TECHNICAL FIELD

The present invention relates to specific complexes comprising heavy metal ions having an atomic number of 29 or higher and 83 or lower (preferably 81 or lower) and one or more ligand(s) selected from the group consisting of specific xanthene derivatives, preferably eosin Y and erythrosin B. In particular, the invention relates to the use of the complexes as contrast agents for a computed tomography scanning of a biological sample. Moreover, the invention relates to specific ex vivo methods for investigating a biological sample by means of computed tomography scanning methods, wherein the method comprises staining the biological sample with a solution comprising one or more of the complex(es); or (herein sometimes referred to as in situ staining) wherein the method comprises staining the biological sample with a staining solution comprising one or more specific xanthenes derivatives (e.g. eosin Y and/or erythrosin B), and separately contacting the biological sample with one or more staining solution(s) comprising one or more heavy metal ions having an atomic number of 29 or higher and 83 or lower (preferably 81 or lower).

BACKGROUND

The study of tissue is known as histology or, in connection with disease, histopathology. The conventional tools for studying tissues are the paraffin block in which tissue is embedded and then sectioned, the histological stain, and the optical microscope. In the last decades, amongst others, the use of frozen tissue sections has enhanced the detail that can be observed in tissues. With these tools, the appearances of tissues can be examined in health and disease, enabling medical diagnosis and prognosis.

Many conventional histological methods require specific staining of the cell cytoplasm (in particular, bind to the proteins and/or peptides present within the cell cytoplasm), which provides instrumental details for diagnosis. Currently, the eosin Y-based stain is the most commonly used cell cytoplasm contrast agent (CA), which is used as counter stain in case of hematoxylin-based staining in conventional histology. Recently, the erythrosine B stain is also used as CA for cell cytoplasm staining. A summary of the aforementioned staining methods and their respective characteristics is given by Mulisch and Welsch (M. Mulisch and U. Welsch, Romeis Mikroskopische Technik, 19th Ed. Springer Spektrum, Heidelberg, 2015, p. 189, table 10.1). One major limitation of conventional histological methods, which are frequently used for diagnostic purposes in a clinical setting, is the production of two-dimensional (2D) images obtained by destructive preparation of a three-dimensional (3D) tissue sample. The destructive preparation in conventional histological methods is obligatory since thin tissue sections (generally 2 to ΙΟμιη thick) have to be prepared since only these sections can be adequately assessed by (light) microscopic methods. The preparation of the afore-mentioned tissue sections is not only time-consuming, but also interrelated to an information loss which considerably limits the diagnostic potential of conventional histology. As an illustrative example, reference is made to a 1 x 1 x 1mm histological sample. The slices produced in conventional histology can vary but a thickness of about 2μιη is commonly used. This, however, were to result in about 500 sections if the entire biological sample were to be investigated. When looking at the daily reality of a histologist/pathologist who has to inspect about 500 biological samples per day, it is evident that only a few microscopic slices per biological sample are prepared and can be investigated.

X-ray imaging techniques such as computed tomography (CT), in particular Micro-Computed Tomography (μΟΤ), allow for a non- destructive investigation of a 3D biological sample enabling sample screening, and thus, aid to determine regions of interest for further histological examinations. Within a short period of time (usually about 2 hours) a complete tomography is obtained which provides 3D information for the entire biological sample. During this period of time about 1000 slices are produced for each viewing plane (xy, yz, xz).

At present, however, the application of CT (in particular μΟΤ) for biological samples is severely limited by the missing contrast of many biological samples (in particular soft tissue samples). A sufficient contrast, however, is important to visualize morphological details. While there are some CAs for CT applications described in the prior art such as iodine potassium iodide (IKI), iodine in ethanol (¾E) and phosphotungstic acid (PTA) (Metscher, B. D. (2009). MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non- mineralized animal tissues. BMC Physiology, 9, 1-14; Martins de Souza e Silva, J. et al. (2015). Three-dimensional non- destructive soft- tissue visualization with X-ray staining micro-tomography. Scientific Reports, 5, 14088.), the quality of the tomography/tomographic images obtainable with the prior art CAs is limited. In particular, it is very challenging to obtain a homogeneous staining result with the prior art CAs/staining methods, amongst others, due to diffusion problems of the prior art CAs in particular in case of voluminous biological samples. For example, osmium tetroxide has been proposed as a staining agent for producing a microCT image of certain specimens (cf. WO 2007/089641). However, said staining agent is of limited suitability for routine laboratory investigations due to its toxicity and has limitations as regards homogenous staining results due to poor diffusion.

Moreover, compatibility of the prior art CAs with conventional histological methods is not always given so that (i) a combination of both methods is not possible, or (ii) a time-consuming removal (if possible at all) of the CAs is necessary. In view of this, there is a need for further improvement of the existing CAs and the staining methods described in the prior art.

In view of the above, it is an object of the present invention to overcome one or more of the disadvantages of the prior art CAs/staining methods.

SUMMARY

The present inventors surprisingly found novel CAs as well as novel staining methods which can provide a contrast enhancement in CT investigations of biological samples. This, in turn, can facilitate the provision of highly detailed 3D structural information of the investigated biological sample, which - amongst others - aid to determine regions of interest. Moreover, the inventive CAs are compatible with conventional histological methods so that a further investigation of regions of interest with conventional histological methods is possible without the need to perform a time and cost consuming removal of the CAs. In preferred embodiments, it is possible to obtain complete and homogeneous staining results throughout the whole biological sample. Moreover, in particularly preferred embodiments a synergistic staining result can be obtained.

In particular, the present invention relates to the following items 1 to 53:

1. A complex comprising:

one or more heavy metal ion(s) M and one or more ligand(s) R, wherein

at least one M is a heavy metal ion having an atomic number of 29 or higher and 83 or lower (preferably 81 or lower), and

at least one R is a xanthene derivative (preferably selected from the group consisting of eosin Y and erythrosin B).

2. The complex according to item 1, wherein the complex is represented by the following formula (I):

M _m R _n (I),

in which at least one M is a heavy metal ion having an atomic number of 29 or higher and 83 or lower (preferably 81 or lower),

at least one R is a xanthene derivative (preferably selected from the group consisting of mono-, di-, tribromofluorescein; mono-, di, triiodofluorescein; eosin B; eosin Y and erythrosin B), and m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The complex according to item 1 or 2, wherein

at least one of the one or more heavy metal ions(s) M is a heavy metal ion having an atomic number of 47 or higher and 83 or lower (preferably 81 or lower),

at least one of the one or more ligand(s) R is eosin Y, and

m and n are each independently 1, 2 or 3. The complex according to any one of items 1 to 3, wherein

m and n are both 1. The complex according to any one of items 1 to 4, wherein

at least one of the one or more heavy metal ion(s) M is an ion of a heavy metal selected from the group consisting of silver (Ag), barium (Ba), gadolinium (Gd), lutetium (Lu), gold (Au), lead (Pb) and bismuth (Bi), (or selected from the group consisting of silver (Ag), barium (Ba), lutetium (Lu), gold (Au), lead (Pb) and bismuth (Bi)). The complex according to item 5, wherein

at least one of the one or more heavy metal ion(s) M is selected from the group consisting of silver(I) (Ag ⁺ ), barium(II) (Ba ²⁺ ), gadolinium(III) (Gd ³⁺ ), lutetium(III) (Lu ³⁺ ), gold(III) (Au ³⁺ ), lead(II) (Pb ²⁺ ) and bismuth(III) (Bi ³⁺ ) (or selected from the group consisting of silver(I) (Ag ⁺ ), barium(II) (Ba ²⁺ ), lutetium(III) (Lu ³⁺ ), gold(III) (Au ³⁺ ), lead(II) (Pb ²⁺ ) and bismuth(III) (Bi ³⁺ )). The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is selected from the group consisting of Ag ⁺ , Ba ²⁺ , Gd ³⁺ , Lu ³⁺ and Au ³⁺ (or selected from the group consisting of Ag ⁺ , Ba ²⁺ , Lu ³⁺ and Au ³⁺ ). The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is selected from the group consisting of Ag ⁺ , Ba ²⁺ and Gd ³⁺ (or selected from the group consisting of Ag ⁺ and Ba ²⁺ ). The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is Ag ⁺ . The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is Ba ²⁺ . The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is Gd ³⁺ . The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is Lu ³⁺ . The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is Au ³⁺ . The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is Pb ²⁺ . The complex according to item 6, wherein

at least one of the one or more heavy metal ion(s) M is Bi ³⁺ . An ex vivo method for investigating a biological sample comprising

(aspect 1)

(al) staining the biological sample with a solution (AB) comprising one or more complex(es) as defined in any one of items 1 to 15; and

(b) subjecting the stained biological sample to a computed tomography scanning method; or (aspect 2)

(a2) staining the biological sample with

(i) a solution (A) comprising a xanthene derivative (preferably mono-, di-, tribromofluorescein; mono-, di, triiodofluorescein; eosin B; eosin Y and/or erythrosin B), and

(ii) one or more solution(s) (B) comprising one or more heavy metal ion(s) as defined in any one of items 1 to 3 and 5 to 15,

wherein the biological sample is contacted with solution (A) separately from the one or more solution(s) (B); and

(b) subjecting the stained biological sample to a computed tomography scanning method. The method according to item 16 (aspect 1 / aspect 2), wherein the computed tomography scanning method is an absorption-based scanning method. The method according to item 16 (aspect 1 / aspect 2) or 17, wherein the computed tomography scanning method is a X-ray absorption-based scanning method. The method according to any one of items 16 (aspect 1 / aspect 2), 17 and 18, wherein the computed tomography scanning method is selected from the group consisting of Micro- Computed Tomography (μΟΤ) and Nano-Computed Tomography (nanoCT). The method according to item 19, wherein the computed tomography scanning method is μΟΤ. The method according to item 19, wherein the computed tomography scanning method is nanoCT. The method according to any one of items 16 (aspect 1 / aspect 2) and 17 to 21, wherein the biological sample is a soft tissue sample. The method according to any one of items 16 (aspect 1 / aspect 2) and 17 to 22, wherein the biological sample is a human soft tissue sample. The method according to any one of items 16 (aspect 1 / aspect 2) and 17 to 23, wherein the biological sample originates from lung, kidney, liver, brain, spleen, heart or cartilage. The method according to any one of items 16 (aspect 1 / aspect 2) and 17 to 24, wherein the biological sample is subjected to chemical fixation by means of one or more chemical fixative(s) prior to staining. The method according to item 25, wherein the one or more chemical fixative(s) is/are a water- based formaldehyde solution or a water-based glutaraldehyde solution, or mixtures thereof. The method according to item 25, wherein the one or more chemical fixative(s) is/are a formaldehyde solution in Dulbecco's phosphate buffered saline or a formaldehyde solution in phosphate buffered saline. The method according to any one of items 25 to 27, wherein the one or more chemical fixative(s) comprise one or more acid(s). The method according to item 28, wherein the one or more acid(s) is glacial acetic acid. The method according to any one of items 16 (aspect 1 / aspect 2) and 17 to 29, wherein the method comprises, in addition to staining step (al) or staining step (a2) an additional step of staining with an additional staining agent, preferably hematein. The method according to any one of items 16 (aspect 2) and 17 to 30, wherein solution (A) comprises eosin Y.

The method according to item 31 , wherein the concentration of eosin Y in solution (A) is in the range of about 10 to about 50 wt/vol-%. The method according to item 31, wherein the concentration of eosin Y in solution (A) is in the range of about 20 to about 40 wt/vol-%. The method according to item 31, wherein the concentration of eosin Y in solution (A) is in the range of about 25 to about 35 wt/vol-%. The method according to item 31, wherein the concentration of eosin Y in solution (A) is about 30 wt/vol-%. The method according to any one of items 16 (aspect 2) and 17 to 35, wherein the time period of contacting the biological sample with solution (A) or the one or more solution(s) (B) is 3 hour or more. The method according to item 36, wherein the time period is 6 hours or more. The method according to item 36, wherein the time period is 12 hours or more. The method according to item 36, wherein the time period is 24 hours or more. The method according to item 36, wherein the time period is 48 hours or more. The method according to item 36, wherein the time period is 72 hours or more. The method according to item 36, wherein the time period is 96 hours or more. The method according to any one of items 16 (aspect 2) and 17 to 42, wherein the biological sample is contacted with the one or more solution(s) (B) before the biological sample is contacted with solution (A). The method according to any one of items 16 (aspect 2) and 17 to 43, wherein the main solvent of solution (A) and the main solvent of the one or more solution(s) (B) is a water-based solution. The method according to any one of items 16 (aspect 1 / aspect 2), 17 to 29 and 31 to 44, wherein the method does not comprise, in addition to staining step (al) or staining step (a2), an additional step of staining with an additional staining agent. 46. The method according to any one of items 16 (aspect 2) and 17 to 45, wherein the one or more solution(s) (B) comprise one or more heavy metal ion(s) M selected from the group consisting of Ag ⁺ , Ba ²⁺ and Gd ³⁺ (or selected from the group consisting of Ag ⁺ and Ba ²⁺ ).

47. The method according to item 46, wherein the one or more solution(s) (B) comprise Ba ²⁺ .

48. Use of a complex according to any one of items 1 to 15 as an ex vivo contrast agent for a computed tomography scanning of a biological sample.

49. The use according to item 48, wherein the computed tomography scanning is an absorption-based scanning method.

50. The use according to item 48 or 49, wherein the computed tomography scanning is an X-ray absorption-based scanning method.

51. The use according to any one of items 48 to 50, wherein the computed tomography scanning method is selected from the group consisting of μΟΤ and nanoCT.

52. The use according to item 51, wherein the computed tomography scanning method is μΟΤ.

53. The use according to item 51 , wherein the computed tomography scanning method is nanoCT. In a particularly preferred embodiment, the present invention relates to:

An ex vivo method for investigating a biological sample comprising

(a) staining the biological sample with

(i) a solution (A) comprising a xanthene derivative (preferably eosin Y), and

(ii) one or more solution(s) (B) comprising one or more heavy metal ion(s) M, wherein the biological sample is contacted with solution (A) separately from the one or more solution(s) (B); and

(b) subjecting the stained biological sample to a computed tomography scanning method wherein the time period of contacting the biological sample with solution (A) or the one or more solution(s) (B) is 3 hour or more,

wherein the biological sample is a human soft tissue sample,

wherein the biological sample is subjected to chemical fixation by means of one or more chemical fixative(s) comprising one or more acid(s) prior to staining,

wherein the method comprises, in addition to staining step (a) an additional step of staining with an additional staining agent, preferably hematein, and wherein is a heavy metal ion selected from the group consisting of Ag' , Ba ²⁺ , Gd ^, , Lu ^?+ and Au ^:,÷ (or Ag , Ba ^2÷ , Lu ^J+ and Au ^' ),

preferably Ag\ Ba ²⁺ and Gd ^1÷ (or Ag ⁺ and Ba ^2÷ ).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is also illustrated by the following illustrative figures.

Figure 1: Line plots obtained by micro CT investigations of acidified and non-acidified turkey liver samples which were stained, amongst others, with a barium-eosin Y complex (te/ra-bromo barium eosin complex; 25 mg/mL; c = 31.9m ) for 72 hours as described in Example 1.

Figure 2: (a) Micro CT image of acidified and non-acidified turkey liver samples which were stained, amongst others, with a Ba(OAc) ₂ solution (c = 217 m ) and an eosin Y solution (30 wt/vol- ) for 24 hours each, (b) Line plots obtained by micro CT investigations of the respective samples as described in Example 2.

Figure 3: (a) Micro CT image of acidified and non-acidified turkey liver samples which were stained, amongst others, with a Gd(OAc)i solution (c = 145 mM) and an eosin Y solution (30 wt/vol-%) for 24 hours each, (b) Line plots obtained by micro CT investigations of the respective samples.

Figure 4: Schematic drawing of a biological sample placed in an appropriate container for computed tomography scanning ((A): Stained tissue sample positioned such mat it is not moving within the sample container; (B): Sample container (e.g. Eppendorf tube made from PE) designed that the sample container on top fits perfectly into the container at the bottom. Both sample containers are mounted on top of each other by an appropriate glue (e.g. two component glues from UHU); (C): Sample container such as an Eppendorf tube made form polyethylene (PE); (D): Solvent, e.g. 70% ethanol, is placed at the bottom of the sample container in order to wet the tissue sample).

Figure 5: The chemical formulas of eosin Y derivatives: mono-bromo, cfr-bromo, /n-bromo and leira- bromo eosin derivative. Used abbreviations: EOY = eosin Y, M _w = molecular weight.

Figure 6: The chemical formulas of erythrosine B derivatives: ono-i ^' odo, c//-iodo, rtv ^' -iodo and tetra- ^' iodo erythrosine derivative. Used abbreviations: ERB = erythrosin B, M _vv = molecular weight.

Figure 7: The reaction scheme showing the synthesis of eosin Y derivatives: mono-bromo (A), dr-bromo (B), /n ^' -bromo (C) and leira-bromo (D) eosin derivatives. Used abbreviations: eq. = equivalents; i t = room temperature.

Figure 8: The reaction scheme showing the detailed synthesis of eosin Y derivatives: mono-bromo, di- bromo, m ^' -bromo and tetra -bromo eosin derivatives, which is expressed by a general X being either a bromo, iodo or hydrogen atom. Used abbreviations: NBS = N-bromo-succinimide; NIS = N-iodo- succinimide; eq. = equivalents; rt = room temperature; HCI = hydrochloric acid; NaOH 0 sodium hydroxide; cond. = condition; min = minutes. In black: reaction regarding the halogenation of the fluorescein core. In grey: Reaction schemes for individual steps of the reaction; on the left: reaction step 1) and on the right: reaction step 2).

Figure 9: The logP values demonstrating the tendency of water solubility are shown for all four eosin Y derivatives in comparison to the fluorescein. The grey circle highlights the similar logP value of the dibromo eosin derivative compared to the fluorescein. Used abbreviations: EOY = eosin Y.

Figure 10: Examples of proposed chemical formulas of the heavy metal eosin derivatives in comparison to the sodium eosin derivatives. For simplicity reasons an example with a metal ion in oxidation state two is shown. The examples of heavy metal eosin salts are not restricted to metals of an oxidation state two. The CT neutral (highlighted in black) and CT active components (highlighted in grey) are shown.

Figure 11: The tetrabromo barium eosin salt was tested in a staining experiment and investigated macroscopically. The acidification during fixation results in a complete and homogeneous staining result for the barium tetrabromo eosin salt after 72 hours of staining time.

Figure 12:

The line plots of i z-bromo barium eosin complex and feira-bromo barium eosin complex are displayed. Soft tissue samples have been acidified during fixation. The i z ^' -bromo barium eosin complex performed better than the feira-bromo derivative at the maximum water solubility for the dibromo barium eosin complex of 45 mg/mL.

Legend: Sample 1 = teira-bromo barium eosin derivate (c = 25 mg/mL, with acid); Sample 2 = i z ^' -bromo barium eosin derivate (c = 25 mg/mL, with acid); Sample 3 = i z ^' -bromo barium eosin derivate (c = 45 mg/mL, with acid).

Figure 13: The line plots of dibromo sodium eosin derivatives and dibromo barium eosin derivatives are displayed. Soft tissue samples being acidified during fixation or prior to staining have better contrast enhancement. The barium eosin derivatives have much better contrast enhancement compared to the sodium dibromo derivatives. This effect will be even more pronounced if equimolar concentrations will be used for the μΟΤ measurements.

Legend: Sample 1 = dibromo barium eosin derivate (c = 45 mg/mL, no acid); Sample 2 = dibromo sodium eosin derivate (c = 25 mg/mL, with acid); Sample 3 = dibromo barium eosin derivate (c = 25 mg/mL, with acid); Sample 4 = dibromo barium eosin derivate (c = 25 mg/mL, no acid); Sample 5 = dibromo sodium eosin derivate (c = 45 mg/mL, with acid); Sample 6 = dibromo barium eosin derivate (c = 45 mg/mL, with acid). Figure 14: UV-vis spectra are shown of all eosin Y derivative salts including the heavy metal eosin salts. The chinoid form was seen for all samples. The UV-vis spectra were obtained for all salts with a concentration of c = 1 mM in DMSO and for all lactones with a concentration of c = 12 μΜ in DMSO. (A) UV-vis spectra of eosin Y (EOY), dibromo eosin Y (EOY Di Na), tetrabromo silver eosin (EOY Ag), tetrabromo barium eosin (EOY Ba), dibromo barium eosin (EOY Di Ba), tetrabromo lead eosin (EOY Pb) and tetrabromo copper eosin (EOY Cu). (B) Difference between lactone and sodium eosin (chinoid form). (C) Difference between lactone and barium eosin derivative (chinoid form). (D) Difference between dibromo lactone and dibromo barium eosin derivative (chinoid form). (E) Difference between eosin lactone and dibromo eosin lactone derivative (chinoid form). Other used abbreviations: DMSO = dimethyl sulfoxide, mM = mili molar, μΜ = micro molar, a.u. = arbitrary units.

Figure 15: Histological slide obtained from a turkey liver stained with tetra-bromo-barium eosin salt for 72 h at a concentration of 25 mg/mL. Microscopic slide directly obtained after staining and CT investigations. No further treatments by the histologist were performed. Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). 20x magnification was used to produce this image.

Figure 16: Histological slide obtained from a turkey liver stained with tetra-bromo-barium eosin salt for 72 h at a concentration of 25 mg/mL. Microscopic slide directly obtained after staining and CT investigations. Hematoxylin was applied as counter stain to the tetra-bromo-barium eosin salt. Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). 20x magnification was used to produce this image.

Figure 17: Histological slide obtained from a turkey liver stained with tetra-bromo-barium eosin salt for 72 h at a concentration of 25 mg/mL. Microscopic slide directly obtained after staining and CT investigations. No further treatments by the histologist were performed. Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). 40x magnification was used to produce this image.

Figure 18: Histological slide obtained from a turkey liver stained with tetra-bromo-barium eosin salt for 72 h at a concentration of 25 mg/mL. Microscopic slide directly obtained after staining and CT investigations. Hematoxylin was applied as counter stain to the tetra-bromo-barium eosin salt. Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). 40x magnification was used to produce this image.

Figure 19: Macroscopic images of the staining solutions used for (A) the 1 ^st and (B) the 2 ^nd staining step as described in Example 2. Figure 20: Macroscopic images of the soft tissue sample after the 2 ^nd staining step as described in Example 2. Legend: 1 i) acidified during fixation and ii) Ba(OAc)2 - Stoichiometric; 2 i) acidified during fixation, ii) Ba(OAc)2 - Stoichiometric and iii) Na-Eosin - Stoichiometric; 3 i) acidified during fixation - Control Sample acidified; 4 i) acidified during fixation and ii) Na-Eosin - Eosin Sample acidified; 5 i) fixation and ii) Ba(OAc)2 - Stoichiometric; 6 i) fixation, ii) Ba(OAc)2 - Stoichiometric and iii) Na-Eosin - Stoichiometric; 7 i) fixation - Control Sample and 8 i) fixation and ii) Na-Eosin - Eosin Sample.

Figure 21: All microscopic slides shown in (A) to (C) are directly derived from the 3D stained tissue sample applying the two-step protocol as described in Example 2. (A) Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). The image was produced with lOx magnification. (B) Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). The image was produced with 40x magnification. (C) Hematoxylin as counter stain was additionally used to show full compatibility with the in situ staining protocol. Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). The image was produced with 40x magnification.

Figure 22: Macroscopic images of solutions of the barium-eosin Y complex (Ba-Eo) vs the sodium-eosin Y complex (Na-Eo).

Figure 23: A comparison of a tissue staining with sodium eosin Y complex as compared to the in situ staining (i.e. staining with a barium acetate solution and subsequently with a sodium eosin solution). All microscopic slides shown in (A) to (C) are directly derived from a 3D stained tissue sample. (A) Histological analysis was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). The image was produced with lOx magnification. (B) Histological analyses was performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). The image was produced with 20x magnification. (C) Hematoxylin as counter stain was additionally used to show full compatibility with the in situ staining protocol. Histological analyses were performed using an Axio Imager 2 microscope and AxioVision Software (Zeiss). The image was produced with 20x magnification. The comparison shows that an in situ complex formation of an barium-eosin Y complex as a result from the two-step staining protocol as the tissue is colored with a change to more orange red as compared to the dark pinkish red of the sodium salt complex of eosin.

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to better describe the present invention. Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the skilled person in the art to which this invention belongs. In case of conflict, the present description, including explanations of terms, will control.

Explanation of common terms and methods in the preparation, staining and microscopic investigation of biological samples (in particular tissue samples) may be found in Mulisch and Welsch (M. Mulisch and U. Welsch, Romeis Mikroskopische Technik, 19th Ed. Springer Spektrum Akademischer Verlag, Heidelberg, 2010), Murphy and Davidson (D. B. Murphy and M. W. Davidson, Fundamentals of Light Microscopy and Electronic Imaging, 2 ^nd Ed. John Wiley and Sons, Inc., Hoboken, New Jersey, 2016) or Schnatz et al. (T. Dockland, D. W. Hutmacher, M. Mah-Lee Ng, J.-T. Schantz in Manuals in Biomedical Research: Volume 2 - Techniques in Microscopy for Biomedical Applications, World Scientific Publishing Co. Pte. Ltd., 2006). Explanation of common terms and methods in the preparation of (heavy) metal-complexes comprising one or more ligands may be found in Lawrance (G. A. Lawrance, Introduction to Coordination Chemistry, John Wiley and Sons Ltd., West Sussex, UK, 2010), Gispert (J. R. Gispert, Coordination Chemistry, Wiley- VCH Verlag GmbH & Co. KGaA, Weinheim, 2008), Soni and Soni (P. L. Soni and V. Soni in Coordination Chemistry: Metal Complexes, CRC Press - Taylor and Francis Group, Boca Raton, FL, 2013) or Bhatt (Vasishta Bhatt, Essentials in coordination Chemistry - A simplified approach with 3D visuals, Academic Press - Elsevier, London, 2016). Explanation of common terms, methods and/or devices used in the computed tomography scanning (in particular X-ray absorption-based computed tomography scanning methods) of samples may be found in Haidekker (M. A. Haidekker in Medical Imaging Technologies - Computed Tomography, Springer Briefs in Physics, 2013, p. 37-53), KachelrieB (M. KachelrieB in Molecular Imaging I, Handbook of Experimental Pharmacology - Micro-CT, W. Semmler and M. Schwaiger (Eds.), Springer Verlag Berlin Heidelberg, 2008, Volume 185/1, p.23-52.) or Stock (S. R. Stock in MircoComputed Tomography: Methodology and Applications, CRC Press - Taylor and Francis Group, Boca Raton, FL, 2011). The above citations are incorporated by reference.

The term "heavy metal" refers to metals having a density of more than 5 g/cm ³ and additionally includes the chemical elements strontium (Sr) and barium (Ba) as well as selenium (Se), rubidium (Rb), yttrium (Y) and caesium (Cs). Specifically, the term heavy metal refers to chemical elements selected from the group consisting of Cu (copper), Zn (zinc), Ga (gallium), Ge (germanium), As (arsenic), Se (selenium), Rb (rubidium), Sr (strontium), Y (yttrium), Zr (zirconium), Nb (niobium), Mo (molybdenum), Tc (technetium), Ru (ruthenium), Rh (rhodium), Pd (palladium), Ag (silver), Cd (cadmium), In (indium), Sn (tin), Sb (antimony), Te (tellurium), Cs (caesium), Ba (barium), La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu, (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), Hf (hafnium), Ta (tantalum), W (tungsten), Re (rhenium), Os (osmium), Ir (iridium), Pt (platinum), Au (gold), Hg (mercury), Tl (thallium), Pb (lead) and Bi (bismuth). The terms "ligand" and "optional ligands" generally refers to an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's electron pairs. Ligands are viewed as Lewis bases (although rare cases are known to involve Lewis acidic ligand).

The term "staining" as used herein refers to a procedure suitable for enhancing the contrast of images of a biological sample, in particular of images obtained by a computed tomography scanning method. Accordingly, staining of the biological sample does not necessarily require that the biological sample is colored after the staining procedure.

The term "water-based solution" or "aqueous solution" refers to solutions in which the main solvent is water. A given solvent is considered to be the "main solvent" if the solvent in question accounts for at least 55%, preferably at least 70%, more preferably at least 90%>, particular preferably at least 99%, and most preferably at least 99.9% of all solvents contained in the solution.

As used herein, the term "about" refers to ±10%> of the indicated numerical value, and in particular to ±5%) of the indicated numerical value. Whenever the term "about" is used, a specific reference to the exact numerical value indicated is also included. If the term "about" is used in connection with a parameter that is quantified in integers, the numbers corresponding to ±10%> or ±5% of the indicated numerical value are to be rounded to the nearest integer. For example, in the context of integers the expression "about 25" refers to the range of 23 to 28, in particular the range of 24 to 26, and preferably refers to the specific value of 25.

If not indicated otherwise, concentrations given in percentages [%] refer to [weight/volume-%>] in volume.

HEAVY METAL ION COMPLEXES

The inventive complex comprises one or more heavy metal ion(s) M and one or more ligand(s) R, wherein at least one M is a heavy metal ion having an atomic number of 29 or higher and 83 or lower (preferably 29 or higher and 81 or lower), and at least one R is a xanthene derivative (preferably selected from the group consisting of eosin Y and erythrosin B). In particular, the inventive complex is represented by the following formula (I):

M _m R _n (I),

in which at least one M is a heavy metal ion having an atomic number of 29 or higher and 83 or lower (preferably 29 or higher and 81 or lower), at least one R is a xanthene derivative (preferably selected from the group consisting of eosin Y and erythrosin B), and m and n are each independently an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. It is to be noted that specific integers for m and n do not necessarily restrict the inventive heavy metal ion complex to exactly said composition of the complex but rather can also define the ratio of the heavy metal ion(s) M to the ligand R (i.e. m and n are integers said to be multiplicatively independent if their only common integer power is 1).

The inventive heavy metal ion complex, in addition to the one or more ligand(s) (i.e. the one or more xanthene derivative(s) such as e.g. eosin Y and/or erythrosin B), optionally contains further ligand(s) (herein sometimes referred to as "optional ligand(s)").

In accordance with the present invention, at least one M is a heavy metal ion having an atomic number of 29 or higher and 83 or lower (preferably 29 or higher and 81 or lower), preferably 47 or higher and 83 or lower (preferably 47 or higher and 81 or lower). Accordingly, in the heavy metal complexes of the present invention, the metal can be present in different oxidation states, e.g. +1, +2, +3, etc. (also referred to as M(I), M(II), M(III), etc). Specifically, M is an ion of a chemical element selected from the group consisting of Cu (copper), Zn (zinc), Ga (gallium), Ge (germanium), As (arsenic), Se (selenium), Rb (rubidium), Sr (strontium), Y (yttrium), Zr (zirconium), Nb (niobium), Mo (molybdenum), Tc (technetium), Ru (ruthenium), Rh (rhodium), Pd (palladium), Ag (silver), Cd (cadmium), In (indium), Sn (tin), Sb (antimony), Te (tellurium), Cs (caesium), Ba (barium), La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), Hf (hafnium), Ta (tantalum), W (tungsten), Re (rhenium), Os (osmium), Ir (iridium), Pt (platinum), Au (gold), Hg (mercury), Tl (thallium), Pb (lead) and Bi (bismuth). Preferably, M is an ion of the chemical elements selected from the group consisting of Ag, Ba, Gd, Lu, Au, Pb and Bi (e.g., Ag ⁺ , Ba ²⁺ , Gd ³⁺ , Lu ³⁺ , Au ³⁺ , Pb ²⁺ and Bi ³⁺ ). More preferably, M is a heavy metal ion selected from the group consisting of Ag ⁺ , Ba ²⁺ , Gd ³⁺ , Lu ³⁺ and Au ³⁺ . Particularly preferable, M is a heavy metal ion selected from the group consisting of Ag ⁺ , Ba ²⁺ and Gd ³⁺ . Most preferably, M is Ba ²⁺ .

In some embodiments, M is preferably an ion of the chemical elements selected from the group consisting of Ag, Ba, Lu, Au, Pb and Bi (e.g., Ag ⁺ , Ba ²⁺ , Lu ³⁺ , Au ³⁺ , Pb ²⁺ and Bi ³⁺ ). More preferably, M is a heavy metal ion selected from the group consisting of Ag ⁺ , Ba ²⁺ , Lu ³⁺ and Au ³⁺ . Particularly preferable, M is a heavy metal ion selected from the group consisting of Ag ⁺ and Ba ²⁺ . Most preferably, M is Ba ²⁺ .

The heavy metal ion M contained in the inventive heavy metal complexes, amongst others, provides properties resulting in X-ray attenuation, and therefore enhances the contrast of the imaged biological sample. Properties of interest are the atomic number, the density, the atomic mass and response to the applied energy. Moreover, it will be appreciated that the heavy metal M may comprise isotopes within one metal species, e.g. different isotopes of barium, etc. In accordance with the present invention, specific xanthene derivatives are used for the preparation of the heavy metal ligand complex (in particular eosin Y and/or erythrosin B can be used for the preparation of the heavy metal ligand complex). The specific xanthene- derivatives are represented by the Chemical Formula (I) below:

wherein

Xi is each independently NO ₂ , halogen or H,

X ₂ is each independently Halogen or H,

Ri, R ₂ , R3 and R4 are each independently H, halogen (independently selected from the group consisting of CI, Br and I) or a water solubility enhancing group selected from OH, NH ₂ , SH, NO ₂ , SO ₃ H, SO ₄ H ₂ , PO ₃ H ₂ diol and polyols such as polyethyleneglycols, and

R ₅ and R6 are each independently H or a water solubility enhancing group selected from NH ₂ , SH, NO ₂ , SO ₃ H, SO ₄ H ₂ , PO ₃ H ₂ diol and polyols such as polyethyleneglycols.

Preferred examples of the xanthene derivatives used in the present invention are mono-, di-, tri- and tetrachlorofluorescemes; mono-, di-, tri-, and tetrabromofluorescemes; Solvent Red 72; mono-, di, tri- and tetraiodofluoresceines; eosin B; eosin Y; ethyleosin; erythrosin B; phloxine B and Rose bengal (4,5,6,7- tetrachloro-2',4',5',7'-tetraiodofluorescein). The following compounds are preferred:

wherein M is metal ion (such as Na or Ba ) which can be present in different oxidation states (e.g. +1 , +2 etc) and X is a halogen (independently selected from the group consisting of CI, Br and I). More preferred are the compounds shown in Figures 5 and 6. Particular preferred are mono-, di-, tribromofluorescein; mono-, di, triiodofluorescein; eosin B; eosin Y and erythrosin B. Most preferred are eosin Y and erythrosin B.

The afore-mentioned xanthene derivatives can be prepared by methods described herein and/or standard synthesis procedures known to the person skilled in the art. For example, the dibromo eosin derivative was synthesized as follows: A Schlenk flask was charged with 1.00 g of fluorescein (lactone form 0.1 M), which was dissolved in methanol and cooled to 0 °C. N-bromo succinimide or N-iodo succinimide was added in corresponding equivalents of 1.1, 2.2 and 3.3 equivalents to the solution. The resulting suspension was stirred vigorously for 2 hours at room temperature. The solvent was removed in vacuum and the residue was taken up in an aqueous sodium hydroxide solution (1 M, 10 ml) and stirred for 30 min. The deep red solution was treated with a 1 M aqueous hydrogen chloride solution until no further precipitation was observed. The remaining reaction mixture was stirred for another 3 hours. The precipitate was filtered off and purified with MPLC (medium pressure liquid chromatography: stationary phase: reversed phase CI 8 column, 12 g dry weight, column from Revelis; mobile phase: acetonitrile/water gradient: 35/65 for 0-5 min; 40/60 for 5-20 min and 100/0 for 20-45 min) to yield the brominated derivatives of eosin or the iodinated derivatives of erythrosine B.

As a further example, the dibromo barium eosin derivative was synthesized as follows: To a suspension of an eosin Y / erythrosine B derivative (lactone form, 1.00 equivalent) in 250 ml of water (milli-Q quality) was added a heavy metal salt (M = Pb ²⁺ , Cu ²⁺ , Ba ²⁺ with 1.00 equivalent and Na ⁺ with 2 equivalents). The reaction mixture was stirred at room temperature and after 12 hours a deep red suspension was observed. The reaction mixture was filtered, and the solvents were removed in vacuum. The remaining residue was dried in vacuum for 12 hours to yield a crystalline coloured (from orange over deep red to purple.)

Moreover, some of the afore-mentioned xanthene derivatives such as eosin Y and erythrosin B are commercially available and can be used for the preparation of the heavy metal ligand complex. Eosin Y and erythrosin B are both commercially available in the form of the respective sodium salts as well as in the form of the closed lactones, which are shown in Chemical Formula (II) below:

eosin Y erythrosin B

In general, the coordination number of a metal ion complex refers to the number of ligands (i.e. donor atoms) attached to the metal ion. For example, in the lead(II) complex of the present invention, the lead has a coordination number of at least two, for example it may be coordinated by or bonded to at least two groups of the ligand(s).

In some embodiments, lead may have a higher coordination number, and it may for example additionally be coordinated by a neutral molecule such as H ₂ 0. For instance, the lead(II) ligand complex may consist of lead(II) and one eosin Y and/or erythrosin B ligands (or other xanthene derivatives as defined above).

In above formula (I), the number of ligands is indicated by "n", which depends on several aspects such as the oxidation state of the heavy metal ion, the concentrations and equivalents used within the reaction mixture. Where the heavy metal ligand complex (e.g. the heavy metal eosin Y and/or erythrosine B complex) has a net overall charge (for example, not all coordination numbers of the metal centre are substituted by an ionic ligand), the heavy metal-ligand complex may be present in the form of a salt. In principle, the counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent complex.

The "optional ligand(s)" in the inventive heavy metal complexes are not particularly limited as long as a formation of the inventive complex with one or more xanthene derivative ligands (e.g. eosin Y and/or erythrosin B ligands) is not hindered. Non-limiting examples of optional ligand(s) include H ₂ 0, NO, hydrogen carbonate, hydrogen phosphate, carboxylates (e.g., formate, acetate, lactate, oxalate), chloride, etc.

Moreover, in some embodiments, the heavy metal complexes described herein may include solvates, hydrates, isomers, tautomers, racemates, stereoisomers, enantiomers or diastereoisomers of those complexes. Asymmetric centers may exist in the complexes disclosed herein. These centers can be designated by the symbols "R" or "S" depending on the configuration of substituents around the chiral carbon atom. It should be understood that the present invention encompasses all stereochemical isomeric forms, including diastereomeric, enantiomeric, and epimeric forms, as well as D-isomers and L-isomers, and mixtures thereof. Additionally, the complexes disclosed herein may exist as geometric isomers. The present invention includes all cis, trans, syn, anti, (E), and (Z) isomers as well as the appropriate mixtures thereof. Additionally, complexes may exist as tautomers, which are also included in the scope of the present invention. Moreover, the complexes disclosed herein may exist in polymorphic forms, which are also encompassed by the present invention.

In some embodiments, the heavy metal complexes may be present in the form of small particles, e.g. nanoparticles. Without wishing to be bound by any theory, it is considered that the diagnostic properties of the complexes may be improved where the complex is present in a form such that surface area is maximized, particularly where solubility of the complex is low, in order to maximize contact of the complex with the environment. Thus, in some embodiments, the inventive heavy metal complex is present in particulate form wherein the particles have a mean diameter (measured e.g. by laser diffraction) of less than ΙΟΟΟμηι, less than 500μηι, less than 200μιη, less than ΙΟΟμηι, less than 50μηι, less than 20μιη, less than ΙΟμιηηι, less than 5μιη, less than 2μιη, or less than Ι μιη. In some embodiments the heavy metal complex is present in particulate form wherein the particles have a mean diameter in the range of from ΙΟΟΟμιη to 500μηι, from 500μιη to 200μιη, from 200μιη to ΙΟΟμιη, from ΙΟΟμιη to 50μηι, from 50μιη to 20μιη, from 20μιη to ΙΟμιη, from ΙΟμιη to 5μιη, from 5μιη to 2μηι, or from 2μιη to Ι μιη.

In further embodiments, the present invention provides a product, device, material, solution or composition comprising a heavy metal complex as defined above. In some embodiments the product, device, material, solution or composition is a medical product, device, material, solution or composition. In some embodiments the inventive heavy metal complex is distributed within the product, device, material, solution or composition. In some embodiments the product, device, material, solution or composition is a polymerizable and/or curable product, device, material, solution or composition.

Preparation of the heavy metal ion complexes

The inventive heavy metal ion complexes preferably comprise eosin Y. The inventive complexes can be prepared by methods generally known to those skilled in the art. For instance, the following exemplary method can be used:

To a suspension of the closed lactone form of eosin Y and/or erythrosine B (or of another xanthene derivative) in an appropriate solvent such as distilled water, alcohols (such as methanol and ethanol) or acetone, a suitable (e.g. stoichiometric) amount of heavy metal salt is added. The heavy metal ion source is usually a salt of the respective heavy metal(s), which is sufficiently soluble in the respective solvent. Suitable salts of the heavy metals mentioned above are well known in the art and are commercially available. The suspension is stirred for a sufficient period of time (e.g. 12 hours or more) while kept at room temperature. Complex formation is usually indicated by a color change of the solution after addition of the heavy metal source. Moreover, the color of the inventive heavy metal complex can depend on the nature of the metal ion and e.g. on the solvent utilized. For instance, in the case of barium(II) the water- based reaction mixture with eosin Y turns orange-red in color after addition of the barium(II) salt. After complex formation, the reaction mixture is filtered and the solvent should be removed in vacuo. Afterwards, the inventive heavy metal complex can be obtained by drying in vacuo for a sufficient period of time (e.g. 12 hours or more). For instance, the resulting isolated solid in the case of barium(II), the complex with eosin Y has an orange-red color. For use in the inventive ex vivo method, the complex obtained by the afore-mentioned method is solved in a suitable solvent (e.g. water, alcohols (such as methanol and ethanol) or acetone), which can then be directly used for staining of the biological sample.

As mentioned above, the inventive heavy metal ion complexes are in particular useful as CAs for a computed tomography scanning of biological samples. The term "computed tomography", in brief "CT", includes all computed tomography scanning methods available to those skilled in the art. Non limiting examples of CT scanning methods are X-ray absorption-based CT, X-ray propagation phase-contrast CT and X-ray grating interferometry phase-contrast CT. Preferred CT scanning methods are X-ray absorption-based scanning methods. In general, a CT scan makes use of computer-processed combinations of many X-ray images taken from different angles to produce cross-sectional (tomographic) images (virtual "slices") of specific areas of a scanned sample. Digital geometry processing is used to generate a three-dimensional image of the inside of the sample from a large series of two-dimensional radiographic images taken around a single axis of rotation. Medical imaging is the most common application of X-ray CT. Non-limiting examples of X-ray absorption-based scanning methods are attenuation-based imaging, dual-energy imaging, K-edge imaging and spectral decomposition. More preferred CT scanning methods are X-ray absorption-based Micro-Computed Tomography ^CT) and Nano-Computed Tomography (NanoCT). The prefix micro- (symbol: μ) is used in the term "Micro- Computed Tomography" to indicate that the pixel sizes of the cross-sections are in the micrometer range. These pixel sizes have also resulted in terms such as high-resolution x-ray tomography and X-ray microtomography. The prefix nano- is used in the term "Nano-Computed Tomography" to indicate that the pixel sizes of the cross-sections are in the nanometer range. Suitable devices for carrying out the aforementioned CT scanning methods are commercially available. For instance, non-limiting examples of commercially available μCT devices are V|tome|X (designed and developed by GE) and XRadia (designed and developed by Zeiss).

In addition to the above-described use as an ex vivo CA for a CT scanning of a biological sample, the inventive complexes may have a plethora of further applications. Non-limiting examples thereof are the use as diagnostic additives for coating surfaces of products, in a curable composition (e.g., an adhesive compositions), in a coating composition for coating the surface of an object (e.g. a composition comprising a film former/binder, the inventive complex, and optionally other components such as diluents(s), pigment(s), and/or filler(s)), and in a monomer composition comprising a polymerizable monomer which is intended for polymerisation to produce a polymerised product and in a polymeric material (e.g., the inventive complex may be dispersed as a separate component within the bulk of the polymeric material; alternatively, the inventive complex may be present in the polymeric material as an integral part of the polymer (i.e. covalently bound to the polymer)).

Further, a product may be coated with a diagnostic surface coating comprising the inventive complex. For example, the exterior of a product may be covered by spraying or coating with a composition comprising the inventive complex. In some embodiments, for example where the inventive complex has low solubility, the composition may take the form of a suspension composition comprising the inventive complex suspended in a liquid carrier.

The amount of the inventive complex used in products, compositions and the like will depend on the nature of the material and the intended application. In some embodiments, the inventive complex is present in an amount of up to 25, up to 20, up to 15, up to 10, up to 5, up to 2, up to 1, up to 0.5, up to 0.2, up to 0.1, up to 0.05, up to 0.02, up to 0.01, up to 0.005, up to 0.002, or up to 0.001% by weight of the product, material, device or composition which comprises the inventive complex.

The diagnostic properties of the inventive complexes make them particularly suitable for use in the hospital/medical environment. Accordingly, in some embodiments the product, device, material, solution or composition comprising the inventive complex is a medical product, device, material, solution or composition, such as a wound dressing, a suture, a surgical implement or a medical implant. For example sutures or stitches (e.g. stitches formed of polymers having glycolic acid and/or lactic acid monomer units, such as polygalactin 910) may be coated with a coating composition comprising the inventive complex, or the stitches formed from monomer compositions containing the inventive complex. Surgical equipment and implements, and medical implants such as dental implants, stents and components used in joint arthroplasty, may be coated with a coating composition comprising the inventive complex or, where appropriate, formed of a composition comprising the inventive complex. The inventive complex may also or alternatively find use in wound dressings. For example the product may be a wound dressing comprising a woven material made of synthetic and/or natural polymer (e.g. polyur ethane, polyester, cotton) coated or impregnated with a composition comprising the inventive complex, or, in the case of a synthetic polymer, formed of a monomer composition containing the inventive complex. The medical product, device, composition or material comprising the inventive complex may also be a curable medical adhesive (e.g. a cyanoacrylate composition), a bone or dental cement (e.g. methyl methacrylate / polymethyl methacrylate compositions), a dental primer or dental adhesive. Medical consumables (where diagnostic properties are desirable) may also comprise the inventive complex.

As discussed above, the inventive complex may be used to impart diagnostic properties to a variety of medical products. For example wound dressings coated or impregnated with the inventive complex, in particular with the inventive complex comprising silver ions, may be applied to the skin of a patient, or the inventive complex may be present in a bone cement composition to visualize the attachment to the human body following joint replacement surgery.

Moreover, the inventive complex may also find use in applications outside the medical environment. For example, the inventive complex may find use in products, devices, materials or compositions used in building construction, renovation and/or maintenance. Examples of compositions which may comprise the inventive complex include curable and/or polymerizable compositions, such as coating compositions (e.g. lacquer compositions, varnish compositions, or paint compositions comprising a binder or film former, and optionally other components such as pigment(s) and/or diluents(s)), adhesive or sealant compositions (e.g. a silicone or polyurethane sealant composition), cement compositions (e.g. a Portland- type cement composition comprising calcium silicates), concrete (e.g. compositions comprising cement, aggregates and water), or grout, mortar or stucco compositions (e.g. compositions comprising water, cement and sand).

EX VIVO METHOD FOR INVESTIGATING A BIOLOGICAL SAMPLE

In a first aspect, the present invention relates to an ex vivo method for investigating a biological sample comprising: (al) staining the biological sample with a solution (AB) comprising one or more complex(es) as defined above.

In a second aspect (herein sometimes referred to as "in situ" staining), the present invention relates to an ex vivo method for investigating a biological sample comprising: (a2) staining the biological sample with (i) a solution (A) comprising one or more xanthene derivatives (e.g. eosin Y and/or erythrosin B), and (ii) one or more staining solution(s) (B) comprising one or more heavy metal ion(s) as defined above, wherein the biological sample is contacted with staining solution (A) separately from the one or more staining solution(s) (B).

In both aspects of the inventive ex vivo method, the biological sample is subjected to a CT scanning method after staining (and is therefore "investigated"). Herein below, the inventive ex vivo method is explained in more detail. If not indicated otherwise, these explanations equally apply to the first as well as the second aspect of the inventive ex vivo method.

The term "biological sample" refers to any material of biological origin to be analyzed. Non-limiting examples of biological samples are organs and tissues, preferably tissues, more preferably soft tissues, most preferably human soft tissues. In biology, tissue is a cellular organizational level intermediate between cells and a complete organ. A tissue is an ensemble of similar cells from the same origin that together carry out a specific function. Organs are then formed by the functional grouping together of multiple tissues. It is to be understood that the biological sample to be analyzed can also be part of an organ or tissue, or can be an aggregation of organs and/or tissues.

The term "soft tissue" refers to tissues that connect, support, or surround other structures and organs of the body, not being hard tissue such as bone. Non-limiting examples of soft tissue are tendons, ligaments, fascia, skin, fibrous tissues, fat, and synovial membranes (which are connective tissue), and muscles, nerves and blood vessels (which are not connective tissue). Preferred soft tissues samples used in the present invention originate from lung, kidney, liver, brain, spleen, heart and cartilage.

The biological sample originates from an animal such as birds and mammals, wherein mammals are preferred. The mammal may be, e.g., a rodent (such as, e.g., a guinea pig, a hamster, a rat or a mouse), a canine (such as, e.g., a dog), a feline (such as, e.g., a cat), a porcine (such as, e.g., a pig), an equine (such as, e.g., a horse), a primate, a simian (such as, e.g., a monkey or an ape), a monkey (such as, e.g., a marmoset or a baboon), an ape (such as, e.g., a gorilla, a chimpanzee, an orang-utan or a gibbon), or a human. It is envisaged that the biological sample is also obtained from non-human mammals, which are economically, agronomically or scientifically important. Scientifically important mammals include, e.g., mice, rats and rabbits. Non-limiting examples of agronomically important mammals are sheep, cattle and pigs. Economically important mammals include, e.g., cats and dogs. Most preferably, the biological sample is obtained from a human.

The biological sample used in accordance with the invention is obtainable by suitable methods for obtaining biological samples generally known in the art. For example, the biological sample can be obtained by excision (cutting out), puncture (also called centesis) followed by aspiration, and scraping or swiping.

The biological sample used in the staining method according to the present invention can be used directly (e.g., fresh or frozen), or can be manipulated prior to staining. Suitable manipulation procedures for biological samples are well known in the art, and non-limiting example procedures are for instance described in Mulisch and Welsch (M. Mulisch and U. Welsch, Romeis Mikroskopische Technik, 19th Ed. Springer Spektrum Akademischer Verlag, Heidelberg, 2010) and Lang (G. Lang, Histotechnik: Praxislehrbuch fur die Biomedizinische Analytik, 2nd Ed. Springer- Verlag Wien, 2013). Preferably, the biological sample is subjected to chemical fixation by means of chemical fixatives prior to staining. Chemical fixatives are, amongst others, used to preserve the biological sample from degradation, and to maintain the structure of the cell and of sub-cellular components. Examples of chemical fixatives include, but are not limited to, water-based solutions of formaldehyde or glutaraldehyde (or mixtures thereof), methanol, ethanol and acetone. A preferred chemical fixative according to the present invention is a water-based formaldehyde solution, more preferably a formaldehyde solution in Dulbecco's phosphate buffered saline (DPBS) or a formaldehyde solution in phosphate buffered saline, particularly preferable is a formaldehyde solution in Dulbecco's phosphate buffered saline, most preferably a formaldehyde solution in Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium. The constituents of Dulbecco's phosphate buffered saline can be taken from VWR - Amresco LifeScience (137 mM sodium chloride, 2.7 mM potassium chloride and 10 mM phosphate buffer, without calcium and magnesium).

The concentration of the aldehyde-based chemical fixative (e.g. formaldehyde) in the water-based solution can vary considerably but is usually in the range of about 0.5 to about 15%, preferably about 0.8 to about 10%, more preferably about 1 to about 5%, particularly preferably about 2 to about 4%, and most preferably about 1%> or about 4%. Preferably, an acid (e.g., glacial acetic acid) is additionally present in the water-based formaldehyde solution since this may improve the diagnostic value of the inventive staining method. Examples of suitable acids are glacial acetic acid and citric acid. Preferably, glacial acetic acid is used. The concentration of the acid in the chemical fixative is usually in the range of about 0.5 to about 15%), preferably about 1 to about 10%>, more preferably about 2 to about 6%>, particularly preferably about 3 to about 5%, and most preferably about 5%>.

Acidification of the tissue allows for an optimal preparation of the tissue for staining by the CA. Without wishing to be bound by any theory, it is assumed that amino acid residues containing an amino group in

As a result, the obtained ammonium functionalities are interacting with endogenous anions such as carboxylates, (hydrogen) phosphates, (hydrogen) carbonates, etc. Eosin Y and erythrosine B are commercially available as the disodium salts, i.e. an ionic substance. If eosin Y and/or erythrosine B enter the tissue, the stronger (more stable) salt replaces the weaker (less stable) salt, i.e. eosin Y and erythrosine B interact better with the peptides and / or proteins (and / or other protonated groups present within the tissue).

The time period of the chemical fixation procedure can vary considerably, but is usually in the range of about 3 to about 120 hours, preferably about 6 to about 96 hours, more preferably about 9 to about 84 hours, particularly preferably about 12 to about 72 hours, and most preferably about 18 to about 48 hours. After the fixation period, the biological sample is preferably washed 1 or more times, preferably 2 or more times, more preferably 3 or more times, particularly preferably 4 or more times, and most preferably 5 or more times with a suitable water-based solution. Preferably, the washing is carried out with a suitable buffer (e.g., Dulbecco's phosphate buffered saline or phosphate buffered saline). Subsequently, the biological sample can be subjected directly to staining, can be stored for a desired amount of time (preferably under cooling) prior to staining, or can be further manipulated prior to staining.

Subject to the specific nature of the biological sample (e.g., biological origin, sample size, etc.), the skilled person can adopt the characteristics of the fixation protocol (such as nature and concentration of the chemical fixative, fixation time, temperature, etc.) in a suitable manner.

In the first aspect of the inventive ex vivo method, the method comprises a step of staining the biological sample with a solution (AB) comprising one or more complex(es) as defined above. An exemplary method for the preparation of solution (AB) has already been described above. Preferably, the concentration of the heavy metal complex in solution (AB) is in the range of about 1 to about 200mM, or about 5 to about 1 OOmM. More preferably, the concentration of the heavy metal complex in solution (AB) is in the range of about 10 to about 50mM. Particularly preferably, the concentration of the heavy metal complex in solution (AB) is in the range of about 20 to about 40mM. Most preferably, the concentration of the heavy metal complex in solution (AB) is about 30mM.

The incubation time of the biological sample with the solution (AB) (staining time) is preferably a time period of 12 hours or more, more preferably 24 hours or more. Particular preferably, the time period is 48 hours or more. Most preferably, the time period is 72 hours or more.

For the staining of the biological sample according to the first aspect of the inventive ex vivo method, the biological sample is brought into contact with the solution (AB), e.g. by submerging it in solution (AB). The contact time between solution (AB) and the biological sample is not particularly limited and for instance depends on the specific nature of the biological sample (e.g., biological origin, sample size, etc.).

In the second aspect of the inventive ex vivo method (in situ staining), the biological sample is contacted with solution (A) separately from the one or more solution(s) (B). An exemplary method for the preparation of solutions (A) and the one or more solution(s) (B) is described below.

The exemplary method starts with the preparation of the two solutions (A) and (B). Solution (A) contains eosin Y and/or erythrosin B (preferably eosin Y) in an appropriate solvent such as distilled water, alcohols (such as methanol and ethanol) and acetone.

The concentration of eosin Y in solution (A) is preferably in the range of about 10 to about 50 wt/vol-%. More preferably, the concentration of eosin Y in solution (A) is in the range of about 20 to about 40 wt/vol-%. Particularly preferably, the concentration of eosin Y in solution (A) is in the range of about 25 to about 35 wt/vol-%. Most preferably, the concentration of eosin Y in solution (A) is about 30 wt/vol-%. The concentration of erythrosin B in solution (A) is preferably in the range of about 1 to about 25 wt/vol- %. More preferably the concentration of erythrosine B in solution (A) is in the range of about 5 to about 20 wt/vol-%. Particularly preferably, the concentration of erythrosine B in solution (A) is in the range of 7.5 to about 15 wt/vol-%. Most preferably, the concentration of erythrosine B in solution (A) is about 10 wt/vol-%). The preferred concentrations of the remaining xanthene derivatives of the present invention can be chosen from the ranges indicated above for eosin Y, or can be determined by the skilled person depending on the specific circumstances such as the solvent used, the sample to be stained, etc. Moreover, the total concentration of the xanthene derivatives (e.g. eosin Y and/or erythrosin B) in solution (A) is preferably in the range of about 10 to about 50 wt/vol-%. More preferably, the total concentration is in the range of about 20 to about 40 wt/vol-%). Particularly preferably, the total concentration is in the range of about 25 to about 35 wt/vol-%. Most preferably, the total concentration is about 30 wt/vol-%. In other words, it can be preferable that solution (A) has a total concentration of the xanthene derivatives (e.g. eosin Y and/or erythrosin B) near, or equal to, the maximum solubility of the xanthene derivatives. For instance, about 30g of eosin Y are dissolved in about 100 mL of distilled water.

Solution (B) is prepared by solving the heavy metal ion source in a suitable solvent such as distilled water, alcohols (such as methanol and ethanol) and acetone. The one or more heavy metal ion(s) to be used in accordance with the method of the present invention are selected from the heavy metal ions listed above with regard to the inventive heavy metal ion complexes. Accordingly, the one or more heavy metal ion(s) derive from a chemical element selected from the group consisting of Cu (copper), Zn (zinc), Ga (gallium), Ge (germanium), As (arsenic), Se (selenium), Rb (rubidium), Sr (strontium), Y (yttrium), Zr (zirconium), Nb (niobium), Mo (molybdenum), Tc (technetium), Ru (ruthenium), Rh (rhodium), Pd (palladium), Ag (silver), Cd (cadmium), In (indium), Sn (tin), Sb (antimony), Te (tellurium), Cs (caesium), Ba (barium), La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), Hf (hafnium), Ta (tantalum), W (tungsten), Re (rhenium), Os (osmium), Ir (iridium), Pt (platinum), Au (gold), Hg (mercury), Tl (thallium), Pb (lead) and Bi (bismuth). Preferably, the one or more heavy metal ion(s) derive from the group consisting of Ag, Ba, Gd, Lu, Au, Pb and Bi (e.g., Ag ⁺ , Ba ²⁺ , Gd ³⁺ , Lu ³⁺ , Au ³⁺ , Pb ²⁺ and Bi ³⁺ ). More preferably, the one or more heavy metal ion(s) derive from the group consisting of Ag ⁺ , Ba ²⁺ , Gd ³⁺ , Lu ³⁺ and Au ³⁺ . Particularly preferable, the one or more heavy metal ion(s) derive from the group consisting of Ag ⁺ , Ba ²⁺ and Gd ³⁺ , most preferably Ba ²⁺ .

In some embodiments, the one or more heavy metal ion(s) preferably derive from the group consisting of Ag, Ba, Lu, Au, Pb and Bi (e.g., Ag ⁺ , Ba ²⁺ , Lu ³⁺ , Au ³⁺ , Pb ²⁺ and Bi ³⁺ ). More preferably, the one or more heavy metal ion(s) derive from the group consisting of Ag ⁺ , Ba ²⁺ , Lu ³⁺ and Au ³⁺ . Particularly preferable, the one or more heavy metal ion(s) derive from the group consisting of Ag ⁺ and Ba ²⁺ , most preferably The heavy metal ion source is usually a salt of the respective heavy metal(s) which is sufficiently soluble in the respective solvent. Suitable salts of the above heavy metals are well known in the art and are commercially available. The maximum concentration of the one or more heavy metal ion(s) in the solution(s) (B) of course depends on the solubility of the respective heavy metal ion salt used to prepare the one or more solution(s) (B). In general, it can be beneficial to use higher concentrated solutions.

For the staining of the biological sample according to the second aspect of the inventive ex vivo method, the biological sample is contacted with solution (A) separately from the one or more solution(s) (B), e.g. by submerging it in the respective solutions separately. The time of contacting the biological sample with solution (A) or the one or more solution(s) (B) is the incubation time (staining time) of the biological sample in the respective solution. The incubation time (staining time) of solution (A) and the incubation time (staining time) of the one or more solution(s) (B) are preferably 12 hours or more, more preferably 24 hours or more. Particular preferably, the incubation time (staining time) is 48 hours or more. Most preferably, the incubation time (staining time) is 72 hours or more. The incubation time (staining time) of solution (A) and the incubation time (staining time) of the one or more solution(s) (B) are independent from each other, i.e. can be the same or different.

The present inventors surprisingly found that an in situ staining of the biological sample can result in particularly favorable staining results in terms of homogeneity throughout the biological sample and/or CT contrast enhancement. In particular, it is noted that the results can be further improved if the chemical fixative, which is prior to staining for the preparation of the biological sample (e.g. a water-based formaldehyde solution), additionally contains an acid (e.g., glacial acetic acid) as described above. Moreover, as regards contrast enhancement, it has been found that the quality of the CT results can be further improved if the biological sample is contacted with the one or more solution(s) (B) before the biological sample is contacted with solution (A). Preferably, the main solvent of solutions (A) and the main solvent of the one or more solution(s) (B) is a water-based solution.

Moreover, in a further aspect the present invention relates to the staining of a biological sample with a solution of the xanthene derivative(s). The definitions of the preferred xanthene derivatives (such as eosin Y and erythrosin B e.g. in the form of the respective sodium salts), as well as further details of preferred embodiments (e.g. with respect to the biological sample which preferably is a human sample etc), given above and below also apply to this aspect of the present invention. For example, also in this aspect of the present invention acidification of the tissue - preferably during the chemical fixation step - allows for an optimal preparation of the tissue for staining which in turn may improve the diagnostic value of the inventive staining method. Examples of suitable acids are glacial acetic acid and citric acid. Preferably, glacial acetic acid is used. The concentration of the acid in the chemical fixative is usually in the range of about 0.5 to about 15%, preferably about 1 to about 10%, more preferably about 2 to about 6%, particularly preferably about 3 to about 5%, and most preferably about 5%. Additionally, also in this aspect of the present invention, the concentration of the xanthene derivative in the staining solution is high (e.g. near, or equal to, the maximum solubility of the respective xanthene derivative in the respective solvent used for the preparation of the staining solution). Also in this aspect of the present invention it is more preferably that staining with the afore-said high concentrated staining solution is carried out after acidification of the tissue sample.

As mentioned above, the inventive ex vivo method (first as well as the second aspect) are compatible with conventional histological methods. This means that the contrast enhancement achieved by the inventive ex vivo method with regard to CT does not preclude a further investigation of regions of interest with conventional histological methods, if desired. Furthermore, in preferred embodiments, the inventive ex vivo method comprises, in addition to staining step (al) or staining step (a2) an additional step of staining with an additional staining agent. Preferably, the additional staining agent is hematein. Hematein (3,4,6a,10-Tetrahydroxy-6,7-dihydroindeno[2,l-c]chromen-9-on e), having a structure represented by below Chemical Formula (III),

is an oxidized derivative of hematoxylin (7,1 lb-Dihydroindeno[2,l-c]chromene-3,4,6a,9,10(6H)-pentol), which is represented by below Chemical Formula (IV)

The aforementioned oxidation of hematoxylin can for instance be carried out by contacting a solution/suspension of hematein in absolute ethanol to air over a time period of several weeks (sometimes referred to as "ripening"). Alternatively, in order to speed up the oxidation process, oxidizing additives such as potassium iodate (KIO ₃ ), sodium iodate (NalOs) or mercury oxide (HgO) can be added to a hematoxylin solution. Moreover, hematein and hematoxylin are both commercially available.

The additional staining can be carried out before subjecting the stained biological sample to a computed tomography scanning method, or thereafter. It is noted that an additional staining with conventional stains such as hematein can be advantageous in terms of a further investigation with conventional histological methods. It is, however, also noted that the additional staining with a conventional stain such as hematein can also be carried out with the three-dimensional biological sample, i.e., it is not obligatory that the additional staining is carried out with sliced tissue sections used in conventional histological investigations.

In other embodiments, the inventive ex vivo method does not comprise, in addition to staining step (al) or staining step (a2), an additional step of staining with an additional staining agent.

The inventive ex vivo method comprises a step of subjecting the stained biological sample to a CT scanning method. Suitable CT scanning methods/devices have already been described above. For instance, the biological sample may be imaged in a commercially available X-ray μCT setup, e.g. the V|tome|X.

In the step of subjecting the stained biological sample to a CT scanning method, for instance, the biological sample is placed in an appropriate container that holds the sample in such a fashion that the biological sample is not subjected to moving (cf, Figure 4). Moreover, for instance, the following parameters may be used to acquire the CT of the stained biological sample of the present invention:

(i) V|tome|X: U = 30 - 60 kV, I = 100 - 250 μΑ, average = 3, skip = 1, exposure time depended on nature of CA used, pixel size depended on sample size, overall scan time depended on number of projections for tomography.

(ii) XRadia: U = 30 - 60 kV, P = 1.8 - 4.5 W, average = 3, skip = 1, exposure time depended on nature of CA used, pixel size depended on sample size and objective chosen for measurement, overall scan time depended on number of projections for tomography.

USE OF THE INVENTIVE HEAVY METAL ION COMPLEXES AS A CONTRAST AGENT FOR CT SCANNING OF A BIOLOGICAL SAMPLE

Moreover, the present invention relates to the use of the inventive heavy metal complexes as a contrast agent for a CT scanning of a biological sample. The above explanations with regard to the heavy metal complexes, suitable CT scanning methods/devices and the potential biological samples to be investigated equally apply, mutatis mutandis, to the use of the disclosed heavy metal complexes.

It is to be understood that the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments. In particular, the invention specifically relates to all combinations of preferred features (including all degrees of preference) of the methods and products provided herein. Moreover, those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all obvious variations and modifications of said disclosure.

In this specification, a number of documents including scientific literature are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Moreover, it will be understood that, although a number of documents are referred to herein, this reference does not - in itself - constitute an admission that any of these documents forms part of the common general knowledge of the skilled person.

The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

Animals Used.

Animal housing was carried out at the Klinikum rechts der Isar, Technical University of Munich in accordance with the European Union guidelines 2010/63. Organ removal was approved from an internal animal protection committee of the Center for Preclinical Research (ZPF) of Klinikum rechts der Isar, Munich, Germany (internal reference number 4-005-09). All procedures were in accordance with relevant guidelines and regulations. All laboratories are inspected for accordance with the OECD principles of good laboratory practice. We prepared a whole mouse kidney using the final version of the eosin-staining procedure. The soft-tissue sample was then used to evaluate structural preservation and to assess stain quality, identify morphological structures, compare with conventional histological methods and evaluate for further histological staining. The remaining organs (mainly mouse liver for sample screening during staining method development and optimization and mouse kidney for final optimization and final data acquisition) were used for the development of the staining protocol and the optimization of parameters (the other organs such as the heart, lung, spleen and brain will be used for future studies).

Sample Screening.

We purchased all reagents from Sigma-Aldrich unless otherwise indicated. Whole mouse organs were fixated and preserved under conditions described below. Cuboidal soft-tissue samples from mouse liver (2 -3 mm edge length) were used for stain development and optimization. The small cuboidal tissue samples were cut with a scalpel (Aesculap). Temperature was controlled by placing samples in a refrigerator (4°C) or in ambient conditions of the laboratory. Incubations were done in sample holders with a flat bottom, which were replaced after each step but not after rinse or dehydration steps. For stain development and optimization several parameters such as fixative, concentration of fixative or staining agent, incubation time or pH of fixative or staining agents were tested. The stained soft-tissue samples were investigated on the phoenix v|tome|x s 240 CT scanner with typical settings of 50kV peak voltage, 6.0W and with 1001 projections distributed over 360° . The low-resolution CT data were acquired with an exposure time of 1 s per projection with an effective pixel size of ca. 30 μιη. The microCT data were reconstructed with the integrated phoenix datos x CT software and analyzed for (i) completeness of staining, (ii) appearance of diffusion rings, (iii) contrast enhancement, (iv) appearance of CT artifacts as streaks and (v) homogeneity of the staining.

Example 1

[BARIUM-EOSIN Y COMPLEX] Fixation step without the addition of acid:

Turkey liver (soft tissue sample) was surgically removed and immediately placed in a 50-ml Falcon Centrifuge Tube (neoLab), which was filled with a fixative solution containing 10 ml of 1 vol/vol-% formaldehyde solution (FA, derived from a 37% acid free FA solution stabilized with ca. 10% methanol from Carl Roth). The soft tissue sample was refrigerated for 24-72 hours and then washed with Dulbecco's phosphate buffered saline solution (DPBS without calcium and magnesium) for 1 hour. The fixated turkey liver was cut into 3 mm ³ cubes, which were transferred into a sample container containing 1 mL of a staining solution comprising a barium-eosin Y complex in distilled water (25 mg/mL; c = 31.9 mM). It is noted that the barium-eosin Y complex in solution appears more orange-red in color as compared to the eosin complex with sodium, which has a deep pink red color (cf. Figure 22).

The soft tissue sample was stained for 72 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the staining solution comprising the barium-eosin Y complex, the turkey liver was removed and the remaining staining solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The soft tissue sample was stored over 70 vol/vol-%) ethanol vapor prior to CT measurement.

In an analogous manner further soft tissue samples were stained with (i) eosin Y solution only (c = 31.9 mM), or (ii) with a solution containing neither barium acetate nor eosin Y (control sample).

Fixation step with the addition of acid:

Turkey liver (soft tissue sample) was surgically removed and immediately placed in a 50-ml Falcon Centrifuge Tube (neoLab), which was filled with a fixative solution containing 9.5 ml of 1 vol/vol- %> formaldehyde solution (FA, derived from a 37% acid free FA solution stabilized with ca. 10%> methanol from Carl Roth) and 0.5 ml glacial acetic acid (AA, Alfa Aesar). The soft tissue sample was refrigerated for 24-72 hours and then washed with DPBS without calcium and magnesium for 1 hour. The fixated and acidified turkey liver was cut into 3 mm ³ cubes, which were transferred into a sample container containing 1 mL of a staining solution comprising a barium-eosin Y complex in water (25 mg/mL; c = 31.9 mM). The soft tissue sample was stained for 72 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the staining solution comprising the barium-eosin Y complex, the turkey liver was removed and the remaining staining solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The soft tissue sample was stored over 70 vol/vol-% ethanol vapor prior to CT measurement.

In an analogous manner further soft tissue samples were stained with (i) eosin Y solution only (c = 31.9 mM), or (ii) with a solution containing neither barium acetate nor eosin Y (control sample).

An overview of the various samples is shown in Table 1 below:

[Table 1]

All samples were subjected to a micro computed tomography scanning method having the following measurement parameters: Phoenix V|tome|X s 240 CT scanner from GE; U = 50 kV; 1 = 110 μΑ; P = 5.5 W; Bin lxl ; exposure time: 2s; filter: air; average =3, skip = 1 ; projections: 1601 ; pixel size: 40.056 μιη; total scan time: 214 min.

The results of these measurements are summarized in Figure 1, which shows a line plot of the various samples. Staining of the turkey liver samples with a solution comprising a barium-eosin Y complex being acidified during fixation (cf. sample 1) resulted in a homogeneously and completely stained soft tissue sample. In general, the acidified samples showed a higher contrast enhancement compared to the non- acidified samples.

Example 2

[STAINING WITH BARIUM ACETATE SOLUTION AND EOSIN Y SOLUTION]

Fixation step without the addition of acid:

Turkey liver (soft tissue sample) was surgically removed and immediately placed in a 50-ml Falcon Centrifuge Tube (neoLab), which was filled with a fixative solution containing 10 ml of 1 vol/vol-% formaldehyde solution (FA, derived from a 37% acid free FA solution stabilized with ca. 10% methanol from Carl Roth). The soft tissue sample was refrigerated for 24-72 hours and then washed with DPBS without calcium and magnesium for 1 hour. The fixated turkey liver was cut into 3 mm ³ cubes, which were transferred into a sample container containing 1 mL of a water-based barium acetate solution (stoichiometric to the disodium salt of eosin Y; c = 217 mM). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the barium acetate solution, the turkey liver was removed and the remaining barium acetate solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The turkey liver was transferred to a new sample container containing 1 mL of a water-based eosin Y solution (eosin Y disodium salt, 30 wt/vol-%). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the eosin Y solution, the turkey liver was removed and the remaining eosin Y solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The soft tissue sample was stored over 70 vol/vol-%> ethanol vapor prior to CT measurement.

In an analogous manner further soft tissue samples were stained with (i) barium acetate solution only, (ii) eosin Y solution only or (iii) with respective solutions containing neither barium acetate nor eosin Y (control sample).

Fixation step with the addition of acid:

Turkey liver (soft tissue sample) was surgically removed and immediately placed in a 50-ml Falcon Centrifuge Tube (neoLab), which was filled with a fixative solution containing 9.5 ml of 1 vol/vol- % formaldehyde solution (FA, derived from a 37% acid free FA solution stabilized with ca. 10% methanol from Carl Roth) and 0.5 ml glacial acetic acid (AA, Alfa Aesar). The soft tissue sample was refrigerated for 24-72 hours and then washed with DPBS without calcium and magnesium for 1 hour. The fixated and acidified turkey liver was cut into 3 mm ³ cubes, which were transferred into a sample container containing 1 mL of a water-based barium acetate solution (stoichiometric to the disodium salt of eosin Y; c = 217 mM). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the barium acetate solution, the turkey liver was removed and the remaining barium acetate solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The turkey liver was transferred to a new sample container containing 1 mL of a water-based eosin Y solution (eosin Y disodium salt, 30 wt/vol-%). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the eosin Y solution, the turkey liver was removed and the remaining eosin Y staining solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The soft tissue sample was stored over 70 wt/vol-%) ethanol vapor prior to CT measurement. In an analogous manner further soft tissue samples were stained with (i) barium acetate solution only, (ii) eosin Y solution only or (iii) with respective solutions containing neither barium acetate nor eosin Y (control sample).

An overview of the various samples is shown in Table 2:

[Table 2]

A macroscopic image showing all staining solutions used for the staining step are shown in Figure 19(A) while all staining solutions used for the 2 ^nd staining step are shown in Figure 19(B). Further, macroscopic images of the soft tissue sample after the 2 ^nd staining step are shown in Figure 20. As can be seen from Figure 20, the tissue sample changed its color upon incubation with Eosin Y in the 2 ^nd step. The incubation with Ba(OAc)2 did not result in a color change. This evidence of color change from deep pink-red for the eosin y complex to an orange-red for the barium-eosin y complex is further shown in Figure 22, where the solution of the barium-eosin y complex was compared with the eosin y complex in the sample holder, which clearly hihglights the orange-red color of the barium-eosin complex. Therefore, only the combination of Ba(OAc)2 and Eosin Y resulted in a "colored" stained tissue sample.

All samples were subjected to a micro computed tomography scanning method having the following measurement parameters: Phoenix V|tome|X s 240 CT scanner from GE; U = 50 kV; 1 = 110 μΑ; P = 5.5 W; Bin lxl ; exposure time: 2s; filter: air; projections: 1601 ; pixel size: 38.770 μιη; total scan time: 267 min.

The results of these measurements are summarized in Figures 2(a) and 2(b), which show a CT image and a line plot of the various samples. Staining of the turkey liver samples with a Ba(OAc)2 solution and an eosin Y solution (cf. samples 2 and 6) resulted in a homogeneously and completely stained soft tissue sample. In general, the acidified samples showed a higher contrast enhancement compared to the respective non-acidified samples. Moreover, in the acidified samples an over-additive contrast enhancement was observed in the sample which was stained with a Ba(OAc)2 solution and an eosin Y solution (cf. sample 2) compared to samples which were stained with the Ba(OAc)2 solution only (cf. sample 1) or the eosin Y solution only (cf. sample 4).

To further evaluate the staining, histological investigations were performed which are shown in Figures 21(A) to 21(C). The histological investigations show that a Barium-eosin Y complex has formed in situ as the tissue is colored with a change to more orange-red (as compared to the dark pinkish-red of the sodium salt of eosin Y). This evidence of color change is further shown in Figure 22, where the solution of the barium-eosin y complex was compared with the eosin y complex in the sample holder, which clearly hihglights the orange-red color of the barium-eosin complex. The compatibility with conventional 2D histology allows for further counter staining with hematoxylin as shown in Figure 21(C). Also these results evidence an in situ complex formation of an barium-eosin Y complex as a result from the two-step staining protocol.

Example 3

[STAINING WITH GADOLINIUM ACETATE SOLUTION AND EOSIN Y SOLUTION]

Fixation step without the addition of acid:

Turkey liver (soft tissue sample) was surgically removed and immediately placed in a 50-ml Falcon Centrifuge Tube (neoLab), which was filled with a fixative solution containing 10 ml of 1 wt/vol-% formaldehyde solution (FA, derived from a 37% acid free FA solution stabilized with ca. 10% methanol from Carl Roth). The soft tissue sample was refrigerated for 24-72 hours and then washed with DPBS without calcium and magnesium for 1 hour. The fixated turkey liver was cut into 3 mm ³ cubes, which were transferred into a sample container containing 1 mL of a water-based gadolinium acetate solution (stoichiometric to the disodium salt of eosin Y; c = 145 mM). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the gadolinium acetate solution, the turkey liver was removed and the remaining gadolinium acetate solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The turkey liver was transferred to a new sample container containing 1 mL of a water-based eosin Y solution (eosin Y disodium salt, 30 wt/vol-%)). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the eosin Y solution, the turkey liver was removed and the remaining eosin Y solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The soft tissue sample was stored over 70 wt/vol-% ethanol vapor prior to CT measurement.

In an analogous manner further soft tissue samples were stained with (i) gadolinium acetate solution only, (ii) eosin Y solution only or (iii) with respective solutions containing neither gadolinium acetate nor eosin Y (control sample). Fixation step with the addition of acid:

Turkey liver (soft-tissue sample) was surgically removed and immediately placed in a 50-ml Falcon Centrifuge Tube (neoLab), which was filled with a fixative solution containing 9.5 ml of 1 wt/vol-% formaldehyde solution (FA, derived from a 37% acid free FA solution stabilized with ca. 10% methanol from Carl Roth) and 0.5 ml glacial acetic acid (AA, Alfa Aesar). The soft tissue sample was refrigerated for 24-72 hours and then washed with DPBS without calcium and magnesium for 1 hour. The fixated and acidified turkey liver was cut into 3 mm ³ cubes, which were transferred into a sample container containing 1 mL of a water-based gadolinium acetate solution (stoichiometric to the disodium salt of eosin Y; c = 145 mM). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the gadolinium acetate solution, the turkey liver was removed and the remaining gadolinium acetate solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The turkey liver was transferred to a new sample container containing 1 mL of a water-based eosin Y solution (eosin Y disodium salt, 30 wt/vol-%). The soft tissue sample was stained for 24 hours while placed on a shaker (horizontal shaking with 60 rpm). After staining with the eosin Y solution, the turkey liver was removed and the remaining eosin Y staining solution was carefully padded off the soft tissue sample with a cellulose tissue paper. The soft tissue sample was stored over 70 vol/vol-% ethanol vapor prior to CT measurement.

In an analogous manner further soft tissue samples were stained with (i) gadolinium acetate solution only, (ii) eosin Y solution only or (iii) with respective solutions containing neither gadolinium acetate nor eosin Y (control sample).

An overview of the various samples is shown in Table 3 :

[Table 3]

All samples were subjected to a micro computed tomography scanning method having the following measurement parameters: Phoenix V|tome|X s 240 CT scanner from GE; U = 50 kV; I = 110 μΑ; P = 5.5 W; Bin lxl ; exposure time: 2s; filter: air; projections: 1601 ; pixel size: 38.770 μιη; total scan time: 267 min.

The results of these measurements are summarized in Figures 3(a) and 3(b), which show a CT image and a line plot of the various samples. Staining of the turkey liver samples with a Gd(OAc)3 solution and an eosin Y solution (cf. samples 2 and 6) resulted in a homogeneously and completely stained soft tissue sample. In general, the acidified samples 1 to 4 showed a higher contrast enhancement compared to the respective non-acidified samples 5 to 8. Moreover, an over-additive contrast enhancement was observed in the samples which were stained with a Gd(OAc)3 solution and an eosin Y solution (cf. samples 2 and 6) compared to samples which were stained with the Gd(OAc)3 solution only (cf. samples 1 and 5) or the eosin Y solution only (cf. samples 4 and 8).