Method, device and marker substance kit for multi-parameter x-ray fluorescence imaging

Abstract

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

Description

Technical Field

[0001] This invention relates to a method for multi-parameter X-ray fluorescence imaging of samples, wherein the distribution of labeled substances in the samples is detected. The invention also relates to an imaging apparatus configured for X-ray fluorescence imaging of samples under study, and to a kit of labeled substances configured for introduction into a sample for X-ray fluorescence imaging. This invention can be used for X-ray fluorescence imaging of samples, particularly biological or non-biological samples. Background Technology

[0002] In this specification, reference is made to the following prior art documents that represent the technical background of the present invention:

[0003] [1]DE 10 2017 003 517;

[0004] [2]US 2012 / 0307962 A1;

[0005] [3]US 2016 / 0252471 A1;

[0006] [4] T. Pellegrino et al., “Nano Lett.”, Vol.4, No.4, 2004, pp.703-707;

[0007] [5] HDFiedler et al., “Anal Chem”, 2013, 85(21):10142-8;

[0008] [6] R. Zhang et al., “Am.J.Nucl.Med.Mol.Imaging”, 2018, 8(3):169-188;

[0009] [7] F. Grüner et al., “Sci.Rep.”, Vol.8, 2018, p.16561;

[0010] [8] K. Khrennikov et al., “Phys. Rev. Lett.”, Vol. 114, p. 195003 (2015);

[0011] [9] M. Hossain et al., “Applied Physics Letters”, Vol. 97, 2010, p. 263704-1-263704-3;

[0012]

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

[0013]

[11] Y. Li et al., “Contrast Media & Molecular Imaging”, Vol. 2018, 2018, Article ID 8174820, pp. 1-7 (with supplementary materials).

[0014] In pharmacology, it is meaningful to measure the local and / or temporal distribution (pharmacokinetic measurement) of physiologically active substances (here also referred to as active substances or active substance molecules or pharmacologically active substances), particularly biomarkers, antibodies, antibody fragments, biological cells, and / or drugs (drug molecules) in a patient or test animal. Pharmacokinetic measurements are specifically designed to obtain the local concentration of the applied active substance in the body in a time-dependent manner, because the effectiveness of the active substance depends directly on its concentration (and duration) bound at the site of action, for example, through coupling with receptors on the cell surface. Conventional pharmacokinetic methods have limited conclusiveness. For example, if a blood sample is collected after drug administration to measure the drug concentration in the blood, the local distribution of the drug in the body is not obtained, particularly information about whether, when, and at what concentration the drug reaches the desired site of action. If multiple active substances are applied, here often referred to as multiple drugs, this information can only be obtained individually if laborious methodological steps are applied.

[0015] A further important application will be measuring the distribution of different immune cell types, for example, to record the effectiveness of drugs for treating Crohn's disease and / or ulcerative colitis, as well as further immune system-mediated inflammatory diseases. Because multiple different immune cell types are often related in this context, a challenge that has remained unresolved to date arises: measuring the dynamics of different immune cell types in the body, separately but simultaneously and in the same location.

[0016] Positron emission tomography (PET) is a well-known method for recording, for example, the distribution of drugs within the body. PET is based on a radioactive tracer molecule bound to a drug molecule. The tracer molecule emits a positron in the body under study; this positron annihilates with an electron, resulting in two X-ray photons with a characteristic energy of 511 keV. These two X-ray photons are detected, and the emission location can be determined through multiple coincidence measurements, thus pinpointing the location of the drug. However, PET has a number of drawbacks, including radiation exposure to the body, complex instrumentation techniques, and limited conclusive evidence in the case of multiple drugs.

[0017] In the case of multiple drugs, PET provides limited information because a single measurement can only record the distribution of a single drug (it cannot simultaneously record the pharmacokinetics of multiple active substances). Even when multiple drugs are used simultaneously, they cannot be measured individually. Since only photons with an energy of 511 keV are generated in all annihilation events, even different tracer molecules bound to different drug molecules cannot be distinguished, and therefore no drug-specific emission sites can be detected. Although PET offers the possibility of sequential measurements, allowing for the injection and detection of the first drug followed by the injection and detection of the second, this is impractical because the diagnostic time window is very limited in the case of PET due to the rapid decay of tracer molecules. Therefore, the practical application of PET in the case of multiple drugs is ruled out.

[0018] Another commonly known functional imaging method is single-photon emission computed tomography (SPECT). However, it also cannot be used to detect the distribution of active agents in the case of multiple drugs. Although different tracer molecules can be used in the case of SPECT, and these different tracer molecules can be distinguished by their different emission energies, the tracer molecules can only bind to biomolecules in a very complex manner (by means of radiochemistry). Furthermore, the tracer molecules available for SPECT have very different emission energies and half-lives, making simultaneous detection difficult—the detector efficiency depends heavily on the energy of the incident photons, and the number of emitted photons decreases accordingly depending on the half-life. Finally, the available tracer molecules provide only a limited diagnostic time window, which is often too short for studying the temporal distribution of active substances. SPECT-based solutions would be significantly more complex because the binding of SPECT tracer molecules to drug molecules is difficult, especially when different tracer molecules are intended to be used simultaneously. In such cases, determining activity directly before injection—essential for quantitative analysis—is also challenging.

[0019] Even the combination of PET and SPECT cannot overcome the limitations of PET. While both methods can track two different drugs when using two different tracer molecules simultaneously, this is only possible within their respective diagnostic time windows, which are significantly limited by their corresponding half-lives. Furthermore, no known combined device to date allows for both PET and SPECT simultaneously because the two methods operate differently.

[0020] X-ray fluorescence imaging (XFI) is another method for detecting the distribution of drugs in vivo (see, for example, [1]-[3], [9] and

[11] ). Diagnostic X-ray fluorescence imaging is based on applying a labeled substance, including, for example, multiple nanoparticles, to the body under study and performing detection in a spatially resolved manner by means of induced fluorescence in the X-ray wavelength range. If a ligand binds to a nanoparticle (functionalized nanoparticle), and the ligand is attached to an active substance or they themselves include an active substance, such as an antibody or antibody fragment, biological cell, biomarker, or drug, information relating to the distribution of the active substance in vivo is obtained by means of spatially resolved measurements of X-ray fluorescence. Gold nanoparticles are commonly used in XFI studies because they are readily synthesized and their functionalization is based on well-studied coupling chemistry. More nanoparticles are described in [4], [5], [9],

[10] (related to computed tomography) and

[11] . As can be seen from [6], XFI can be used to detect a variety of different toxic metals that are already present in the organism before measurement, due to their corresponding X-ray fluorescence in the organism.

[0021] However, studies using XFI or invasive methods have shown that even non-functionalized nanoparticles can aggregate at varying concentrations in different organs of biological organisms. Furthermore, nanoparticles exhibit a strongly asymmetric mass ratio compared to typical drug molecules, meaning drug delivery in vivo can be nanoparticle-dominated. These properties of nanoparticles limit the conclusiveness of conventional XFI methods: if XFI is used to measure the concentration of gold nanoparticles in a specific organ, and the gold nanoparticles are drug-functionalized, it cannot be assumed that the concentration is determined by the drug solely based on the measured concentration value. It is possible for gold nanoparticles to reach the same concentration without drug binding.

[0022] Therefore, in [7], for comparative purposes, it was suggested that nanoparticles with ligands be measured in a first organism and nanoparticles without ligands be measured in a second separate organism (in this case, a mouse). However, this method is highly demanding on the test animals and ignores individual differences between individual organisms. Furthermore, it is not medically practical for patients. Therefore, it would be meaningful to conduct comparative measurements simultaneously in a single organism. In addition, such comparative measurements would also allow the detection of non-specific physiological background in the target area. For example, if blood vessels and unbound nanoparticles therein are located there, this “background image” can be subtracted from the XFI image. The difference image then shows only the specifically bound nanoparticles. In addition, such comparative measurements, which cannot be performed using conventional techniques, would also allow the detection of non-specific physiological background in the target area. For example, if blood vessels and unbound nanoparticles therein are located there, this “background image” can be subtracted from the XFI image. The difference image then shows only the specifically bound nanoparticles.

[0023] Another drawback of conventional XFI is that it's impossible to simultaneously track multiple different drugs or biomarkers in the case of multiple drugs. This also applies to the simultaneous imaging of the dynamics of different immune cell types. However, such measurements will be important in a range of medical studies, such as for visualizing inflammatory processes in vivo or for tumor diagnosis using multiple different antibodies. For example, in the case of inflammatory processes, the intention is to record different immune cell types that arrive at the site of inflammation at different times, thus influencing the progression of inflammation. Therefore, in the case of Crohn's disease, it would be meaningful to simultaneously track four different immune cell types. However, different cell types cannot be distinguished using conventional XFI. Measuring pharmacokinetics in the case of multiple drugs is also meaningful for studying drug interactions or comparing drug effects. It is well known that new drugs often fail after being marketed because patients must take multiple drugs simultaneously, which can block each other's binding sites in the body. To date, this goal has been unattainable because conventional techniques cannot identify all the immune cell types to be measured simultaneously with the same sensitivity.

[0024] In the case of conventional XFI, another important issue is that the kinetics of nanoparticles are known to depend on their size. Currently, there is no practical and efficient method for tracking the kinetics of multiple different sizes of nanoparticles together with the same sensitivity, and for directly comparing these nanoparticles with each other.

[0025] To measure pharmacokinetics in multiple drug cases, sequential measurements can be performed using XFI, but this would require unacceptably long measurement times. Different drugs can only be studied sequentially after the drugs under study have been respectively metabolized and excreted. Therefore, under practical, routine conditions, it is impossible to measure multi-parameter pharmacokinetics using conventional XFI.

[0026] In [9], a method for detecting multiple biomarkers of different nanoparticle types using X-ray fluorescence is described. The authors of [9] have determined that different nanoparticle types can be distinguished based on their different spectral fluorescence properties, and that spatially resolved measurements of different nanoparticle types can be performed at different locations. The technique according to [9] is based on a model system in which nanoparticles are irradiated with high-intensity excitation X-ray radiation on a thin aluminum substrate. The high excitation intensity makes it possible to produce sufficiently high X-ray fluorescence intensity, thus enabling sensitive detection. The use of an aluminum substrate largely avoids interference from scattered radiation (background radiation) due to multiple scattering.

[0027] However, the technique according to [9] is not suitable for biomedical imaging because it requires significantly lower intensity excitation X-ray radiation for radiation protection reasons, and produces significantly stronger scattered radiation in biological tissues, as objects (e.g., mice) have significantly more tissue, resulting in more (multiple) scattering. To overcome this problem, monochromatic X-ray radiation is suggested for excitation in [9]; however, in practice this has been found insufficient to achieve adequate sensitivity for X-ray fluorescence detection, especially for objects much larger than the substrates used in [9], because their larger volume causes a large background in the detected spectrum, making it impossible to detect even a weak XFI signal at the minimum radiation dose. Target selection for the labeling kit is also not described in [2].

[0028] In [2], a method for computed tomography XFI is described, wherein XFI can in principle be performed using a combination of various different nanoparticle types. However, in this case, the different emission spectra of different nanoparticle types are not used for imaging living organisms when analyzing the detected X-ray fluorescence.

[0029]

[11] describes a library of nanoparticles for X-ray fluorescence tomography. The authors of

[11] show in the supplementary material that, in the case of nanoparticles composed of different elements, the intensity of the scattered radiation varies by several orders of magnitude, respectively, making it impossible for different nanoparticles to be detected at the same sensitivity level. This problem in XFI of biological samples is overcome by adjusting the concentration of nanoparticles, as the concentration of nanoparticles with high background increases by, for example, by 1000 times, which is subject to strict physiological limits. Therefore,

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

[11] , the background in the signal range of the labeled elements studied is generally neither very different nor minimized. Thus, high-sensitivity XFI is not possible. Summary of the Invention

[0030] One object of the present invention is to provide an improved method for X-ray fluorescence imaging of samples, by means of which the disadvantages of conventional techniques are avoided. Another object of the present invention is to provide an improved imaging apparatus for X-ray fluorescence imaging of samples, wherein the disadvantages of conventional techniques are avoided. Another object of the present invention is to provide an improved labeling kit adapted to be introduced into a sample for X-ray fluorescence imaging. The present invention is particularly aimed at providing highly sensitive XFI with increased conclusiveness, allowing the measurement of the spatial and / or temporal distribution of one or more active substances in the organism under study, and / or providing new applications for XFI, particularly under practical application conditions. In this context, in vivo imaging, for example, including the detection of one or more active substances, particularly biomarkers, antibodies, antibody fragments, biological cells such as immune cells, and / or drugs, is particularly meaningful. XFI is particularly designed to allow for studies with multiple drugs while reducing methodological complexity, and / or to prevent the limitations of short diagnostic time windows.

[0031] These objectives are respectively achieved by a method for X-ray fluorescence imaging, an X-ray fluorescence imaging apparatus, and a labeling material kit having the features of aspects according to this application. Preferred embodiments and applications of the invention can be found in other aspects according to this application.

[0032] According to a first general aspect of the invention, the above-mentioned objective is achieved by a method for multi-parameter X-ray fluorescence imaging of a sample, wherein the following steps are provided: The sample to be studied is irradiated with X-ray radiation, preferably with monochromatic or at least narrow-band X-ray radiation, wherein X-ray fluorescence of a first labeled substance is excited. The sample is typically a shape-preserving object, preferably the body (or part of the body) of a biological organism, particularly preferably the body (or part of the body) of a human or animal subject. Alternatively, other unnatural objects, such as synthetic biological objects or artificial organs, such as implants or skin models, or technological objects, may also be studied. The X-ray fluorescence of the first labeled substance is spatially resolved, particularly by scanning the sample. The distribution of the first labeled substance in the sample can be determined based on the detected X-ray fluorescence of the first labeled substance.

[0033] According to the present invention, in addition to a first labeling substance, the sample also contains at least one additional labeling substance, which is excited into X-ray fluorescence by X-ray radiation. If the at least one additional labeling substance is located in the same irradiation region (beam volume at the irradiation position, scan position) as the first labeling substance, the excitation of X-ray fluorescence preferably occurs simultaneously. The labeling substances are molecular or particulate substances, each containing at least one X-ray fluorescent element as a pure element or in a compound. The labeling substance is a foreign substance to the bulk and is specifically provided to the sample before the XFI method and leaves the sample again after the XFI method after a substance-specific residence time, for example, separated by a transport process. Active substance molecules and / or ligand molecules are coupled to at least one of the first and at least one additional labeling substances, which exhibit specific biological and / or chemical interactions with the sample and are transported and / or coupled therein in specific portions of the sample by means of transport processes such as metabolism or blood transport. According to a preferred variant, if the active substance comprises, for example, biological cells for immunotherapy, and particularly in applications aimed at monitoring cells by means of the method according to the invention, the coupled labeling substance is preferably contained in the cells or bound to the surface of the cells. The first and each additional labeling substance are characterized by distinct X-ray fluorescence. In the measured spectra, the fluorescence lines of the X-ray fluorescence elements of the first and each additional labeling substance are distinct, and they have maximum values, particularly at different energies and / or different spectral widths. According to the invention, the detection comprises, preferably in a single measurement procedure, for example, a single measurement procedure using an X-ray beam for scanning, spectrally and spatially resolved acquisition of the X-ray fluorescence (X-ray fluorescence emission) of the first and each additional labeling substance. In addition to the distribution of the first labeling substance, the distribution of at least one additional labeling substance in the sample is determined based on the detected X-ray fluorescence of the first and each additional labeling substance.

[0034] Therefore, this multi-parameter method provides multiple specific distributions of the first and each additional labeled substance in the sample. The determined distributions themselves do not typically constitute diagnostic information. Medical diagnosis of the sample can be performed solely according to the method of the invention, using the determined distributions.

[0035] The first labeling material and the at least one other labeling material preferably exhibit equal or similar fluorescence probabilities, X-ray fluorescence attenuation in the sample, and background noise levels in the sample, thereby ensuring comparable statistical significance in X-ray fluorescence detection at the same concentration of labeling material. Furthermore, the irradiation photon energy that excites X-ray radiation is preferably higher than the absorption edges of all labeling materials. The term "concentration" refers to the mass of the labeling material on each (beam) surface of the sample, i.e., "area density." "Fluorescence probability" refers to the effective cross-section of a single atom of the labeling material. The number of fluorescent photons generated by the labeling material that transmits a detectable signal after fluorescence transmission through the sample is determined by the product of the area density of the relevant labeling material and the effective cross-section of the fluorescence. If the effective cross-sections and transmittances of the two labeling materials are equal or similar, and their area densities are equal or similar, this advantageously results in equal or similar detectable signals from the labeling materials.

[0036] The labeling material preferably comprises nanoparticles. The nanoparticles particularly preferably comprise an outer surface that is indistinguishable from the sample, preferably identical, and can be differentiated only by targeted functionalization, and whose interior is composed of different elements, each possessing its characteristic "fluorescent fingerprint." These nanoparticles may have been introduced into the sample from the outside, as is the case with conventional nanoparticle-based XFI.

[0037] In particular, the target selection of X-ray fluorescence elements for labeled materials is advantageous for multi-parameter XFI. This selection preferably meets the following criteria:

[0038] (a) The irradiated photon energy is higher than that of all absorption edges.

[0039] (b) X-ray fluorescent elements exhibit similar fluorescence probabilities.

[0040] (c) The attenuation of the corresponding fluorescence lines of X-ray fluorescent elements should also be similar.

[0041] (d) Due to scattering (single and / or multiple scattering) in the sample, the fluorescence lines of X-ray fluorescent elements should have similar background noise levels, such that, together with (b) and (c), if the surface density (concentration) of the elements in the X-ray volume is the same, the corresponding statistical significance of the fluorescence lines of the detected elements has similar levels.

[0042] For example, the ratio of the number of recorded fluorescence photons to the root of the background photon count, or a variable quantitatively representing said ratio, can be used as a measure of statistical significance, i.e., particularly as a measure of sensitivity for X-ray fluorescence detection. The root of the background photon count, in turn, is a measure of statistical background noise. Both the number of fluorescence photons and the background photon count can be determined using available numerical methods from a mathematical fitting function of the X-ray spectrum measured in the region of the fluorescence line.

[0043] Standards (b) through (d) are a particularly important discovery by the inventors and are especially advantageous for embodiments of the invention—for example, if two X-ray fluorescent elements so far apart in a periodic system that their fluorescence probabilities (i.e., effective cross-sections), attenuation in the sample, and background noise levels are very different, then even if the two elements have the same areal density in the X-ray volume, they will be detected with significantly different sensitivities. This may result in only one of the two types of labeled substances being effectively detected, thus preventing the achievement of multi-parameter XFI.

[0044] The effective cross-section of the atoms of the labeled material can be obtained from standard measurements and published tables, and the attenuation of X-ray fluorescence in the sample can be determined by reference measurements and / or simulations. The background is formed both by the scattering of incident photons in the sample and by detector effects. The background behavior of X-ray fluorescent elements can be determined by numerical simulation or measurement (see, for example, [7]), thereby enabling optimal selection of X-ray fluorescent elements according to standards (b) through (d).

[0045] Introducing labeled substances into samples is not considered part of this invention because introduction requires invasive intervention of biological materials, such as injection.

[0046] According to a preferred embodiment of the invention, the statistical significance level of the labeled material is maximized because the irradiation photon energy of the X-ray radiation is selected to be one spacing above the highest absorption edge of the labeled material in the sample, such that the background noise level of the labeled material is minimized and equal or approximately equal, while the effective cross-section and the transmittance of X-ray fluorescence through the sample are maximized and equal or approximately equal. If the difference in background noise level or effective cross-section and transmittance has a negligible effect on the detection of X-ray fluorescence, then the aforementioned variables are preferably approximately equal.

[0047] The selection of the X-ray irradiation photon energy and the selection of the labeling material are preferably performed by means of optimization, wherein the irradiation photon energy is selected to be as large as possible compared to the highest absorption edge of the labeling material in the sample, so that the incident photons must be scattered as much as possible and suffer corresponding energy loss until they fall within the energy range of the X-ray fluorescence line (minimizing background noise through necessary multiple scattering, making it unlikely that the total energy loss from the irradiation energy down to the fluorescence energy range will require more scattering). At the same time, the irradiation photon energy should not be selected too high, thereby reducing the effective cross-section excessively.

[0048] This invention provides for the first time multi-parameter X-ray fluorescence imaging of samples suitable for practical applications. In [9], although two different nanoparticles have been used as described above, they were only used on very thin substrates in vitro. In contrast, this invention allows for reliable XFI of real objects, such as at least mouse-sized objects, by means of the selection of labeling materials not described in [9]. The selection of labeling materials according to the invention is also not described in [2] or

[11] , and therefore the XFI labeling materials mentioned in

[11] , for example, have very different imaging sensitivities, excluding effective multi-parameter XFI.

[0049] Unlike the methods described above in [7], the present invention allows for comparative measurements of individual samples, since simultaneous comparative measurements have the same imaging sensitivity as actual measurements, thus yielding clear results.

[0050] According to a second general aspect of the invention, the above-mentioned objective is achieved by an imaging apparatus configured for studying multi-parameter X-ray fluorescence imaging of a sample, and comprising an X-ray radiation source device, a detector device, and an analytical processing device. The X-ray radiation source device is configured to irradiate the sample with X-ray radiation, wherein X-ray fluorescence of a first labeled substance and at least one additional labeled substance is excited in the sample. The detector device, comprising at least one spectrally resolved X-ray detector, preferably multiple spectrally resolved X-ray detectors, is configured to perform spectrally resolved detection of the X-ray fluorescence of the first labeled substance and the at least one additional labeled substance in the sample, wherein the X-ray fluorescence of the first labeled substance and the at least one additional labeled substance has different fluorescence lines. The analytical processing device is configured to determine the spatial distribution of the first labeled substance and the at least one additional labeled substance in the sample based on the detected X-ray fluorescence emission. The imaging apparatus is preferably configured to perform a method according to the first general aspect of the invention or an embodiment thereof.

[0051] According to a third general aspect of the invention, the above-mentioned object is achieved by a labeling material kit configured to be introduced into a sample for multiparameter X-ray fluorescence imaging, and the labeling material kit contains at least two labeling materials that emit X-ray fluorescence upon irradiation with X-ray radiation, wherein the fluorescence lines of the labeling materials are different. The labeling material kit is preferably provided for use in methods according to a first general aspect of the invention or embodiments thereof, particularly for use in samples. The labeling material kit may be provided in liquid or solid form. The composition (substance, concentration, particle size) of the labeling material kit and the preferred photon energy of the irradiated X-ray radiation can be determined by testing or reference measurements and / or numerical simulations.

[0052] Spatial-resolved detection of X-ray fluorescence of a first marker and at least one other marker in a sample includes detecting the X-ray fluorescence of the marker only in at least one spatially confined region of the sample, such as in the region of at least one organ and / or in the region of at least one other part of an organism, and / or spatial-resolved detection of the X-ray fluorescence of the marker over the entire sample.

[0053] The distribution of the first labeled substance and the at least one additional labeled substance respectively includes assigning quantities—such as the concentration (or areal density) of the labeled substance, absolute mass, and / or the relative prevalence of different labeled substances—to positional coordinates within at least one spatially confined region and / or volume. The distribution of at least two labeled substances arises from their transport in the sample, for example by means of diffusion and / or transport in the sample by a carrier fluid, such as blood, and their bio / chemical interactions with the sample. The quantities can be determined directly from the amplitude of the X-ray fluorescence emission of the labeled substance, since amplitude is a measure of the number of X-ray fluorescent elements detected. Furthermore, the amplitude of the detected X-ray fluorescence emission also depends on the transmittance of emitted fluorescent photons in the sample, which is known in itself. The spatial and / or temporal distribution of the labeled substances can be determined.

[0054] The spatial distribution of the marker substance in the sample accordingly includes assigning the amount of marker substance at a specific time to at least one spatially confined region and / or to the location coordinates in the body.

[0055] The temporal distribution of the marker substance in the sample accordingly includes the assignment of the marker substance's magnitude to at least one spatially confined region and / or the temporal dependence on its positional coordinates within the body.

[0056] Therefore, the distribution includes, for example, the average concentration value (and / or its time function) in the organ, and / or the mapping of the mass value to specific location coordinates (such as a specific scan line or a specific scan surface or a specific scan volume).

[0057] This invention advantageously extends XFI, enabling the simultaneous measurement in vivo of the specific distribution of multiple different labeled substances, such as molecularly labeled elements or nanoparticles, with minimal radiation dose and the same sensitivity. These different labeled substances respectively carry different active substance molecules (active substances), such as drugs or antibodies, or whole biological cells, which may or may not contain active substance molecules. This is not, in principle, possible with conventional XFI or PET. The preferred simultaneous detection of the distribution of multiple different labeled substances (“multiparametric X-ray fluorescence imaging”) provides additional information related to the sample under study, such as pharmacokinetic information and / or information for diagnostic analytical processing, compared to conventional XFI methods that include a single labeled substance. Thus, for example, the measurement of multiparametric pharmacokinetics and multiparametric tumor diagnosis become new applications of XFI. This invention makes it possible for the first time to determine, for example, the effects on the kinetics of nanoparticles that can be used as labeled substances, because at least one control group comprising nonfunctionalized nanoparticles is also measured simultaneously.

[0058] For example, it can be determined for the first time in vivo whether a drug is present at a sufficient concentration at a specific location in a sample, and whether another drug that interacts with it (e.g., with an inhibitory effect) is also present at a significant concentration at the same location.

[0059] Further applications include simultaneously measuring the pharmacokinetics of drugs, such as new drugs and already approved drugs, or alternative drugs, or generic drugs.

[0060] According to the present invention, not only can the efficacy of drugs be studied, but also their interactions in the case of multiple drugs, i.e., when multiple drugs are administered simultaneously. The distribution of different drugs can be determined by binding different labeled substances and detecting their distribution. Therefore, the locations of interactions between different drugs at specific concentrations in a sample, which may interfere with each other's corresponding effects, can be accurately detected. This is a significant advantage for drug development.

[0061] For example, a method for multiparametric tumor diagnosis could be implemented where different markers are conjugated to different antibodies to identify the subtype of the tumor under study. This is a significant advantage for subsequent treatment, as optimal treatment depends on the tumor subtype. Especially in cases where biopsy is not feasible (e.g., tumors in the respiratory center of the brain), knowing the subtype would be a decisive advantage for subsequent treatment.

[0062] A key feature of this invention is the application of XFI in conjunction with different labeled substances, referred herein to as the first and additional labeled substances. The various labeled substances are inherently different, for example, inherently different within themselves, by producing different fluorescent elements (X-ray fluorescent elements) that produce different fluorescent lines. As a further important feature, in their non-functionalized state, i.e., without coupled active substances, the different labeled substances can be identical in their interactions with the sample, thus possessing the same biological, chemical, physiological, and / or physical effects on the sample, i.e., identical to the sample. In their non-functionalized state, the labeled substances are therefore indistinguishable from their surrounding environment, particularly biologically and / or chemically.

[0063] Furthermore, the varying degrees of fluorescence in the labeled material allow a detector with limited spectral resolution (energy resolution) to distinguish the corresponding X-ray fluorescent elements in the measured X-ray spectrum of the labeled material's X-ray fluorescence emission. Preferably, the X-ray fluorescent elements of the labeled material are selected such that the spectral spacing of the K- and L-α / β lines of said element allows the detector device to distinguish these lines in the measured X-ray spectrum. Additionally, the X-ray fluorescent elements of the labeled material preferably have equal or similar fluorescence probabilities (effective cross-sections) and transmittance through the sample, as well as comparable minimum background noise levels.

[0064] These characteristics are realized and preferably used for the elements cited in the following examples, where the K-α lines of two adjacent elements in a periodic system typically overlap, but not those of more distant elements, and thus appear separately in the spectrum. Since all four lines have similar energies, they are absorbed similarly in the sample, and therefore all four lines can be measured with the same or comparable accuracy.

[0065] Spectral-resolved detection of X-ray fluorescence provides an additional superimposed X-ray spectrum of the fluorescent lines of the labeled substance. The individual quantitative contributions of the fluorescent lines can be determined from the X-ray spectrum by numerical deconvolution and / or by comparison with predetermined reference measurements. The amount of the desired labeled substance (e.g., concentration, absolute mass, and / or relative prevalence of different labeled substances) is derived from the quantitative contributions of the fluorescent lines. Even if all types of labeled substances are present in the X-ray volume, their relative prevalence can be determined from the X-ray spectrum because the X-ray fluorescence lines of all X-ray fluorescent elements are clearly distinguishable from each other in the spectrum.

[0066] In practice, the specific sample under consideration can be optimized through numerical simulation of background noise and / or test measurements under different irradiation photon energies of X-ray radiation.

[0067] According to another preferred embodiment of the invention, a common excitation beam (or interrogation beam) of X-ray radiation is used to excite and simultaneously detect the X-ray fluorescence emission of the first labeled substance and the at least one other labeled substance. Advantageously, the radiation exposure of the sample and the duration of the method are thus minimized.

[0068] According to an alternative embodiment of the invention, X-ray fluorescence emission of the first labeled substance and the at least one other labeled substance is simultaneously or sequentially excited and detected using different excitation beams of X-ray radiation, the beams having different energies (irradiation photon energies). In this embodiment, the X-ray radiation source device is configured to generate multiple excitation beams of X-ray radiation, wherein multiple sources, for example, directed onto the sample, operate simultaneously or sequentially (e.g., directly and continuously) at different energies. Advantages can be derived by adapting the energies of the excitation beams accordingly to the absorption of the X-ray fluorescent elements of the labeled substance. Simultaneous excitation and detection are advantageous in minimizing the duration of the method. Sequential excitation and detection refers to exciting different labeled substances and detecting the associated fluorescence in a stepwise manner, preferably directly and continuously. In this variant, direct detection of the labeled substance from the X-ray spectrum of a continuously recorded labeled substance is advantageous.

[0069] Another advantage of this invention is the availability of different types of labeled substances, including nanoparticles (or target particles) and labeled molecules. The first and each additional labeled substance respectively comprise multiple nanoparticles and / or multiple labeled molecules. Nanoparticles are particles with a typical size ranging from 2 nm to 100 nm or greater, the surface of which is adapted or intentionally prepared for coupling ligands and / or active substance molecules. Labeled molecules are monomolecules or molecular aggregates containing X-ray fluorescent elements, suitable for coupling with active substance molecules. All portions of a labeled substance having a specific X-ray fluorescent element may consist entirely of nanoparticles or entirely of labeled molecules, or the labeled substance may comprise nanoparticles and labeled molecules simultaneously containing the same or different X-ray fluorescent elements.

[0070] Labeling substances in nanoparticle form offer particular advantages for the binding of active substances. They can be coupled to active substance molecules or act as ligands for the active substance themselves on the surface of the nanoparticles. The active substance molecules reside on the surface of the nanoparticles. Alternatively, the active substance molecules can also be arranged inside the particle. In this case, the nanoparticles advantageously form active substance carriers, as in conventional drug delivery technologies. Thus, nanoparticles can be used where different ligand molecules are bound to the surface to provide different labeling substances (thus allowing the study of which ligands dock at which location in the body), while the same or other active substance molecules are arranged inside. However, in most applications, the ligand molecules on the particle surface simultaneously form the active substance.

[0071] The labeling material, composed of different nanoparticles, includes, for example, a first plurality of unfunctionalized nanoparticles (or groups or types of nanoparticles), and at least one additional plurality of nanoparticles respectively bound to at least one predetermined drug to be studied. Multiparameter XFI of the labeling material is used to simultaneously or sequentially measure the local concentrations of the unfunctionalized and functionalized nanoparticles, wherein differences in concentration may be attributed to the drug conjugated to the functionalized nanoparticles, since for a sample, the two types of nanoparticles are preferably different only by means of an active substance or ligand molecule. In a further variation, a third type of nanoparticle may be functionalized with a second drug. This multiparameter XFI variation allows the basic distribution of the unfunctionalized nanoparticles to be subtracted from the measured concentration of functionalized nanoparticles, and even allows for the differentiation of two different drugs in the process. The corresponding method can also be implemented using a labeling material comprising a labeling molecule instead of nanoparticles.

[0072] Nanoparticles are particles composed entirely of one X-ray fluorescent element (optionally in a compound) or of a composition of one X-ray fluorescent element (optionally in a compound) and at least one other element. Therefore, according to a variation of the invention, each type of nanoparticle may contain only one of a plurality of X-ray fluorescent elements, optionally in a composition with a non-fluorescent element. According to an alternative variation of the invention, the first labeling material may comprise a first type of nanoparticle predominantly comprising a first X-ray fluorescent element, and the at least one other labeling material may comprise at least one other type of nanoparticle, each correspondingly predominantly comprising at least one other X-ray fluorescent element. Thus, the nanoparticles may each correspondingly comprise at least two X-ray fluorescent elements, wherein one X-ray fluorescent element is decisive for the fluorescence line to be detected. This may be advantageous for the design of nanoparticles, for example, in the case of a core-shell structure mentioned below.

[0073] According to another advantageous embodiment of the invention, the first type of nanoparticles may carry a first type of active substance molecule that has chemical and / or physical interactions with the sample, while each additional type of nanoparticle carries a correspondingly different type of active substance molecule, which may have chemical and / or physical interactions with the sample that deviate from the first type, or may not carry any active substance molecule. Particularly preferably, each type of nanoparticle carries only one specific type of active substance molecule. This advantageously increases the conclusiveness of XFI. Advantageously, the nanoparticles thus provide a high degree of flexibility in adapting to specific XFI investigation tasks.

[0074] According to another preferred embodiment of the invention, at least one type of nanoparticle can have a core-shell structure comprising a particle core and a particle coating (hybrid nanoparticles). This core-shell structure advantageously allows for the separation of two functions of the nanoparticles: firstly, concerning X-ray fluorescence emission, and secondly, concerning interaction with the surrounding environment. Thus, the particle core can be generated by an X-ray fluorescent element having the desired fluorescence line of the corresponding type of nanoparticle, while the particle coating is generated by a material different from the core and forms a surface for coupling ligands and / or active substance molecules, and for providing predetermined biological and / or chemical interactions with the sample. The particle coating can be generated by fluorescent or non-fluorescent elements.

[0075] According to a preferred variant of the invention, the particle coating is formed of a metal, particularly gold, or a non-metallic material, particularly a polymer, liposome, or micelle material. Since the coupling chemistry of active substances with nanoparticles has been studied to date, particularly using metal nanoparticles, especially gold nanoparticles, nanoparticles with a core-shell structure preferably have an X-ray fluorescent element in the particle core, the X-ray fluorescent element having an atomic number comparable to that of gold (so that the X-ray fluorescence is in a fairly high energy range, allowing for high sensitivity measurement of these photons outside the sample), and the particle core is preferably covered by a metal layer, particularly a gold layer, for coupling the active substance (ligand). The thickness of the particle coating is preferably, for example, 1 / 4 or less of the particle diameter. Therefore, the volume fraction of gold is negligible compared to other X-ray fluorescent elements, making the XFI signal appear as if only the X-ray fluorescent element in the particle core is present. Another possibility is to produce nanoparticles from various X-ray fluorescent elements and use polymers instead of metals for the particle coating, as described, for example, in [5]. The corresponding ligands, i.e., drugs or antibodies, can then be bound to the polymer particle coating. Polymer layer nanoparticles can also be combined with suitable internal X-ray fluorescence elements, depending on specific application conditions (e.g., the size and quantity of the drug under study), to implement multi-parameter XFI, where all nanoparticles used have similar sensitivity (the ratio of signal intensity to statistical noise of the relevant background).

[0076] Particularly preferably, the nanoparticles of various labeled substances all have a core-shell structure, wherein the particle core of each labeled substance is made of different X-ray fluorescent elements, and the particle coating of all labeled substances is made of the same element capable of binding at least one of the active substance molecules and ligand molecules. Optionally, the particles of one of the multiple labeled substances may be formed entirely of the elements forming the particle coatings of the remaining labeled substances.

[0077] According to a preferred design, the nanoparticles are indistinguishable externally, while internally they contain different elements, wherein they preferably have the same size. Nanoparticles with a core-shell structure preferably have an external granular coating, which is formed of the same material and is preferably identical. Therefore, unless they are functionalized differently, they cannot be distinguished by the sample, particularly by the bulk of the biological organism under study. Internally, the nanoparticles contain different elements with varying X-ray fluorescence energies. Thus, different nanoparticle types can be distinguished from each other in the measured XFI spectra, and their respective concentrations can be determined simultaneously, whereas they cannot be distinguished by the sample except through functionalization.

[0078] According to a particularly preferred embodiment of the invention, the nanoparticles respectively contain, particularly in the core of the particle, iridium, platinum, gold, or bismuth as X-ray fluorescent elements. Due to their similar but distinguishable X-ray fluorescence energies, these elements can advantageously be detected with comparable levels of sensitivity. Therefore, it is even possible to simultaneously, for example, track up to four different drugs, (immune) cell types, and / or subtype-specific antibodies in vivo within a biological organism. Depending on alternative variations, the nanoparticles may respectively contain different X-ray contrast agent molecules. Advantageously, X-ray contrast agent molecules—such as iodine, barium, or gadolinium—are widely available in practice, and their absorption behavior has been well studied. Iodine and barium are particularly suitable for imaging small animals. Nanoparticles made of silver, palladium, indium, cadmium, or iodine are also advantageous for imaging small animals.

[0079] According to a further preferred embodiment of the invention, if different nanoparticles, i.e., different labeled substances with different X-ray fluorescent elements, have different nanoparticle sizes, further degrees of freedom are provided in multi-parameter XFI. Therefore, the conclusiveness of XFI and / or the behavior of nanoparticles in the sample under study can be advantageously further improved. For example, it is known in practice that nanoparticles of different sizes can have different dynamics. For example, the distribution of nanoparticles including up to four different sizes can be detected by means of X-ray fluorescence. In this case, typical sizes are selected in diameter ranges of 2 nm to 5 nm, 6 nm to 10 nm, 11 nm to 20 nm, and 21 nm to 50 nm. Nanoparticles of different sizes preferably have the same surface type, so that only the size is changed, and the corresponding X-ray fluorescent element is changed according to the size.

[0080] Alternatively or additionally, nanoparticles with different labeled substances can also differ in how they are supplied to the sample. For example, multiparameter XFI can be used to study the effects of different routes of administration, such as oral and intravenous delivery of nanoparticles, on the distribution of the labeled substance in the sample.

[0081] Labeled substances in molecular form offer particular advantages for transport within samples. Since labeled molecules are significantly smaller than nanoparticles, their transport within a sample is more akin to molecular mass transfer, especially in biological organisms. Preferably, the first labeled substance comprises a first type of labeled molecule containing a first X-ray fluorescent element, and each additional labeled substance comprises a further type of labeled molecule containing at least one additional X-ray fluorescent element.

[0082] According to another advantageous embodiment of the invention, the advantages of new applications of XFI are realized if the first labeling material comprises nanoparticles containing a first X-ray fluorescent element, and the at least one additional labeling material comprises labeling molecules each containing at least one additional X-ray fluorescent element. For example, it is possible to combine multiple, for example four, labeling materials with different nanoparticles with one or more additional labeling materials made of labeling molecules in the form of contrast agent molecules.

[0083] For example, three different types of immune cells can be coupled to three different nanoparticles, and additionally, a drug bound to a fluorescently labeled molecule can be studied. Labeling different immune cell types with different nanoparticles can be performed, for example, by pre-removing cells and subsequently loading the nanoparticles and introducing them into a sample, or by using functionalized nanoparticles that bind to specific, distinct immune cell types in vivo. The nanoparticles used for immune cells should be indistinguishable from the cells and the sample, while the drug is distinct from the immune cells. In other words, in this case, the drug does not necessarily need to bind to the nanoparticles as well. This application could have particular advantages in studying immune-based diseases, such as Crohn's disease. The distribution of different immune cell types and the distribution of the drug can be measured simultaneously to study the drug's effectiveness in vivo. In this case, the effect might be that the drug alters the prevalence of immune cell types in inflammatory areas; for example, immune cell types that reduce inflammation may appear more frequently.

[0084] Another advantageous application of the invention is as a so-called drug carrier, in which nanoparticles and labeled molecules serve as different labeling substances. For example, two different labeling substances with different nanoparticles can be used, one set of nanoparticles being unfunctionalized, and the other set being functionalized with a predetermined ligand intended to attach to a target structure in the sample. In this case, both nanoparticles can serve as drug carriers, i.e., the actual drug with a molecular-based label now bound inside. The location and, optionally, timing of the nanoparticles acting as drug carriers can be determined by means of multi-parameter XFI. For example, early release can be identified if the drug distribution does not correspond to the distribution of the nanoparticles. Comparing the distribution of the unfunctionalized nanoparticles with the distribution of the ligand-carrying nanoparticles can show how specifically the ligands find their target sites. If the two distributions are identical, it is found that the drug carrier reaches the target site rather coincidentally than in a targeting manner that should actually be achieved by the ligand.

[0085] The labeled molecule is preferably bound to an active substance molecule, wherein the labeled molecule accordingly includes one of the first and at least one additional X-ray fluorescent element. The X-ray fluorescent elements particularly preferably include medium-heavy elements from zirconium to cerium, whose background noise levels can be significantly reduced, or heavy elements such as iridium, platinum, gold, and bismuth. These two groups of elements are advantageous due to their similar and well-studied fluorescence properties and can be readily conjugated to drugs or ligand molecules.

[0086] According to the present invention, the spatial and / or temporal distribution of the labeled substances can be determined. In this case, a preferred embodiment of the invention is particularly advantageous, wherein the measurements are resolved both spatially and temporally, and the spatial distribution of a first labeled substance and at least one additional labeled substance in the sample is determined as a temporal function. This embodiment has particularly high conclusiveness regarding the transport of the labeled substances in the sample from introduction into the sample to binding within the sample.

[0087] According to another feature of the preferred embodiment, the irradiation photon energy of the X-ray radiation used to excite the labeled material and the X-ray fluorescence characteristics of the labeled material are preferably selected such that the irradiation photon energy of the X-ray radiation is higher than the absorption edge of the X-ray fluorescent elements of all the labeled materials, the fluorescence probability exhibited by the X-ray fluorescent elements of all the labeled materials, the attenuation of X-ray fluorescence in the sample, and the signal and background noise levels in the sample are equal or similar, thereby ensuring that the detection of X-ray fluorescence at the same concentration produces comparable signal intensities. This feature advantageously makes it easier to determine the relative popularity of different labeled materials directly from the detected X-ray spectra. For example, one of several labeled materials may have a significantly lower concentration than the others because, for example, ligands on these nanoparticles are difficult to couple to the target structure in the sample. This behavior can be observed directly from the detected X-ray spectra when X-ray fluorescence is detected with comparable sensitivity. If all labeled materials have the same or similar areal density, their signals will have comparable intensities.

[0088] According to a preferred application of the invention, the sample under study is a human or animal object, or a portion thereof. The labeling substance is introduced into the subject beforehand, for example, via oral or other administration or injection. The first labeling substance and the at least one additional labeling substance can be introduced into the sample in different ways. Preparation steps including the introduction of the labeling substance into the subject via injection are not part of this invention.

[0089] According to another advantageous application of the invention, the active substance comprises biological cells, at least one of the first labeling substance and the at least one additional labeling substance is conjugated to the biological cells, and determining the distribution of the first labeling substance and the at least one additional labeling substance comprises detecting the transport of the biological cells through the sample. Therefore, for example, the transport of different immune cells through the sample to be determined can be advantageous. Attached Figure Description

[0090] Further details and advantages of the invention will now be described with reference to the accompanying drawings, in which:

[0091] Figure 1 This is a schematic view of an imaging apparatus for X-ray fluorescence imaging according to an embodiment of the present invention;

[0092] Figure 2 This is a flowchart of a method for X-ray fluorescence imaging according to an embodiment of the present invention;

[0093] Figure 3 An example of a labeling substance that can be used in X-ray fluorescence imaging according to the method of the present invention is shown;

[0094] Figure 4 This is a schematic diagram of a marker material kit according to an embodiment of the present invention;

[0095] Figure 5 The X-ray spectrum used to illustrate the measurement of X-ray fluorescence emission of two different X-ray fluorescent elements in biological cells is shown.

[0096] Figure 6 The X-ray spectra used to illustrate the measurements of X-ray fluorescence emission of four different X-ray fluorescent elements in the sample are shown; and

[0097] Figure 7 The simulated X-ray spectrum is shown to illustrate the X-ray fluorescence emission of X-ray fluorescent elements in the sample at two different irradiation energies. Detailed Implementation

[0098] Features of preferred embodiments of the invention are described below, for example with reference to XFIs of human subjects using specific X-ray fluorescence elements. It should be emphasized that the implementation of the invention in practice is not limited to the examples described, but may use other samples, such as portions of human subjects, synthetic biological objects, animal objects, or portions thereof, in a corresponding manner. Embodiments of the invention will be described below with particular reference to important features of the configuration of the labeled material, the execution of XFIs, and the structure of the imaging apparatus. Other features of the imaging apparatus may be implemented, for example, as described in [1]. Regarding the structure and function of the imaging apparatus, [1] is incorporated herein by reference. Details of the functionalization of nanoparticles, the coupling of nanoparticles with active substances, the selection of ligands, the coupling of labeled molecules with active substances, the spectral and spatially resolved detection of X-ray fluorescence, and the analysis of superimposed spectra consisting of multiple fluorescence lines are not described, as these are known per se from the prior art. The concentration of the labeled material may be selected as is known per se from conventional XFIs.

[0099] Figure 1 Features of an embodiment of an imaging device 100 for X-ray fluorescence imaging are schematically shown, the imaging device 100 being used to study a sample 10, such as a human subject, arranged on a sample holder 101, such as a bed. Figure 2 The steps of the method according to the invention using imaging device 100 are illustrated schematically, including supplying a labeling material (S1), irradiating the sample with X-ray radiation (S2), detecting X-ray fluorescence (S3), and determining the distribution of the labeling material based on the detected X-ray fluorescence (S4).

[0100] also, Figure 2Step S0, which includes selecting the labeling material and irradiating the photons with energy, is illustrated. For a given sample or group of samples with the same scattering characteristics, such as small animals of a specific species and size, it is sufficient to perform step S0 once, separately from the implementation of the method according to the invention. Alternatively, step S0 may be provided as part of each execution of the method according to the invention.

[0101] For example, according to Figure 7 The fluorescence spectra shown in the example are selected based on the following considerations. In detail, Figure 7 A direct comparison of two simulated spectra after excitation with monochromatic X-rays is shown, first for an irradiation energy of, for example, 85 keV, and second for, for, for example, 53 keV. In the first case, gold nanoparticles (K-edge at approximately 81 keV) can be excited, however, exhibiting a fluorescence line in a high peak region of approximately 65 keV, the peak originating from a photon scattered only once. In contrast, the 53 keV spectrum shows a significant background minimum at the fluorescence line of intermediate-heavy elements in the range of approximately 15 to 28 keV, because for this to occur, the incident photon typically must be scattered 5 times or more. Although the 85 keV spectrum also exhibits a minimum in the same energy range, this value is higher, and higher irradiation energies would imply lower fluorescence yields for intermediate-heavy elements.

[0102] Therefore, lower irradiation energies are more efficient for labeling kits of medium-heavy elements, while higher energies are preferred for heavy elements like gold. Variations in the detector position relative to the beam direction (150° in this case) can slightly expand the minimum in the background range, but the determining parameter for the minimum level is the irradiation energy.

[0103] In the specific example of mouse XFI, iodine can be chosen as the X-ray fluorescent element for the first labeling substance, and 53 keV can be chosen as the irradiation photon energy (see [link to relevant documentation]). Figure 7 The irradiation photon energy of 53 keV is significantly higher than that of the iodine edge at 33 keV. After multiple scatterings, the incident photon falls into the energy range of iodine's X-ray fluorescence (approximately 29 keV), thus requiring multiple successive Compton scatterings, specifically approximately five. With each subsequent Compton scattering after the previous one, the overall probability decreases, minimizing the background. Meanwhile, iodine exhibits a sufficiently high fluorescence probability at 53 keV excitation. In this example, elements closely adjacent to iodine in the PTE, such as indium, are provided as additional labeling materials, which are excited at the same irradiation photon energy of 53 keV.

[0104] Based on the above optimization, for example, the selection of labeling materials and irradiation photon energies is performed by simulating the scattering behavior of the sample and / or test, such that the irradiation photon energy of the X-ray radiation is at a distance from the highest absorption edge of all labeling materials in the sample, at which the background noise level of the labeling materials is minimized and the effective cross-sections are similar.

[0105] In another example of XFI on a larger sample, elements closely adjacent to gold in PTE, such as platinum, will be used as X-ray fluorescent elements, and the irradiation photon energy of X-ray radiation, such as 85 keV, will be used.

[0106] Step S1 includes, for example, oral and / or injectable labeling substances, and if an intervention in the body is provided, it may be considered not part of the present invention.

[0107] Imaging apparatus 100 includes an X-ray radiation source device 110 configured to irradiate sample 10 with X-ray radiation 1 (step S2) and emit photon energies of, for example, 50 keV or 100 keV. The X-ray radiation source device 110 is preferably a compact laser-based Thomson source (an X-ray radiation source that generates X-ray radiation based on Thomson scattering of a laser at relativistic electrons), as described, for example, in [8], but may also include a synchrotron source or a conventional X-ray source, such as an X-ray tube, which produces X-ray radiation with sufficiently low divergence and high intensity, particularly in the energy range above the K edge of the X-ray fluorescent element of the labeled material, and should be as monochromatic as possible to improve the optimization of the aforementioned irradiation energy.

[0108] X-ray radiation 1 can be generated in the form of a parallel radiation beam with a diameter covering the entire cross-section of the sample 10 under investigation. In this case, all areas of the sample are irradiated simultaneously, and the labeled material present is excited into X-ray fluorescence 2. Alternatively, X-ray radiation 1 can be generated as a pencil beam, particularly a pencil beam with a diameter smaller than the cross-section of the sample transverse to the beam direction, and can be moved (scanned) relative to the sample 10 by means of X-ray optics (not shown). In this case, areas of the sample are continuously irradiated (“scanned”), and the labeled material present is excited into X-ray fluorescence 2. Since the scanning motion of the pencil beam over the sample 10 can occur within a scan duration that is negligible compared to the typical transport time of the labeled material in the sample 10, a snapshot of X-ray fluorescence 2 is also effectively obtained in the case of scanning X-ray radiation 1.

[0109] The imaging apparatus 100 further includes a detector device 120 arranged for spectral and spatially resolved detection of the X-ray fluorescence 2 of the labeled substance in the sample 10 (step S3). The detector device 120 includes a plurality of detector elements (not shown) that acquire the X-ray spectrum of the X-ray fluorescence 2. The corresponding solid angle range covering a predetermined geometric portion of the sample 10 may be limited by collimators of the individual detector elements or on the group of detector elements. The detector device 120 is constructed, for example, as described in [1]. A collimator may be arranged between the detector device and the sample, and the collimator may reduce scattered radiation, as described in [7].

[0110] With Figure 1 In a different manner, detector device 120 may consist of a single detector element, movable relative to sample 10 and arranged for spectrally resolved detection of X-ray fluorescence 2 of the labeled material in sample 10. If the collimator only cuts out certain regions of the sample within the solid angle range of the detector element, sample 10 can be sampled (scanned) by means of the movable detector element to obtain the spatial distribution of the labeled material. According to another alternative, a single detector element may be arranged to be fixed in place relative to sample 10 and arranged for spectrally resolved detection of X-ray fluorescence 2 of the labeled material in a specific portion of sample 10. In this case, if a collimator is used, the X-ray fluorescence 2 is also obtained in a manner spatially confined to a defined portion of sample 10, such as an organ.

[0111] Alternatively, the marker material can be located by scanning an X-ray beam 1 without a collimator, for example as described in [7].

[0112] Sample 10 contains at least two labeled substances, each with a different X-ray fluorescent element, which are excited by X-ray radiation 1 to produce X-ray fluorescence 2. Detector device 120 transmits an output signal in the form of X-ray spectra, which are correlated with predetermined portions (geometric locations) in sample 10 and include superpositions of fluorescence lines 3 of the X-ray fluorescent elements (see [reference]). Figure 1 Spectral diagram in the image, and Figure 5 , 6 (Example measurement results in the text).

[0113] Furthermore, the imaging device 100 also includes an analysis processing unit 130 for receiving the output signal (spatially resolved X-ray spectrum) of the detector device 120 and for determining the spatial distribution 4 of the labeled substance in the sample 10 based on the detected X-ray fluorescence 2 (step S4). The analysis processing unit 130 includes, for example, a computer device coupled to the detector device 120. The analysis processing unit 130 is configured to execute a computer program, by means of which, preferably taking into account a previously determined background spectrum, the intensity of fluorescence lines at geometric locations in the sample 10 is determined based on the output signal of the detector device 120, and from these, the desired distribution 4 of the labeled substance is determined.

[0114] Distribution of labeled substances 4 (see Figure 1 (The diagram in the diagram) can be output as an image (map) or tabular value, for example. When obtaining the temporal distribution of the labeled substance, a series of motion images, such as video sequences, can be output, representing the movement of the labeled substance in sample 10 and / or the accumulation of at least one of the labeled substances in a portion of sample 10, such as an organ.

[0115] Computer devices may optionally be provided as controllers for imaging device 100, particularly for controlling and / or monitoring X-ray radiation source device 110 and / or detector device 120.

[0116] Sample 10 contains a first labeled substance and at least one additional labeled substance, which differ in their fluorescence lines 3, and will be referenced below. Figure 3 Described through examples. Figure 3 A-3C shows a first type of labeled material in the form of nanoparticles 11 and 12, while Figure 3 D-3E illustrates a second type of labeled material in the form of labeled molecules 14, 15. Nanoparticles 11, 12 may be spherical (as shown in the example) or may be of different shapes, such as angular and / or rod-shaped with multiple sides.

[0117] according to Figure 3 A, Nanoparticles 11 can be generated by a single X-ray fluorescent element, particularly by being entirely composed of X-ray fluorescent elements such as gold or platinum, and have a diameter of, for example, 10 nm. According to Figure 3 B, the nanoparticles 12 can have a core-shell structure including a particle core 13 and a particle coating 14. Just as according to Figure 3 Like nanoparticle 11 of A, the particle core 13 can be generated by a single X-ray fluorescent element, particularly by an X-ray fluorescent element such as platinum. The particle coating 14 is composed of a different material from the particle core 13, for example, gold or a polymer (see [5]). The particle coating 14 has a thickness of, for example, 2 nm. Figure 3 C, Nanoparticles 12 with a core-shell structure and / or a particle coating can be functionalized, i.e., provided with ligands and / or active substance molecules on their surface. The ligands and / or active substance molecules in... Figure 3 In C, triangles are used to illustrate the concept, and in particular, entire biological cells can be included.

[0118] Each labeled substance comprises multiple nanoparticles 11, 12, the amount of which is selected based on the desired concentration in the sample and the desired sensitivity when measuring X-ray spectra using detector device 120. The selection of X-ray fluorescent elements for the nanoparticles and, optionally, the functionalization of the nanoparticles are implemented with the following considerations in mind.

[0119] In the Periodic Table of Elements (PTE), elements that are close to each other have physically very similar properties in terms of the probability of X-ray fluorescence generation and decay. For a given energy of incoming X-ray photons, the first variable depends only on the element. Therefore, the X-ray fluorescent elements of nanoparticles are chosen such that they are directly adjacent or so close in the PTE that the X-ray fluorescence of all elements can be measured with considerable sensitivity. The X-ray fluorescent elements in different nanoparticles include at least two of, for example, iridium, platinum, gold, and bismuth, because these four heavy elements are close to each other in the PTE (Ir, Pt, and gold are practically directly adjacent). Therefore, their properties are very similar, and all four can be used simultaneously for XFI. Another advantageous variation would be medium-heavy X-ray fluorescent elements from zirconium to cerium. In contrast, nanoparticles forming two different labeling materials—for example, some nanoparticles consisting of gold internally while others consist of iodine compounds—are disadvantageous. Gold and iodine are far apart in the PTE, and they can only emit X-ray fluorescence simultaneously when the radiation energy is above the so-called gold edge—if the energy is below the edge, no gold X-ray fluorescence is excited. However, since the iodine edge is far away, the probability of iodine fluorescence is also significantly reduced. In addition, there is a background problem in XFI—(multiple) Compton scattering produces a strong background in the X-ray spectrum of the signal region of the actual fluorescence line (see [1] and [7]), and the background is significantly higher for gold than for iodine, so it is not possible to measure these two elements with similar sensitivity levels overall.

[0120] It is advantageous if the sample, particularly the subject's bulk, cannot distinguish between gold and platinum nanoparticles because they have the same size, the same surface (e.g., the same polymer particle coating), and equal or very similar mass. However, if, for example, only platinum nanoparticles with a gold particle coating are functionalized, while the gold nanoparticles remain unfunctionalized, and both are introduced into the sample, the difference in local concentration measurement can be traced back to the effect of the ligand, since the two nanoparticle types are otherwise indistinguishable from the bulk. Only when the ligand binds purposefully in vivo will the local concentration at the binding site of the platinum nanoparticles be higher than the local concentration of the unfunctionalized gold nanoparticles, thus serving as a reference concentration. These differences in local concentration can be advantageously measured by XFI according to the invention. For this purpose, it is particularly advantageous that the measurement sensitivity of the two internal X-ray fluorescent elements of the nanoparticles is sufficiently high and preferably equal or very similar (possible differences in the analytical processing of the detector output signal can be ignored).

[0121] Another variation using nanoparticles is possible, in which the nanoparticles do not contain any heavy elements internally, but instead contain lighter molecules including X-ray fluorescent elements, such as the two contrast agents mentioned below for computed tomography (CT), and have a polymer shell as a coating on the particles.

[0122] Figure 3 E and 3D relate to variations of the invention in which the active substance, such as a drug molecule, is directly linked to the labeling molecules 15, 16, such as smaller complexes comprising X-ray fluorescent elements, such as triiodobenzene rings or barium atom rings. Triiodobenzene and barium are advantageously available CT contrast agents, wherein the two elements, iodine and barium, are close together in the PTE. Therefore, multiparametric XFI including labeling molecules uses X-ray fluorescent element complexes comprising different X-ray fluorescent elements, to which different drug molecules, for example, can bind. The labeling molecules preferably have chemically equivalent or very similar effects on the sample, such that they do not affect the kinetics of the drug in the sample, or only affect it in a way that is not important to the measurement.

[0123] Regarding the use of different CT contrast agents as markers, it is important to note that conventional CT methods do not allow for multi-parameter measurements because the differences in the absorption of different contrast agents are too small to be measured simultaneously. A significant advantage of this invention compared to CT is that XFI is a spectroscopic method where each element produces its own characteristic line, rather than a purely absorption-based method.

[0124] Figure 4A labeling kit 200 according to the invention for introducing labeled substances into a sample for X-ray fluorescence imaging is illustrated schematically by way of example. The labeling kit 200 includes a container 210, such as a flexible bag, filled with a labeling suspension 220. The labeling suspension 220 includes a physiological fluid, such as a saline solution, in which nanoparticles 11, 12 comprising different X-ray fluorescent elements are dispersed. The design of the nanoparticles 11, 12, the volume of the container 210, and the concentration of the nanoparticles 11, 12 in the labeling suspension 220 are selected according to the specific XFI application. To use the labeling kit 200, the container 210 is connected to a blood vessel of the subject via a tubing and an injection needle, and the labeling suspension 220 comprising the nanoparticles 11, 12 is guided into the blood vessel.

[0125] Alternatively, it can be provided according to Figure 4 The labeling substance kit 200 is intended for oral administration. Alternatively, the labeling substance kit may be provided in a dry form, such as in tablet form, comprising nanoparticles 11, 12 and a physiological binder.

[0126] Through the inventors' testing, biological cells were provided with a predetermined concentration of gold and platinum nanoparticles and arranged in a reagent container (Eppendorf container) with a diameter of 6 mm. The reagent container was then inserted into a piece of animal meat similar in size to a mouse. The sample, including the meat with the inserted reagent container, was irradiated with monochromatic X-rays by the German Electron Synchrotron (DESY) (DESY Hamburg). In further testing, four different fluorescent elements were arranged in a reagent container and irradiated with X-rays from the DESY synchrotron. Tests yielded results demonstrating the distinguishable characteristics and quantitative analytical processing of the measured X-ray fluorescence. Figure 5 and 6 The results are shown in the figure. For example, the techniques described in [1] can also be used to perform corresponding measurements with spatial resolution.

[0127] according to Figure 5 This allows for the clear differentiation of gold and platinum fluorescent lines detected simultaneously with high sensitivity. Analytical processing of the superimposed fluorescent lines—including numerical deconvolution to determine the individual intensities of the fluorescent lines—yields the concentrations of the gold and platinum nanoparticles, taking into account predetermined reference or calibration data.

[0128] Figure 6The X-ray spectrum measured in the presence of a reagent container shows four elements—iridium, platinum, gold, and bismuth—present in solution at predetermined concentrations. The four elements are clearly identifiable in the spectrum. The corresponding concentrations, closely matching the concentrations used, can be determined based on the presence of all fluorescent lines.

[0129] Figure 5 and 6 The background spectra measured when the reagent container contains only water are also shown accordingly. The measurement of the background spectra demonstrates that background knowledge is important for the quantitative analysis of individual fluorescence lines in the processing spectrum in order to infer the corresponding number of fluorescent photons. The background can be measured specifically for each use, or it can be determined using reference or calibration data. In particular, the background can be selected to be approximately the same for all labeled elements and as minimal as possible for all labeled elements.

[0130] When selecting X-ray fluorescence elements, the background curve can also be considered. If an element's fluorescence photon absorption is too strong, making it almost indistinguishable from the background in the spectrum at the fluorescence energy position, then the element cannot be used.

[0131] The features of the invention disclosed in the above description, drawings and claims are of great significance for implementing the invention individually, in combination or sub-combinations, in its various embodiments.

Claims

1. A method for performing multiparameter X-ray fluorescence imaging on a sample (10), said sample (10) comprising at least a portion of the bulk of a biological organism and containing a first labeling substance and at least one additional labeling substance, wherein, At least one of the first labeling substance and the at least one additional labeling substance is coupled to an active substance molecule and / or a ligand molecule, the active substance molecule and / or ligand molecule being provided for a specific interaction with the sample (10), the method comprising the following steps: - Irradiate the sample (10) with monochromatic or narrow-band X-ray radiation (1), wherein the X-ray fluorescence (2) of the first labeled substance and the at least one other labeled substance is excited by the X-ray radiation (1), and the fluorescence lines (3) of the X-ray fluorescence (2) of the first labeled substance and the at least one other labeled substance are different. - Spatial-resolved detection of X-ray fluorescence (2), comprising spectral-resolved detection of X-ray fluorescence (2) of the first labeled substance and the at least one other labeled substance, and -Based on the detected X-ray fluorescence (2), the distribution of the first labeled substance in the sample (10) is determined, and additionally, the distribution of at least one of the at least one other labeled substance in the sample (10) is determined, wherein - The fluorescence probabilities exhibited by the first labeling substance and the at least one other labeling substance, the attenuation of X-ray fluorescence (2) in the sample (10), and the background noise level due to scattering in the sample (10) are equal or similar, so that the detection of X-ray fluorescence (2) has equal sensitivity or such similar sensitivity when the labeling substance concentration is the same: the difference in sensitivity is negligible in relation to the analysis of the detection, and - The irradiation photon energy of X-ray radiation (1) is higher than the absorption edge of all labeled materials, where, The irradiation photon energy of the X-ray radiation is a distance away from the highest absorption edge of all labeled substances in the sample (10), at which the background noise level of the labeled substances is minimal and the fluorescence probability is similar.

2. The method according to claim 1, wherein - The statistical significance level of the labeled material is maximized because the irradiation photon energy of the X-ray radiation (1) is selected at a distance above the highest absorption edge of the labeled material in the sample, thereby minimizing and equalizing or approximately equalizing the background noise level of the labeled material, while maximizing or approximately equalizing the fluorescence probability and the transmittance through the sample.

3. The method according to claim 1 or 2, wherein - Use a common excitation beam of X-ray radiation (1) to excite and simultaneously detect the X-ray fluorescence of the first labeled substance and the at least one other labeled substance (2).

4. The method according to claim 1 or 2, wherein - Use different excitation beams of X-ray radiation (1) with different energies to excite and simultaneously or sequentially detect the X-ray fluorescence (2) of the first labeled substance and the at least one other labeled substance.

5. The method according to claim 1 or 2, wherein - The first labeling substance and the at least one additional labeling substance each comprise one of nanoparticles (11, 12) and labeling molecules (15, 16).

6. The method according to claim 5, wherein - The first labeling substance or the at least one other labeling substance respectively comprises nanoparticles (11, 12), and - The nanoparticles (11, 12) of the first labeling substance and the at least one other labeling substance include surfaces that are indistinguishable from the sample (10), wherein, The nanoparticles (11, 12) contain different elements.

7. The method according to claim 5, wherein - The first labeled material comprises first-type nanoparticles (11) mainly containing a first X-ray fluorescent element, and - The at least one additional labeling material includes at least one additional type of nanoparticle (12), which respectively mainly contains at least one additional X-ray fluorescent element.

8. The method according to claim 7, wherein - Each type of nanoparticle (11, 12) contains only one of the first X-ray fluorescent element and at least one other X-ray fluorescent element.

9. The method according to claim 7 or 8, wherein - The first type of nanoparticles (11) carry first type of active substance molecules and / or ligand molecules provided for chemical and / or physical interaction with the sample (10), and - The at least one other type of nanoparticle (12) carries different types of active substance molecules and / or ligand molecules provided for chemical and / or physical interaction with the sample (10), or does not carry any active substance molecules or any ligand molecules.

10. The method of claim 9, wherein - Each type of nanoparticle (11, 12) carries only one specific type of active substance molecule and / or ligand molecule.

11. The method according to claim 7 or 8, wherein - At least one of the first type of nanoparticles (11) and the at least one other type of nanoparticles (12) has a core comprising a particle core (13) and a particle coating (14). -Shell structure.

12. The method of claim 11, wherein - All nanoparticles (11, 12) have a core-shell structure.

13. The method of claim 11, wherein - The particulate coating (14) may include metal or non-metallic materials.

14. The method of claim 11, wherein - The particle coating (14) of all nanoparticles (11, 12) is made of the same material to which active substance molecules and / or ligand molecules can bind.

15. The method of claim 5, wherein - The nanoparticles (11, 12) respectively contain iridium, platinum, gold, bismuth, silver, iodine, palladium, cadmium or indium.

16. The method of claim 5, wherein - The nanoparticles (11, 12) contain different X-ray contrast agent molecules.

17. The method of claim 5, wherein - Each type of nanoparticle (11, 12) has a different nanoparticle size.

18. The method of claim 5, wherein -The first labeled substance comprises a first type of labeled molecule (15) containing a first X-ray fluorescent element. -The at least one additional marker substance comprises at least one additional type of marker molecule (16) that contains at least one additional X-ray fluorescent element.

19. The method of claim 5, wherein - The first labeling material comprises nanoparticles (11) containing a first X-ray fluorescent element. -The at least one additional marker substance comprises a marker molecule (15) that contains at least one additional X-ray fluorescent element.

20. The method according to claim 18 or 19, wherein - The labeled molecules (15, 16) bind to the active substance molecule and / or ligand molecule, wherein the labeled molecules (15, 16) respectively include the first X-ray fluorescent element and one of the at least one other X-ray fluorescent element.

21. The method according to claim 18 or 19, wherein The X-ray fluorescent elements include silver, indium, palladium, cadmium, iodine, or barium.

22. The method according to claim 1 or 2, wherein - Determine the time function of the spatial distribution of the first marker and the at least one other marker in the sample (10).

23. The method according to claim 1 or 2, wherein - The active substance comprises a biological cell, and at least one of the first labeling substance and the at least one additional labeling substance is coupled to the biological cell, and Determining the distribution of the first marker and the at least one additional marker includes detecting the biological cells transported through the sample.

24. The method according to claim 1 or 2, wherein - The first marker and the at least one other marker were introduced into the sample (10) in different ways.

25. The method according to claim 1 or 2, wherein - The first labeling substance and the at least one additional labeling substance are configured such that they have the same effect on the sample without the presence of coupled active substance molecules and / or ligand molecules.

26. The method according to claim 13, wherein, The metal in question is gold.

27. The method according to claim 13, wherein, The non-metallic material is a polymer, liposome, or micelle.

28. An imaging apparatus (100) configured for multi-parameter X-ray fluorescence imaging to study a sample (10) by means of the method according to any one of the preceding claims, wherein, The sample comprises at least a portion of the bulk of a biological organism and contains a first labeling substance and at least one additional labeling substance, wherein at least one of the first labeling substance and the at least one additional labeling substance is coupled to an active substance molecule and / or a ligand molecule, the active substance molecule and / or the ligand molecule being provided for a specific interaction with the sample (10), and the imaging device (100) comprises: - An X-ray radiation source device (110) is arranged to irradiate the sample (10) with monochromatic or narrow-band X-ray radiation (1), wherein the X-ray fluorescence (2) of the first labeled substance and the at least one other labeled substance is excited, and the fluorescence lines (3) of the X-ray fluorescence (2) of the first labeled substance and the at least one other labeled substance are different, wherein the irradiation photon energy of the X-ray radiation is at a distance from the highest absorption edge of all labeled substances in the sample (10), at which the background noise level of the labeled substances is minimal and the fluorescence probability is similar. - Detector device (120), configured for spatially and spectrally resolved detection of the X-ray fluorescence (2) of the first labeled substance and the at least one other labeled substance, and - An analytical processing apparatus (130) configured to determine the spatial distribution of the first marker in the sample (10) based on the detected X-ray fluorescence (2), and additionally to determine the spatial distribution of the at least one other marker in the sample (10).

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