the detector arrangement 120 (spatially resolved X-ray spectrum) and for determining (step S4) the spatial distribution 4 of each marker substance in the sample 10 from the detected X-ray fluorescence 2. The evaluation arrangement 130 comprises, for example, a computer arrangement coupled to the detector arrangement 120. The evaluation arrangement 130 is configured to execute a computer program, whereby from the output signal of the detector arrangement 120, preferably taking into account a previously determined background spectrum, the intensities of the fluorescence rays at each geometrical position in the sample 10 and, from these (intensities), the respective determined distributions 4 of each marker substance are determined.

[0095] Each distribution 4 of each marker substance (see schematic diagram in FIG. 1) is output, for example as an image (map) or as tabular values. Once the temporal distribution of each marker substance has been captured, it is possible to output a series of moving images, e.g. a video sequence, which represents the movement of each marker substance within the sample 10 and / or the accumulation of at least some of the marker substances in a part of the sample 10, e.g. an organ.

[0096] A computing device may optionally additionally be provided as a controller of the imaging arrangement 100 , in particular for controlling and / or monitoring the X-ray radiation source arrangement 110 and / or the detector arrangement 120 .

[0097] The sample 10 contains a first marker substance and at least one further marker substance, the respective fluorescent lines 3 of which are different from each other, and is described below by way of example with reference to Figure 3. Figures 3A to 3C show a first type of marker substance in the form of nanoparticles 11, 12, while Figures 3D and 3E show the marker molecules 1 5, 16 of 1 shows a second type of marker material in the form: the nanoparticles 11, 12 may be spherical in shape (as shown by way of example) or may have different shapes, for example multi-sided angular (polyhedral) and / or rod-like shapes.

[0098] According to FIG. 3A, the nanoparticle 11 may be made of a single X-ray fluorescent element, in particular may consist entirely of an X-ray fluorescent element, for example gold or platinum, and may have a diameter of, for example, 10 nm. According to FIG. 3B, the nanoparticle 12 may have a core-shell structure, comprising a particle core 13 and a particle coating layer 14. As with the nanoparticle 11 according to FIG. 3A, the particle core 13 may be made of a single X-ray fluorescent element, in particular may consist entirely of an X-ray fluorescent element, for example platinum. The particle coating layer 14 consists of a material different from the particle core 13, for example gold or a polymer (see document [5]). The particle coating layer 14 has a thickness of, for example, 2 nm. According to FIG. 3C, the nanoparticle 12 with a core-shell structure and / or the particle coating layer may be functionalized, i.e. provided with ligands and / or active substance molecules on its surface. The ligands and / or active substance molecules are shown diagrammatically in FIG. 3C as triangles and in particular comprise a plurality of biological (organism) cells entirely.

[0099] Each marker material comprises a number of nanoparticles 11, 12, the amount of which is selected according to the desired concentration in the sample and the desired sensitivity in measuring the X-ray spectrum with the detector device 120. The selection of the X-ray fluorescent element of the number of nanoparticles, and optionally the functionalization of each nanoparticle, is realized taking into account the following considerations:

[0100] Elements that are close to each other in the Periodic Table of Elements (PTE) have very similar physical properties in terms of the probability of generation and attenuation of X-ray fluorescence. At a given energy of the emitted X-ray photon, the first variable depends only on that one element. Therefore, the X-ray fluorescent elements of the nanoparticles are selected to be directly adjacent in the PTE or close enough together that the X-ray fluorescence of all the X-ray fluorescent elements can be measured with equal sensitivity. X-ray fluorescent elements in different nanoparticles include, for example, at least two of iridium, platinum, gold and bismuth, because these four heavy elements are close to each other in the PTE (Ir, Pt and gold are indeed directly adjacent). Therefore, their properties are very similar and all four can be used simultaneously for XFI. Another advantageous variant would be the medium-weight X-ray fluorescent elements from zirconium to cerium. In contrast, it would not be desirable to form nanoparticles of two different marker materials, for example some nanoparticles made of gold on the inside and others made of iodine compounds. The two elements gold and iodine are separated from each other in the PTE in such a way that they can only emit X-ray fluorescence simultaneously if the radiation energy is higher than the so-called gold edge (absorption edge), whereas if the radiation energy is lower than the gold edge, the gold X-ray fluorescence will not be excited. However, since the iodine edge is far away from here, the probability of iodine fluorescence occurring is significantly reduced. Furthermore, there is also the problem of background in XFI, i.e. (multiple) Compton scattering causes a strong background in the X-ray spectrum in the signal region of the actual fluorescence line (see references [1], [7]), which is much higher for gold than for iodine, so that overall the two elements cannot be measured with similar sensitivity levels.

[0101] Since both gold and platinum nanoparticles are the same size, have the same surface (e.g. the same polymer particle coating layer) and are equal or very similar in mass to each other, it is advantageous if the sample, and in particular the body of the subject (subject), cannot distinguish between gold and platinum nanoparticles. However, if both are introduced into a sample, for example only the platinum nanoparticles with a coating layer of gold particles thereon are functionalized, while the gold nanoparticles remain unfunctionalized, the difference in the measured local concentrations can be attributed to the action of the ligand, since otherwise the two nanoparticle types cannot be distinguished by (by) the body. Only if the ligand forms a specific or intended bond in the body will the local concentration at the binding site of the platinum nanoparticle be higher than that (local concentration) of the non-functionalized gold nanoparticle used as the reference concentration. These local concentration differences can be advantageously measured by XFI according to the invention. For this, it is particularly advantageous that the measurement sensitivity of both internal X-ray fluorescent elements of the nanoparticles is both sufficiently high and preferably equal or very similar to each other (possible differences in terms of the evaluation of the detector output signal can be negligible).

[0102] Another variation is possible using nanoparticles that do not contain heavy elements inside but contain lighter molecules that contain X-ray fluorescent elements, such as the two contrast agents for CT (Computerised Tomography) described below, and that carry a polymer shell as a particle coating layer.

[0103] 3E and 3D relate to a variant of the invention in which an active substance, e.g. a drug molecule, is directly linked to a marker molecule 15, 16, e.g. a smaller complex containing an X-ray fluorescent element, e.g. a triiodbenzolring or a ring of barium atoms. Triiodbenzolring and barium are readily available CT contrast agents, and the two elements iodine and barium are located close to each other in the PTE. Thus, multiparametric XFI with marker molecules uses X-ray fluorescent element complexes containing different X-ray fluorescent elements to which, e.g., drug molecules of different phases can be attached. The marker molecules preferably have a chemically equivalent or very similar effect on the sample in a form that does not affect the kinetics of the drug in the sample or has a negligible effect on the measurement.

[0104] Regarding the use of various different CT contrast agents as marker substances, it is pointed out that conventional CT methods would not allow multiparametric measurements since the measurement differences of the different absorptions of the contrast agents would be far too small to measure them simultaneously. An important advantage of the present invention compared to CT is that XFI is a spectroscopic method in which every element produces its own characteristic line, and not a purely absorption method.

[0105] FIG. 4 shows, as an example, a schematic representation of a marker substance kit 200 for introducing a marker substance into a sample for X-ray fluorescence imaging according to the present invention. The marker substance kit 200 comprises a container 210, e.g. a flexible pouch, filled with a marker substance suspension 220. The marker substance suspension 220 comprises a physiological liquid, e.g. saline, in which nanoparticles 11, 12 containing different X-ray fluorescent elements are placed. 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. To use the marker substance kit 200, the container 210 is connected to a blood vessel of a subject via a line and a syringe needle, and the marker substance suspension 220 containing the nanoparticles 11, 12 is introduced into the blood vessel.

[0106] Alternatively, the marker substance kit 200 according to Fig. 4 can be provided for oral ingestion. According to another alternative, the marker substance kit can be provided in dry form, for example in the form of a tablet, comprising the nanoparticles 11, 12 and a physiological binder.

[0107] In the test measurements made by the inventors, living cells were fed with gold and platinum nanoparticles at the respective defined concentrations and placed in a reagent vessel (Eppendorf vessel) with a diameter of 6 mm. The reagent vessel was then pushed into a piece of meat from an animal of the same size as a mouse. The specimen, including the piece of meat in which the reagent vessel was inserted, was irradiated with monochromatic X-ray radiation by the Deutsche Elektronen Synchrotron (DESY) (DESY Hamburg). For other test measurements, four different fluorescent elements were placed in the reagent vessel and irradiated with X-ray radiation at the DESY synchrotron. Test measurements aimed at the distinguishability and quantitative assessment of the measured X-ray fluorescence were carried out, leading to the results shown in figures 5 and 6. Corresponding measurements of the spatial resolution can also be made, for example, using the techniques described in reference [1].

[0108] According to Figure 5, the fluorescence lines of gold and platinum, detected simultaneously with high sensitivity, can be clearly distinguished. Evaluation of the superimposed fluorescence lines, including numerical deconvolution to determine the individual intensities of the fluorescence lines, resulted in the respective concentrations of gold and platinum nanoparticles, taking into account predefined reference or calibration data.

[0109] Figure 6 shows the X-ray spectrum of four elements, iridium, platinum, gold and bismuth, dissolved in a reagent vessel at the indicated concentrations. From this spectrum, the four elements can be clearly identified. Their concentrations can be determined from all the fluorescent lines present and correspond very well to the concentrations used.

[0110] Figures 5 and 6 also show the background spectrum measured with only water in each reagent vessel. The measurement of the background spectrum shows 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 estimate the number of corresponding fluorescence photons. The background can be measured specifically for each use or can be determined using standards or calibration data. In particular, the background can be chosen to be approximately the same for all marker elements and to have as many equal minima as possible for all.

[0111] The background curve can also be considered when selecting an X-ray fluorescent element: if the absorption of the fluorescence photons of an element is so strong that it is barely distinguishable from the background in the spectrum at the location of the fluorescence energy, the element will not be usable.

[0112] The features of the invention disclosed in the foregoing description, in the drawings and in the claims can be of importance both individually and in any combination or subcombination for realizing the invention in its various embodiments.

Claims

1. A method for multi-parametric X-ray fluorescence imaging on a sample (10) comprising at least a part of a body of a biological organism and comprising a first marker substance and at least one further marker substance, comprising: said first marker substance and at least one of said at least one further marker substance are bound to active substance molecules and / or ligand molecules provided for specific interaction with said sample (10); - irradiating said sample (10) with monochromatic or narrowband X-ray radiation (1), which excites X-ray fluorescence (2) of said first marker material and of said at least one further marker material, the respective fluorescence lines (3) of said X-ray fluorescence (2) of said first marker material and of said at least one further marker material being different from one another; a spatially resolved detection of said X-ray fluorescence (2), comprising a spectrally resolved detection of said X-ray fluorescence (2) of said first marker material and of said at least one further marker material; - determining from the detected X-ray fluorescence (2) the distribution of said first marker material in said sample (10) and also at least one distribution of said at least one further marker material; Including, - the first marker substance and the at least one further marker substance exhibit comparable or similar fluorescence probability, attenuation of the X-ray fluorescence (2) in the sample (10) and background noise level due to scattering in the sample (10) such that detection of the X-ray fluorescence (2) at the same concentrations of the marker substances results in a comparable level of statistical significance; the emitted photon energy of the X-ray radiation (1) is higher than the absorption edges of all the marker substances, the emitted photon energy of the X-ray radiation (1) being located away from the highest absorption edges of all the marker substances in the sample (10), at which simultaneously the background noise level of the marker substances is minimal and the fluorescence probabilities are close to each other; method.

2. 2. The method of claim 1, wherein the statistical significance level of the marker material is maximized by selecting the emitted photon energy of the X-ray radiation (1) to be above the highest absorption edge of the marker material in the sample, such that the background noise levels of the marker material are both minimal and equal or approximately equal, and at the same time the fluorescence probability and the transmittance through the sample are both equal or approximately equal and maximum.

3. 3. The method according to claim 1 or 2, wherein the X-ray fluorescence (2) of the first marker material and the at least one further marker material is excited and detected simultaneously using a common excitation beam of the X-ray radiation (1).

4. 3. The method according to claim 1 or 2, wherein the X-ray fluorescence (2) of the first marker material and the at least one further marker material are excited with different excitation beams of the X-ray radiation (1) exhibiting different energies and are detected simultaneously or sequentially.

5. The method according to any of the preceding claims, wherein the first marker substance and the at least one further marker substance comprise nanoparticles (11, 12) and marker molecules (15, 16), respectively.

6. - said first marker substance or said at least one further marker substance each comprise nanoparticles (11, 12); the nanoparticles (11, 12) of the first marker material and of the at least one further marker material comprise a surface which is indistinguishable with respect to the sample (10), the nanoparticles (11, 12) comprising different elements in their interiors; The method according to claim 5.

7. - said first marker material comprises a first group of nanoparticles (11) containing mainly a first X-ray fluorescent element; said at least one further marker substance comprises at least one further group of nanoparticles (12) each mainly comprising at least one further X-ray fluorescent element; The method according to claim 5 or 6.

8. 8. The method of claim 7, wherein each group of nanoparticles (11, 12) contains only one element from among the first X-ray fluorescent element and the at least one further X-ray fluorescent element.

9. said first group of nanoparticles (11) carrying a first group of active substance molecules and / or ligand molecules provided for chemical and / or physical interaction with said sample (10); said at least one further group of nanoparticles (12) each carrying a distinct group of active substance molecules and / or ligand molecules or no active substance molecules or ligand molecules provided for chemical and / or physical interaction with said sample (10); A method according to any one of claims 5 to 8.

10. 10. The method according to claim 9, wherein each group of nanoparticles (11, 12) carries only one particular group of active substance molecules and / or ligand molecules.

11. The method according to any one of claims 5 to 10, wherein the first group of nanoparticles and at least one group of nanoparticles among the at least one further group of nanoparticles (11, 12) have a core-shell structure comprising a particle core (13) and a particle coating layer (14).

12. The method according to claim 11, wherein all said nanoparticles (11, 12) have a core-shell structure.

13. The method of claim 11 or 12, wherein the particulate coating layer (14) comprises a metallic or non-metallic material.

14. The method of claim 13, wherein the metal is gold and the non-metallic material is a polymer or a liposome material or a micelle material.

15. 13. The method according to claim 11 or 12, wherein the particle coating layer (14) of all the nanoparticles (11, 12) is made of the same material to which active substance molecules and / or ligand molecules can be bound.

16. The method according to any of claims 5 to 15, wherein the nanoparticles (11, 12) each comprise iridium, platinum, gold, bismuth, silver, iodine, palladium, cadmium or indium.

17. 17. The method according to any one of claims 5 to 16, wherein the nanoparticles (11, 12) each contain different X-ray contrast agent molecules.

18. 18. The method according to any of claims 5 to 17, wherein the nanoparticles (11, 12) of each group have a different nanoparticle size.

19. said first marker material comprises a first group of marker molecules (15) containing a first X-ray fluorescent element; said at least one further marker substance comprises at least one further group of marker molecules (16) each comprising at least one further X-ray fluorescent element; 19. The method according to any one of claims 5 to 18.

20. said first marker material comprises nanoparticles (11) containing a first X-ray fluorescent element; said at least one further marker substance comprises marker molecules (15) each comprising at least one further X-ray fluorescent element; 19. The method according to any one of claims 5 to 18.

21. 21. The method according to claim 19 or 20, wherein the marker molecules (15, 16) are bound to active substances and / or ligand molecules, the marker molecules (15, 16) each comprising one of the first X-ray fluorescent element and the at least one further X-ray fluorescent element.

22. 22. The method of any one of claims 19 to 21, wherein the X-ray fluorescent element comprises silver, indium, palladium, cadmium, iodine or barium.

23. 23. The method of any of the preceding claims, wherein a spatial distribution of the first marker substance and the at least one further marker substance in the sample (10) as a function of time is determined.

24. - said active substance comprises a biological cell, and at least one marker substance among said first marker substance and said at least one further marker substance is bound to said biological cell, - determining the distribution of the first marker substance and the at least one further marker substance comprises detecting the transport of the biological cells in the sample; 24. A method according to any one of claims 1 to 23.

25. 25. The method of any one of claims 1 to 24, wherein the first marker substance and the at least one further marker substance are introduced into the sample (10) by different methods or routes.

26. 26. The method of any one of claims 1 to 25, wherein the first marker substance and the at least one further marker substance are configured to have the same biological, chemical, physiological and / or physical effect on the sample when binding of active substance molecules and / or ligand molecules is not taken into account.

27. 27. An imaging device (100) configured for performing multi-parametric X-ray fluorescence imaging for investigating a sample (10) according to a method according to any one of claims 1 to 26, comprising: The sample comprises at least a portion of a body of a biological organism and comprises a first marker substance and at least one further marker substance; said first marker substance and at least one of said at least one further marker substance are bound to active substance molecules and / or ligand molecules provided for specific interaction with said sample (10); an X-ray source device (110) arranged to irradiate the sample (10) with monochromatic or narrowband X-ray radiation (1), in which X-ray fluorescence (2) of the first marker material and of the at least one further marker material is excited, the fluorescence lines (3) of the X-ray fluorescence (2) of the first marker material and of the at least one further marker material are different from one another, the emitted photon energy of the X-ray radiation (1) being located at a position away from the highest absorption edges of all the marker materials in the sample (10), at which simultaneously the background noise level of the marker materials is minimal and the fluorescence probabilities are close to one another; a detector device (120) configured for spatially and spectrally resolved detection of said X-ray fluorescence (2) of said first marker material and of said at least one further marker material; an evaluation device (130) configured to determine, from the detected X-ray fluorescence (2), the spatial distribution of the first marker substance and also of the at least one further marker substance in the sample (10); 1. An imaging device (100) comprising:

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