U.S. patent application number 16/755677 was filed with the patent office on 2021-12-02 for novel heavy metal ion-ligand-complexes useful as ex vivo contrast agent for a computed tomography scanning of a biological sample, ex vivo method for investigating a biological sample, and use of the complexes.
The applicant listed for this patent is TECHNISCHE UNIVERSITAT MUNCHEN. Invention is credited to Madleen BUSSE, Franz PFEIFFER.
Application Number | 20210371436 16/755677 |
Document ID | / |
Family ID | 1000005823217 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210371436 |
Kind Code |
A1 |
BUSSE; Madleen ; et
al. |
December 2, 2021 |
NOVEL HEAVY METAL ION-LIGAND-COMPLEXES USEFUL AS EX VIVO CONTRAST
AGENT FOR A COMPUTED TOMOGRAPHY SCANNING OF A BIOLOGICAL SAMPLE, EX
VIVO METHOD FOR INVESTIGATING A BIOLOGICAL SAMPLE, AND USE OF THE
COMPLEXES
Abstract
The present invention relates to specific complexes comprising
heavy metal ions having an atomic number of 29 or higher and 83 or
lower (preferably 29 or higher and 81 or lower) and one or more
ligand(s) selected from the group consisting of specific xanthene
derivatives, preferably eosin Y and/or erythrosin B ligand(s). In
particular, the invention relates to the use of the complexes as ex
vivo 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 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
29 or higher and 81 or lower).
Inventors: |
BUSSE; Madleen; (Eching,
DE) ; PFEIFFER; Franz; (Unterfohring, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TECHNISCHE UNIVERSITAT MUNCHEN |
Munchen |
|
DE |
|
|
Family ID: |
1000005823217 |
Appl. No.: |
16/755677 |
Filed: |
October 12, 2018 |
PCT Filed: |
October 12, 2018 |
PCT NO: |
PCT/EP2018/077958 |
371 Date: |
April 13, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2001/305 20130101;
C07F 3/00 20130101; G01N 2001/302 20130101; C07F 5/00 20130101;
G01N 1/30 20130101 |
International
Class: |
C07F 3/00 20060101
C07F003/00; G01N 1/30 20060101 G01N001/30; C07F 5/00 20060101
C07F005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 13, 2017 |
EP |
17196491.9 |
Oct 13, 2017 |
EP |
17196492.7 |
Feb 22, 2018 |
EP |
18158168.7 |
Claims
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, and at
least one R is a xanthene derivative.
2. The complex according to claim 1, wherein the complex is
represented by the following formula (I): M.sub.mR.sub.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, at least one R is a xanthene
derivative, and m and n are each independently 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11 or 12.
3. The complex according to claim 1, 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).
4. An ex vivo method for investigating a biological sample
comprising (aspect 1) (a1) staining the biological sample with a
solution (AB) comprising one or more complex(es) as defined in
claim 1; 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, and (ii) one or more solution(s) (B) comprising one or
more heavy metal ion(s) as defined in claim 1, 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.
5. The method according to claim 4 (aspect 1/aspect 2), wherein the
computed tomography scanning method is an X-ray absorption-based
scanning method.
6. The method according to claim 4, wherein the biological sample
of either aspect 1 or aspect 2 is a human soft tissue sample.
7. The method according to claim 4, wherein the biological sample
of either aspect 1 or aspect 2 is subjected to chemical fixation by
means of one or more chemical fixative(s) prior to staining.
8. The method according to claim 7, wherein the one or more
chemical fixative(s) comprise one or more acid(s).
9. The method according to claim 4, wherein the method of either
aspect 1 or aspect 2 comprises, in addition to staining step (a1)
or staining step (a2) an additional step of staining with an
additional staining agent.
10. The method according to claim 4 (aspect 2), wherein solution
(A) comprises eosin Y.
11. The method according to claim 10, wherein the concentration of
eosin Y in solution (A) is in the range of about 10 to about 50
wt/vol-%.
12. The method according to claim 4 (aspect 2), wherein the time
period of contacting the biological sample with solution (A) or the
one or more solution(s) (B) is 3 hours or more.
13. The method according to claim 4 (aspect 2), wherein the
biological sample is contacted with the one or more solution(s) (B)
before the biological sample is contacted with solution (A).
14. The method according to claim 4 (aspect 2), wherein the one or
more solution(s) (B) comprise one or more heavy metal ion(s) M
selected from the group consisting of Ag.sup.+, Ba.sup.2+ or
Gd.sup.3+.
15. (canceled)
16. The complex according to claim 1, wherein the xanthene
derivative is selected from the group consisting of eosin Y and
erythrosin B.
17. The complex according to claim 2, wherein the xanthene
derivative is selected from the group consisting of mono-, di-,
tribromofluorescein; mono-, di, triiodofluorescein; eosin B; eosin
Y and erythrosin B.
18. The complex according to claim 3, wherein the 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 Ag.sup.+, Ba.sup.2+ or
Gd.sup.3+.
19. The method according to claim 5, wherein the computed
tomography scanning method is Micro-Computed Tomography (.mu.CT) or
Nano-Computed Tomography (nanoCT).
20. The method according to claim 9, wherein the additional
staining agent is hematein.
21. The method according to claim 13, wherein the main solvent of
solution (A) and the main solvent of the one or more solution(s)
(B) is a water-based solution.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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.
[0003] 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 afore-mentioned 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).
[0004] 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 10
.mu.m 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.times.1.times.1 mm
histological sample. The slices produced in conventional histology
can vary but a thickness of about 2 .mu.m 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.
[0005] X-ray imaging techniques such as computed tomography (CT),
in particular Micro-Computed Tomography (.mu.CT), 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).
[0006] At present, however, the application of CT (in particular
.mu.CT) 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 (I.sub.2E) 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.
[0007] 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.
[0008] 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
[0009] 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.
[0010] In particular, the present invention relates to the
following items 1 to 53: [0011] 1. A complex comprising: [0012] one
or more heavy metal ion(s) M and one or more ligand(s) R, wherein
[0013] 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
[0014] at least one R is a xanthene derivative (preferably selected
from the group consisting of eosin Y and erythrosin B). [0015] 2.
The complex according to item 1, wherein the complex is represented
by the following formula (I):
[0015] M.sub.mR.sub.n (I), [0016] 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), [0017] 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 [0018] m and n are each
independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. [0019] 3.
The complex according to item 1 or 2, wherein [0020] 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), [0021] at least one of the one or more ligand(s) R is
eosin Y, and [0022] m and n are each independently 1, 2 or 3.
[0023] 4. The complex according to any one of items 1 to 3, wherein
[0024] m and n are both 1. [0025] 5. The complex according to any
one of items 1 to 4, wherein [0026] 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)). [0027] 6. The complex
according to item 5, wherein [0028] at least one of the one or more
heavy metal ion(s) M is selected from the group consisting of
silver(I) (Ag.sup.+), barium(II) (Ba.sup.2+), gadolinium(III)
(Gd.sup.3+), lutetium(III) (Lu.sup.3+), gold(III) (Au.sup.3+),
lead(II) (Pb.sup.2+) and bismuth(III) (Bi.sup.3+) (or selected from
the group consisting of silver(I) (Ag.sup.+), barium(II)
(Ba.sup.2+), lutetium(III) (Lu.sup.3+), gold(III) (Au.sup.3+),
lead(II) (Pb.sup.2+) and bismuth(III) (Bi.sup.3+)). [0029] 7. The
complex according to item 6, wherein [0030] at least one of the one
or more heavy metal ion(s) M is selected from the group consisting
of Ag.sup.+, Ba.sup.2+, Gd.sup.3+, Lu.sup.3+ and Au.sup.3+ (or
selected from the group consisting of Ag.sup.+, Ba.sup.2+,
Lu.sup.3+ and Au.sup.3+). [0031] 8. The complex according to item
6, wherein [0032] at least one of the one or more heavy metal
ion(s) M is selected from the group consisting of Ag.sup.+,
Ba.sup.2+ and Gd.sup.3+ (or selected from the group consisting of
Ag.sup.+ and Ba.sup.2+). [0033] 9. The complex according to item 6,
wherein [0034] at least one of the one or more heavy metal ion(s) M
is Ag.sup.+. [0035] 10. The complex according to item 6, wherein
[0036] at least one of the one or more heavy metal ion(s) M is
Ba.sup.2+. [0037] 11. The complex according to item 6, wherein
[0038] at least one of the one or more heavy metal ion(s) M is
Gd.sup.3+. [0039] 12. The complex according to item 6, wherein
[0040] at least one of the one or more heavy metal ion(s) M is
Lu.sup.3+. [0041] 13. The complex according to item 6, wherein
[0042] at least one of the one or more heavy metal ion(s) M is
Au.sup.3+. [0043] 14. The complex according to item 6, wherein
[0044] at least one of the one or more heavy metal ion(s) M is
Pb.sup.2+. [0045] 15. The complex according to item 6, wherein
[0046] at least one of the one or more heavy metal ion(s) M is
Bi.sup.3+. [0047] 16. An ex vivo method for investigating a
biological sample comprising [0048] (aspect 1) [0049] (a1) 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 [0050] (b)
subjecting the stained biological sample to a computed tomography
scanning method; or [0051] (aspect 2) [0052] (a2) staining the
biological sample with [0053] (i) a solution (A) comprising a
xanthene derivative (preferably mono-, di-, tribromofluorescein;
mono-, di, triiodofluorescein; eosin B; eosin Y and/or erythrosin
B), and [0054] (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, [0055] wherein the biological sample is contacted with
solution (A) separately from the one or more solution(s) (B); and
[0056] (b) subjecting the stained biological sample to a computed
tomography scanning method. [0057] 17. The method according to item
16 (aspect 1/aspect 2), wherein the computed tomography scanning
method is an absorption-based scanning method. [0058] 18. 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. [0059] 19. 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 (.mu.CT) and Nano-Computed Tomography
(nanoCT). [0060] 20. The method according to item 19, wherein the
computed tomography scanning method is .mu.CT. [0061] 21. The
method according to item 19, wherein the computed tomography
scanning method is nanoCT. [0062] 22. 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. [0063] 23. 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. [0064]
24. 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. [0065] 25. 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. [0066] 26. 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 [0067] 27. 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. [0068] 28. 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). [0069] 29. The method according to
item 28, wherein the one or more acid(s) is glacial acetic acid.
[0070] 30. 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 (a1) or staining step (a2) an additional step of
staining with an additional staining agent, preferably hematein.
[0071] 31. The method according to any one of items 16 (aspect 2)
and 17 to 30, wherein solution (A) comprises eosin Y. [0072] 32.
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-%.
[0073] 33. 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-%. [0074] 34. 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-%. [0075] 35. The method
according to item 31, wherein the concentration of eosin Y in
solution (A) is about 30 wt/vol-%. [0076] 36. 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. [0077] 37. The
method according to item 36, wherein the time period is 6 hours or
more. [0078] 38. The method according to item 36, wherein the time
period is 12 hours or more. [0079] 39. The method according to item
36, wherein the time period is 24 hours or more. [0080] 40. The
method according to item 36, wherein the time period is 48 hours or
more. [0081] 41. The method according to item 36, wherein the time
period is 72 hours or more. [0082] 42. The method according to item
36, wherein the time period is 96 hours or more. [0083] 43. 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). [0084] 44. 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. [0085] 45. 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 (a1) or
staining step (a2), an additional step of staining with an
additional staining agent. [0086] 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.sup.+, Ba.sup.2+ and Gd.sup.3+ (or
selected from the group consisting of Ag.sup.+ and Ba.sup.2+).
[0087] 47. The method according to item 46, wherein the one or more
solution(s) (B) comprise Ba.sup.2+. [0088] 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. [0089]
49. The use according to item 48, wherein the computed tomography
scanning is an absorption-based scanning method. [0090] 50. The use
according to item 48 or 49, wherein the computed tomography
scanning is an X-ray absorption-based scanning method. [0091] 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 .mu.CT and nanoCT. [0092] 52. The use according to
item 51, wherein the computed tomography scanning method is .mu.CT.
[0093] 53. The use according to item 51, wherein the computed
tomography scanning method is nanoCT.
[0094] In a particularly preferred embodiment, the present
invention relates to:
[0095] An ex vivo method for investigating a biological sample
comprising [0096] (a) staining the biological sample with [0097]
(i) a solution (A) comprising a xanthene derivative (preferably
eosin Y), and [0098] (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 [0099] (b) subjecting the stained biological
sample to a computed tomography scanning method [0100] 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, [0101]
wherein the biological sample is a human soft tissue sample, [0102]
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, [0103] wherein the method comprises, in
addition to staining step (a) an additional step of staining with
an additional staining agent, preferably hematein, and [0104]
wherein M is a heavy metal ion selected from the group consisting
of Ag.sup.+, Ba.sup.2+, Gd.sup.3+, Lu.sup.3+, and Au.sup.3+ (or
Ag.sup.+, Ba.sup.2+, Lu.sup.3+ and Au.sup.3+), [0105] preferably
Ag.sup.+, Ba.sup.2+ and Gd.sup.3+ (or Ag.sup.+ and Ba.sup.2+).
BRIEF DESCRIPTION OF THE DRAWINGS
[0106] The invention is also illustrated by the following
illustrative figures.
[0107] FIG. 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 (tetra-bromo
barium eosin complex; 25 mg/mL; c=31.9 mM) for 72 hours as
described in Example 1.
[0108] FIG. 2: (a) Micro CT image of acidified and non-acidified
turkey liver samples which were stained, amongst others, with a
Ba(OAc).sub.2 solution (c=217 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 as described in Example
2.
[0109] FIG. 3: (a) Micro CT image of acidified and non-acidified
turkey liver samples which were stained, amongst others, with a
Gd(OAc).sub.3 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.
[0110] FIG. 4: Schematic drawing of a biological sample placed in
an appropriate container for computed tomography scanning ((A):
Stained tissue sample positioned such that 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).
[0111] FIG. 5: The chemical formulas of eosin Y derivatives:
mono-bromo, di-bromo, tri-bromo and tetra-bromo eosin derivative.
Used abbreviations: EOY=eosin Y, M.sub.w=molecular weight.
[0112] FIG. 6: The chemical formulas of erythrosine B derivatives:
mono-iodo, di-iodo, tri-iodo and tetra-iodo erythrosine derivative.
Used abbreviations: ERB=erythrosin B, M.sub.w=molecular weight.
[0113] FIG. 7: The reaction scheme showing the synthesis of eosin Y
derivatives: mono-bromo (A), di-bromo (B), tri-bromo (C) and
tetra-bromo (D) eosin derivatives. Used abbreviations:
eq.=equivalents; rt=room temperature.
[0114] FIG. 8: The reaction scheme showing the detailed synthesis
of eosin Y derivatives: mono-bromo, di-bromo, tri-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; HCl=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).
[0115] FIG. 9: The log P 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 log P
value of the dibromo eosin derivative compared to the fluorescein.
Used abbreviations: EOY=eosin Y.
[0116] FIG. 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.
[0117] FIG. 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.
[0118] FIG. 12:
[0119] The line plots of di-bromo barium eosin complex and
tetra-bromo barium eosin complex are displayed. Soft tissue samples
have been acidified during fixation. The di-bromo barium eosin
complex performed better than the tetra-bromo derivative at the
maximum water solubility for the dibromo barium eosin complex of 45
mg/mL.
[0120] Legend: Sample 1=tetra-bromo barium eosin derivate (c=25
mg/mL, with acid); Sample 2=di-bromo barium eosin derivate (c=25
mg/mL, with acid); Sample 3=di-bromo barium eosin derivate (c=45
mg/mL, with acid).
[0121] FIG. 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 .mu.CT measurements.
[0122] 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).
[0123] FIG. 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 .mu.M 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, .mu.M=micro molar, a.u.=arbitrary units.
[0124] FIG. 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).
20.times. magnification was used to produce this image.
[0125] FIG. 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). 20.times. magnification was used to
produce this image.
[0126] FIG. 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).
40.times. magnification was used to produce this image.
[0127] FIG. 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). 40.times. magnification was used to
produce this image.
[0128] FIG. 19: Macroscopic images of the staining solutions used
for (A) the 1.sup.st and (B) the 2.sup.nd staining step as
described in Example 2.
[0129] FIG. 20: Macroscopic images of the soft tissue sample after
the 2.sup.nd staining step as described in Example 2. Legend: 1 i)
acidified during fixation and ii) Ba(OAc).sub.2--Stoichiometric; 2
i) acidified during fixation, ii) Ba(OAc).sub.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).sub.2--Stoichiometric; 6 i) fixation, ii)
Ba(OAc).sub.2--Stoichiometric and iii) Na-Eosin--Stoichiometric; 7
i) fixation--Control Sample and 8 i) fixation and ii)
Na-Eosin--Eosin Sample.
[0130] FIG. 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 10.times.
magnification. (B) Histological analysis was performed using an
Axio Imager 2 microscope and AxioVision Software (Zeiss). The image
was produced with 40.times. 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 40.times. magnification.
[0131] FIG. 22: Macroscopic images of solutions of the barium-eosin
Y complex (Ba-Eo) vs the sodium-eosin Y complex (Na-Eo).
[0132] FIG. 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 10.times.
magnification. (B) Histological analyses was performed using an
Axio Imager 2 microscope and AxioVision Software (Zeiss). The image
was produced with 20.times. 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 20.times. 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
[0133] 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.
[0134] 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.sup.nd Ed. John Wiley
and Sons, Inc., Hoboken, N.J., 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, U K, 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, Fla., 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), Kachelriel.beta. (M. Kachelriel.beta. 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, Fla., 2011). The above
citations are incorporated by reference.
[0135] The term "heavy metal" refers to metals having a density of
more than 5 g/cm.sup.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).
[0136] 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).
[0137] 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.
[0138] 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.
[0139] 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.
[0140] If not indicated otherwise, concentrations given in
percentages [%] refer to [weight/volume-%] in volume.
Heavy Metal Ion Complexes
[0141] 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.sub.mR.sub.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.
[0142] 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).
[0143] 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)").
[0144] 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.sup.+, Ba.sup.2+, Gd.sup.3+, Lu.sup.3+, Au.sup.3+,
Pb.sup.2+ and Bi.sup.3+). More preferably, M is a heavy metal ion
selected from the group consisting of Ag.sup.+, Ba.sup.2+,
Gd.sup.3+, Lu.sup.3+ and Au.sup.3+. Particularly preferable, M is a
heavy metal ion selected from the group consisting of Ag.sup.+,
Ba.sup.2+ and Gd.sup.3+. Most preferably, M is Ba.sup.2+.
[0145] 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.sup.+, Ba.sup.2+, Lu.sup.3+, Au.sup.3+, Pb.sup.2+
and Bi.sup.3+). More preferably, M is a heavy metal ion selected
from the group consisting of Ag.sup.+, Ba.sup.2+, Lu.sup.3+ and
Au.sup.3+. Particularly preferable, M is a heavy metal ion selected
from the group consisting of Ag.sup.+ and Ba.sup.2+. Most
preferably, M is Ba.sup.2+.
[0146] 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.
[0147] 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:
##STR00001##
wherein X.sub.1 is each independently NO.sub.2, halogen or H,
X.sub.2 is each independently Halogen or H, R.sub.1, R.sub.2,
R.sub.3 and R.sub.4 are each independently H, halogen
(independently selected from the group consisting of Cl, Br and I)
or a water solubility enhancing group selected from OH, NH.sub.2,
SH, NO.sub.2, SO.sub.3H, SO.sub.4H.sub.2, PO.sub.3H.sub.2 diol and
polyols such as polyethyleneglycols, and R.sub.5 and R.sub.6 are
each independently H or a water solubility enhancing group selected
from NH.sub.2, SH, NO.sub.2, SO.sub.3H, SO.sub.4H.sub.2,
PO.sub.3H.sub.2 diol and polyols such as polyethyleneglycols.
[0148] Preferred examples of the xanthene derivatives used in the
present invention are mono-, di-, tri- and
tetrachlorofluoresceines; mono-, di-, tri-, and
tetrabromofluoresceines; 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:
##STR00002##
wherein M is metal ion (such as Na.sup.+ or Ba.sup.2+) 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 Cl, Br
and I). More preferred are the compounds shown in FIGS. 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.
[0149] 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.degree. 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 C18 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.
[0150] 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.sup.2+, Cu.sup.2+, Ba.sup.2+ with 1.00 equivalent and
Na.sup.+ 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.)
[0151] 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:
##STR00003##
[0152] 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).
[0153] 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.sub.2O. 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).
[0154] 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.
[0155] 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.sub.2O, NO,
hydrogen carbonate, hydrogen phosphate, carboxylates (e.g.,
formate, acetate, lactate, oxalate), chloride, etc.
[0156] 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.
[0157] 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 1000 .mu.m, less
than 500 .mu.m, less than 200 .mu.m, less than 100 .mu.m, less than
50 .mu.m, less than 20 .mu.m, less than 10 .mu.m, less than 5
.mu.m, less than 2 .mu.m, or less than 1 .mu.m. In some embodiments
the heavy metal complex is present in particulate form wherein the
particles have a mean diameter in the range of from 1000 .mu.m to
500 .mu.m, from 500 .mu.m to 200 .mu.m, from 200 .mu.m to 100
.mu.m, from 100 .mu.m to 50 .mu.m, from 50 .mu.m to 20 .mu.m, from
20 .mu.m to 10 .mu.m, from 10 .mu.m to 5 .mu.m, from 5 .mu.m to 2
.mu.m, or from 2 .mu.m to 1 .mu.m.
[0158] 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
[0159] 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:
[0160] 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.
[0161] 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 (.mu.CT) and
Nano-Computed Tomography (NanoCT). The prefix micro- (symbol: .mu.)
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 .mu.CT
devices are V|tome|X (designed and developed by GE) and XRadia
(designed and developed by Zeiss).
[0162] 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)).
[0163] 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.
[0164] 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.
[0165] 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. polyurethane, 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.
[0166] 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.
[0167] 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
[0168] In a first aspect, the present invention relates to an ex
vivo method for investigating a biological sample comprising: (a1)
staining the biological sample with a solution (AB) comprising one
or more complex(es) as defined above.
[0169] 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).
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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).
[0176] 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%.
[0177] 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 the tissue are acidified as
illustrated in the Scheme below:
##STR00004##
[0178] 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).
[0179] 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.
[0180] 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.
[0181] 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 200 mM, or about 5 to about 100 mM. More preferably, the
concentration of the heavy metal complex in solution (AB) is in the
range of about 10 to about 50 mM. Particularly preferably, the
concentration of the heavy metal complex in solution (AB) is in the
range of about 20 to about 40 mM. Most preferably, the
concentration of the heavy metal complex in solution (AB) is about
30 mM.
[0182] 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.
[0183] 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.).
[0184] 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.
[0185] 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.
[0186] 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 30 g of
eosin Y are dissolved in about 100 mL of distilled water.
[0187] 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.sup.+, Ba.sup.2+,
Gd.sup.3+, Lu.sup.3+, Au.sup.3+, Pb.sup.2+ and Bi.sup.3+). More
preferably, the one or more heavy metal ion(s) derive from the
group consisting of Ag.sup.+, Ba.sup.2+, Gd.sup.3+, Lu.sup.3+ and
Au.sup.3+. Particularly preferable, the one or more heavy metal
ion(s) derive from the group consisting of Ag.sup.+, Ba.sup.2+ and
Gd.sup.3+, most preferably Ba.sup.2+.
[0188] 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.sup.+, Ba.sup.2+, Lu.sup.3+, Au.sup.3+, Pb.sup.2+
and Bi.sup.3+). More preferably, the one or more heavy metal ion(s)
derive from the group consisting of Ag.sup.+, Ba.sup.2+, Lu.sup.3+
and Au.sup.3+. Particularly preferable, the one or more heavy metal
ion(s) derive from the group consisting of Ag.sup.+ and Ba.sup.2+,
most preferably Ba.sup.2+.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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 (a1) 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[2,1-c]chromen-9-one),
having a structure represented by below Chemical Formula (III),
##STR00005##
is an oxidized derivative of hematoxylin
(7,11b-Dihydroindeno[2,1-c]chromene-3,4,6a,9,10(6H)-pentol), which
is represented by below Chemical Formula (IV)
##STR00006##
[0194] 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.sub.3), sodium iodate (NaIO.sub.3) or mercury
oxide (HgO) can be added to a hematoxylin solution. Moreover,
hematein and hematoxylin are both commercially available.
[0195] 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.
[0196] In other embodiments, the inventive ex vivo method does not
comprise, in addition to staining step (a1) or staining step (a2),
an additional step of staining with an additional staining
agent.
[0197] 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 .mu.CT setup, e.g. the V|tome|X.
[0198] 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., FIG.
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 .mu.A, 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
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.degree. 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 50 kV
peak voltage, 6.0 W and with 1001 projections distributed over
360.degree.. The low-resolution CT data were acquired with an
exposure time of 1 s per projection with an effective pixel size of
ca. 30 .mu.m. 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]
[0205] Fixation Step without the Addition of Acid:
[0206] 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.sup.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. FIG. 22).
[0207] 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.
[0208] 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:
[0209] 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.sup.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.
[0210] 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).
[0211] An overview of the various samples is shown in Table 1
below:
TABLE-US-00001 TABLE 1 Sample Acidification Staining 1 Yes
barium-eosin Y complex (c = 31.9 mM) 2 Yes eosin Y (c = 31.9 mM) 3
Yes Control 4 No barium-eosin Y complex (c = 31.9 mM) 5 No eosin Y
(c = 31.9 mM) 6 No Control 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 .mu.A; P = 5.5 W; Bin 1 .times. 1; exposure
time: 2 s; filter: air; average = 3, skip = 1; projections: 1601;
pixel size: 40.056 .mu.m; total scan time: 214 min.
[0212] The results of these measurements are summarized in FIG. 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
[0213] [Staining with Barium Acetate Solution and Eosin Y Solution]
Fixation Step without the Addition of Acid:
[0214] 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.sup.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.
[0215] 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:
[0216] 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.sup.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.
[0217] 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).
[0218] An overview of the various samples is shown in Table 2:
TABLE-US-00002 TABLE 2 Sample Acidification Staining 1 Yes
Ba(OAc).sub.2 2 Yes Ba(OAc).sub.2 and eosin Y 3 Yes Control 4 Yes
eosin Y 5 No Ba(OAc).sub.2 6 No Ba(OAc).sub.2 and eosin Y 7 No
Control 8 No eosin Y
[0219] A macroscopic image showing all staining solutions used for
the 1.sup.st staining step are shown in FIG. 19(A) while all
staining solutions used for the 2.sup.nd staining step are shown in
FIG. 19(B). Further, macroscopic images of the soft tissue sample
after the 2.sup.nd staining step are shown in FIG. 20. As can be
seen from FIG. 20, the tissue sample changed its color upon
incubation with Eosin Y in the 2.sup.nd step. The incubation with
Ba(OAc).sub.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 FIG.
22, where the solution of the barium-eosin y complex was compared
with the eosin y complex in the sample holder, which clearly
highlights the orange-red color of the barium-eosin complex.
Therefore, only the combination of Ba(OAc).sub.2 and Eosin Y
resulted in a "colored" stained tissue sample.
[0220] 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 .mu.A;
P=5.5 W; Bin 1.times.1; exposure time: 2 s; filter: air;
projections: 1601; pixel size: 38.770 .mu.m; total scan time: 267
min.
[0221] The results of these measurements are summarized in FIGS.
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).sub.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).sub.2
solution and an eosin Y solution (cf. sample 2) compared to samples
which were stained with the Ba(OAc).sub.2 solution only (cf. sample
1) or the eosin Y solution only (cf. sample 4).
[0222] To further evaluate the staining, histological
investigations were performed which are shown in FIGS. 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 FIG. 22, where the solution of the barium-eosin y complex
was compared with the eosin y complex in the sample holder, which
clearly highlights 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 FIG.
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
[0223] [Staining with Gadolinium Acetate Solution and Eosin Y
Solution] Fixation Step without the Addition of Acid:
[0224] 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.sup.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.
[0225] 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:
[0226] 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.sup.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.
[0227] 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).
[0228] An overview of the various samples is shown in Table 3:
TABLE-US-00003 TABLE 3 Sample Acidification Staining 1 Yes
Gd(OAc).sub.3 2 Yes Gd(OAc).sub.3 and eosin Y 3 Yes Control 4 Yes
eosin Y 5 No Gd(OAc).sub.3 6 No Gd(OAc).sub.3 and eosin Y 7 No
Control 8 No eosin Y 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 .mu.A; P = 5.5 W; Bin 1 .times. 1; exposure time: 2 s;
filter: air; projections: 1601; pixel size: 38.770 .mu.m; total
scan time: 267 min.
[0229] The results of these measurements are summarized in FIGS.
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).sub.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).sub.3 solution and an
eosin Y solution (cf. samples 2 and 6) compared to samples which
were stained with the Gd(OAc).sub.3 solution only (cf. samples 1
and 5) or the eosin Y solution only (cf. samples 4 and 8).
* * * * *