3D-Non-destructive Imaging through Heavy-Metal Eosin Salt Contrast Agents

Article abstract of DOI:10.1002/chem.202005203

Conventional histology is a destructive technique based on the evaluation of 2D slices of a 3D biopsy. By using 3D X-ray histology these obstacles can be overcome, but their application is still restricted due to the inherently low attenuation properties of soft tissue. In order to solve this problem, the tissue can be stained before X-ray computed tomography imaging (CT) to enhance the soft tissue X-ray contrast. Evaluation of brominated fluorescein salts revealed a mutual influence of the number of bromine atoms and the cations applied on the achieved contrast enhancement. The dibromo fluorescein barium salt turned out to be the ideal X-ray contrast agent, allowing for 3D imaging and subsequent complementing counterstaining applying standard histological techniques.

Full text of DOI:10.1002/chem.202005203

Accepted Article
Accepted Article
Title: 3D-Non-Destructive Imaging through Heavy Metal-Eosin Salt
Contrast Agents
Authors: Madleen Busse, Jaroslaw P. Marciniszyn, Simone Ferstl,
Melanie A. Kimm, Franz Pfeiffer, and Tanja Gulder
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202005203
Link to VoR: https://doi.org/10.1002/chem.202005203
01/2020

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3D-Non-Destructive Imaging through Heavy Metal-Eosin Salt
Contrast Agents
Madleen Busse,+*[b] Jaroslaw P. Marciniszyn,+[a] Simone Ferstl,[b] Melanie A. Kimm,[c] Franz Pfeiffer,[b,c]
Tanja Gulder*[a,d]
Dedication ((optional))
[a]
[b]
[c]
J. P. Marciniszyn, Prof. Dr. T. Gulder
Department of Chemistry and Catalysis Research Center (CRC)
Technical University Munich
85748 Garching, (Germany)
Dr. M. Busse, Prof. Dr. F. Pfeiffer
Department of Physics and Munich School of BioEngineering
Technical University Munich
85748 Garching, (Germany)
M. A. Kimm, Prof. Dr. F. Pfeiffer
Department of Diagnostic and Interventional Radiology
Klinikum Rechts der Isar
Technical University Munich
81675 Munich, (Germany)
[d]
Prof. Dr. T. Gulder
Institute of Organic Chemistry
Leipzig University
04103 Leipzig, (Germany)
E-mail: tanja.gulder@uni-leipzig.de
Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))
Abstract: Conventional histology is a destructive technique based on
the evaluation of 2D slices of a 3D biopsy. By using 3D X-ray histology
these obstacles can be overcome, but their application is still
the resulting reconstructions are often incomplete or do not
represent the 3D structure of the imaged sample in a reliable way.
To overcome these limitations, X-ray microscopic computed
restricted due to the inherently low attenuation properties of soft tissue. tomography (μCT)[5] and nanoscopic CT (nanoCT)[5a,6] imaging
In order to solve this problem, the tissue can be stained before X-ray
computed tomography (CT) imaging to enhance the soft tissue X-ray
contrast. Evaluation of brominated fluorescein salts revealed a mutual
influence of the number of bromine atoms and the cations applied on
the achieved contrast enhancement. The dibromo fluorescein barium
salt turned out to be the ideal X-ray contrast agent, allowing for 3D
imaging and subsequent complementing counterstaining applying
standard histological techniques.
have proven to provide valuable 3D information of biological
samples in a fast, convenient and non-destructive way (Figure
1A). Here, non-destructive refers to the ability of X-ray CT to
investigate biological samples using a multiscale approach
ranging from whole organisms over whole organs to pieces of
organs of animals without the need to embed or destruct the
biological material. As a result, a 3D data set is received, which
allows for virtually extracting any desired slice under any arbitrary
angle throughout the entire volume (as an example see video in
the SI).
Medical diagnosis providing microscopic information on
(sub)-cellular level is currently realized through conventional 2D
histopathology, which is mainly based on light microscopy
techniques. In combination with a large diversity of dyes and
staining methods that have been developed over time various
specific biological structures can be individually targeted.
However, the current techniques are limited to the staining of only
thin (2 µm – 20 µm) 2D microscopic slides that originate from a
3D biopsy sample. As the demand to extend biological and
medical investigations to three dimensions has grown significantly
over the last decade, several approaches for 3D imaging.[1]
Beyond serial-sectioning based approaches, these imaging
methods include confocal and light sheet laser-scanning
microscopy[2] and block-face imaging (episcopic microscopy).[3,4]
Even though these methods have improved considerably over the
years, they still require salt correction and registration steps and
X-ray CT devices can be found at large synchrotron facilities[7] but
also in a laboratory environment[6b,6d,6e,8] capable of providing
resolutions comparable to conventional 2D histology. Because of
the intrinsically low attenuation properties of soft tissue at hard X-
ray energies, the use of stains bearing high atomic number
elements is inevitable in order to reach sufficient contrast (Figure
1B and 1C). Nonetheless, the availability of easy to handle X-ray
staining agents is currently very limited that are (i) speedily
penetrating the tissue without creating artefacts, (ii) targeting a
specific biological morphology by staining the probe
homogenously and completely, and (iii) at the same time suitable
for large and dense tissue samples. In addition, staining
procedures need to be introduced that are fully compatible with
standard histology and thus allow further investigations of the
region of interest (ROI) by the histologist, if necessary. First
attempts to develop such dyes as X-ray contrast agents included
1
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modifications of elemental iodine I2 for whole organ morphology[9]
and eosin Y disodium salt (2a) to target specifically the cell
cytoplasm.[5a,6d] Eosin y is a versatile, non-toxic compound[10,11]
and readily available as the disodium salt 2a (Scheme 1), but its
contrast enhancement is limited despite the four covalently bound
bromine atoms (Z = 35). Best results for soft-tissue visualization
employing 2a are achieved only by using very high concentrations
of 2a (30% (w/v)) and cannot be further enhanced because of its
limited water solubility (300 mg/mL).[5a]
Scheme 1. A) Synthesis of eosin y salts 2 and (B) macroscopic investigations
of stained tissue. The tissue samples were pre-treated with a 4% formaldehyde
solution and 0.5 mL glacial acetic acid. Staining was performed using a 31.9
mM aq. solution of eosin y salt 2a or 2c – 2e. For each condition, an individual
number of three turkey liver pieces (ca. 27 mm3) was stained to ensure
reproducibility; one of these pieces was investigated macroscopically.
In a first, preliminary solubility study, all eosin y salts 2b – 2f
showed a significantly lowered maximum concentration in water
when compared to the disodium compound 2a (cf. SI, chapter 4).
The Ag(I) salt 2b as well as the Gd(III) salt 2f did not meet the
sensitivity threshold needed for μCT measurements (< 10 mg/mL)
and were thus not further investigated. The other compounds 2c
– 2e were then studied with regard to their staining properties.
The use of X-ray contrast agents to stain ex vivo biological
samples often suffers from a spatially and temporally anisotropic
stain penetration into soft tissue, leading to an artificial contrast
gradient between the surface and the sample core. We thus
chose turkey liver samples as soft tissue test pieces for staining
as liver tissue is one of the densest soft tissues present in both
birds and mammalians. If the liver tissue can be completely and
homogeneously stained within a reasonable time frame the dye
should be capable of successfully staining other tissue types as
well. Cuboidal turkey liver pieces of ca. 3 mm edge length were
therefore treated in triplicates with 31.9 mM aqueous solutions of
each dye 2c – 2e whereby the maximum water solubility of the
barium-eosin y salt (2c) was used as the concentration of the
staining agents. Macroscopic investigations showed the best
results using the Ba(II) eosin-salt 2c with respect to uniform
staining. In addition, no physical deformation of the tissue cubes,
Figure 1. (A) Schematic representation of a microCT setup showing the X-ray
source on the left and the detector on the right. In the middle is a rotation stage
holding the soft-tissue sample. The Lambert-Beer Law describes the reduction
of the X-ray intensity by passing through a material. Here the linear absorption
coefficient µi, which is unique for each material, is directly proportional to the
atomic number Z to the power of 4.[12] (B) Unstained soft tissue is mainly made
of hydrogen atoms (H), carbon atoms (C), oxygen atoms (O) and nitrogen atoms
(N), which consist of low atomic numbers resulting in an inherent low contrast
for soft tissue seen on the CT slice. (C) Stained tissue on the other hand has
accumulated contrast agent that holds elements of high atomic number Z (see
stained CT slice). The contrast agent used here was dibromo fluorescein 9c
(see chemical structure) holding two bromine atoms (ZBr = 35, highlighted in
apricot) and a barium atom (ZBa = 56, highlighted in green).
Based on recent reports on 2a as contrast agent for μCT,[5a,6d,6e]
we investigated the possibility to further improve the contrast
within the soft tissue by using heavy metal-eosin y salts 2b – 2f.
Because of the high Z value of heavy metals (Figure 1), their
implementation should allow to lower the concentration of
contrast agent needed to reach a suitable contrast enhancement
comparable to that of 2a. As water solubility might be impaired by
the involvement of heavy metal atoms, we furthermore
investigated the influence of the bromine atoms at the fluorescein
core in 2 in response to solubility in aqueous media, tissue
penetration, staining properties, and contrast enhancement, in
order to implement a new ‘gold standard’ for μCT of biopsy
samples.
such as shrinking, was visible.
An inhomogeneous and
incomplete staining was observed for the samples treated with the
Cu(II) 2d and Pb(II) salts 2e[13] even after an incubation time of
144 h (see Scheme 1B and SI, chapter 8). To allow for an X-ray
contrast agent to enhance the soft-tissue contrast, the standard
histological staining protocol had to be tailored towards X-ray μCT
by means of pH adjustment of the tissue prior to staining,
increasing the incubation times and the concentration of the
contrast agent. As such acidification of the tissue sample prior to
staining turned out to be crucial for 2a and 2c. The enhanced
accumulation of both 2a and 2c in the cell cytoplasm can be
explained by increased ionic interactions between the contrast
agent and basic protein residues that are protonated at acidic pH
values.[5a] Under these conditions, a macroscopically complete
and homogeneous staining for the barium-eosin y salt (2c) was
achieved after incubation of the tissue for 72 hours, while the
tissue samples treated with 2a suffered from inhomogeneities (cf.
SI, chapter 8).
The heavy metal-eosin y salts 2b – 2f were synthesized by
treating the lactone form of eosin y (1) with the corresponding
metal hydroxide and metal acetate, respectively (Scheme 1A and
SI, chapter 2). The salts 2b – 2f were obtained as homogenous
solids after recrystallization in 12% - 51% yield.
2
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In order to study the contrast enhancement of the barium-eosin y
salt (2c) in comparison to the eosin y disodium salt (2a) in more
detail, CT measurements at a μCT system were performed for the
tissue samples incubated for 72 h under different conditions. The
obtained µCT data were analyzed and evaluated for (i)
completeness of staining, (ii) presence of diffusion rings, (iii)
contrast enhancement, (iv) appearance of CT artifacts as streaks
and (v) homogeneity of the staining.
staining agents. These negative control experiments show nicely
the mutual influence of the organic anion eosinate y and the heavy
metal barium cation for cytoplasm interaction and contrast
enhancement. Besides this, the variations in the line plots of the
acidified eosin y disodium samples (2 and 6) as well as the non-
acidified barium-eosin y samples (5 and 7) seen at the beginning
or end of the plateau, clearly indicate the formation of a diffusion
ring, indicative of an incomplete and inhomogeneous staining of
the soft tissue sample.
Encouraged by the results obtained above using the barium eosin
y staining agent (2c), we further explored a possible contrast
enhancement by raising the water solubility of the barium
eosinates. We thus developed a directed synthesis to selectively
brominate the xanthene core in 3 to obtain the mono-, di- and tri-
brominated fluorescein derivatives 4 – 6 (Scheme 2). Treatment
of 3 – 6 with NaOH or Ba(OH)2 afforded after recrystallization the
corresponding sodium and barium salts 2, 7 – 10. To get a first
hint at the hydrophilicity of these compounds, we determined the
logP values of the lactones 1 and 3 – 6 as well as of the acid salts
2, 7 and 9 (SI, chapter 3). Here, an explicit correlation of the
number of Br atoms and the partition coefficient was revealed, in
particular for the barium fluoresceins 2c, 7c – 10c.
Figure 2. (A) Representative μCT slices of the equimolar stained turkey liver
pieces (incubation time 72 h) and (B) the corresponding line plots. 1: 2c (c =
31.9 mM), acidified; 2: 2a (c = 31.9 mM), acidified; 3: 2c (c = 31.9 mM), acidified;
4: control sample (no staining agent was applied), acidified; 5: 2c (c = 31.9 mM);
6: 2a (c = 31.9 mM); 7: 2c (c = 31.9 mM); 8: control sample (no staining agent
was applied). Parameters of the microCT setup (V|tome|X, Phönix X-ray/ GE)
used for the measurement: U = 50 kV; I = 110 µA; exposure time: 2s; pixel size:
40 µm. The microCT slices were chosen so that all investigated samples are
visible as they are mounted in different positions within the sample holder. The
gray values were normalized to the sample holder, which was the same for all
stained samples (0.5 mL Eppendorf tube).
The barium-eosin y treated samples (1 and 3), which have been
acidified during fixation, offered a complete and homogeneous
staining of the soft-tissue samples, as indicated by the CT slices
(Figure 2A) and the constant plateau seen in the line plot (Figure
2B). This result clearly confirms the macroscopic observations (cf.
Scheme 1B and SI, chapter 8) and shows the reliability and
reproducibility of the staining method using 2c as the dye. In
addition, these probes (2c, acidified) displayed a significantly
higher gray value (Figure 2B) when compared to the equimolar
eosin y disodium stained samples 2 and 6 and the non-acidified
barium-eosin y samples 5 and 7. As the gray values represent a
measure for contrast enhancement (the higher the gray value the
better the attenuation contrast), this refers to a significantly
increased contrast of the acidified liver samples stained with 2c
compared to 2a and thus demonstrates the impact of the heavy
metal cation. This effect, however, was only observed for the
barium eosin y salt 2c while other Ba salts containing low atomic
number elements, such as Ba(OH)2 or Ba(OAc)2, failed as specific
Scheme 2. Selective bromination of fluorescein (3) and preparation of the
corresponding sodium 7a – 10a and barium salts 7c – 10c.
Because of the significantly lowered lipophilicity of the dibromo
barium fluoresceinate 9c in comparison to the eosin y salt 2c in
combination with its straightforward synthesis, the dibromo
fluorescein 9c was evaluated in more detail. In a second staining
experiment the influence of the halogenation degree on the
fluorescein core was studied with respect to contrast
enhancement using µCT. Since the first staining experiment
proved already a better performance of the barium eosin y salt
(2c) compared to the eosin y disodium salt (2a), we decided to
exclude 2a from the current study. In line with the results
described for 2c before (see Figure 2), the soft-tissue samples
being acidified during fixation or prior to staining with 2c and 9c,
3
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respectively, displayed a better contrast enhancement compared
to the non-acidified samples (for details see SI, chapter 10). The
dibromo barium fluorescein derivative (9c) performed better than
2c when the maximum concentration of each compound in water
was applied (c = 45 mg/mL [c = 72.0 mM] for 9c; c = 25 mg/mL [c
= 31.9 mM] for 2c) offering a homogeneous staining of the whole
tissue sample (cf. SI, chapter 9, Figure S3). The increased
contrast for 9c can be explained by its enhanced water solubility
of the dibromo compound 9c. Overall, the barium xanthene
derivatives 2c and 9c showed much better contrast enhancement
compared to the sodium analogs 2a and 9a at that low
concentrations.
histological section, which is still identical with the µCT slice
shown in Figure 3C.
To allow for comparison with the histological results and to
showcase the compatibility of the developed barium fluorescein
staining protocol with standard histological methods, the cell
nucleus-specific staining with the counter stain Mayr’s
hematoxylin was applied to the histological microscopic slide after
staining with 2c and microCT measurements. As expected, the
cell cytoplasm still appeared pinkish (cf. SI, chapter 11, Figure
S4A) and the cell nuclei were highlighted purple in color (cf. SI,
chapter 12, Figure S4B). The barium dibromo fluoresceinate salt
9c, staining of the cell cytoplasm, as well as the additional Mayr’s
hematoxylin counterstain (H-stain) were therefore not disturbed
by each other and thus standard histological slides (cf. SI, chapter
12, Figure S4A and S4B) were obtained following standard
histological H staining procedures. The resulting images were of
high quality and suitable to undergo further histological analysis,
which contributes to the practicality of our method and
significantly enhances the sample availability.
After the in-depth evaluation on the ability of the barium salts 2c
and 9c to act as X-ray CT tracers, we wanted to compare the
staining quality of our newly developed dye 9c to the recently
introduced X-ray stain 2a.[5a] Therefore, we applied the dibromo
fluorescein barium salt (9c) to mouse kidney-tissue pieces (ca.
5mm edge length). The mouse kidney with its different tissue
types offers various biological structures at multiple scales. After
the acquisition of an overview scan, a volume of interest (VOI)
was chosen and measured with high-resolution as a local
tomography (cf. SI, video). The ability to obtain high-resolution 3D
data with isotropic resolutions of desired VOIs while keeping the
soft-tissue sample intact, are unique features of µCT that cannot
be found among other microscopy techniques such as most light
or electron microscopy techniques.
Our results are displayed in Figure 3 and confirm homogenous
staining throughout the sample without diffusion artefacts. The
perspective view of a representative volume in Figure 3A
highlights the 3D arrangement of inner structures and connectivity
between different tissue types. The provided contrast enabled the
visualization of relevant anatomical structures within this volume
of interest, the cortex and outer medulla region such as
convoluted tubules, vessels, or medulla rays. Representative
individual 2D µCT slices derived from the orthogonal planes of the
volume in Figure 3A are displayed in Figure 3B-D. The validation
of the µCT results was performed through histological analysis.
The representative histological light microscopy image shown in
Figure 3E was obtained from the very same dibromo fluorescein
barium salt (9c) stained mouse kidney tissue piece seen in Figure
3A. Here, the obtained histological section was counterstained
with the standard histological procedure according to Mayr’s sour
hematoxylin to stain the cell nuclei, which were not targeted by
the cytoplasm selective 9c. This resulted in a typical H&E stained
Figure 3. High-resolution µCT data of the cortex and outer medulla region of a
mouse kidney after application of the dibromo fluorescein barium salt (9c). (A)
Representative µCT volume of 1.20 mm x 1.20 mm x 1.20 mm (effective voxel
size ~ 3.3 µm). (B) (C) and (D) showing individual µCT slices derived from the
orthogonal planes through the volume shown in (A). (E) Representative X-ray
barium eosin y stained histological microscopic slide. The cell nuclei were
counter stained with Mayr’s sour hematoxylin. Legend: I proximal convoluted
tubules, II: artery, III: vein, IV: capillary, V: medullary rays and VI loop of Henle.
Different gray values within the sample display varying
concentrations of contrast agent accumulated in the soft-tissue,
i.e. the higher the protonated protein / peptide content in the
cytoplasm the more contrast agent can interact with these
structures and the higher the contrast. This can be seen in the
perspective view of a representative virtual volume in Figure 3A
and the individual CT slices in Figure 3B-D, e.g. labelled
4
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[4]
[5]
W. Denk, H. Hortsmann, PLoS Biol. 2004, 2, e329.
structures such as V appear very bright and III being black
meaning no accumulation of contrast agent. The CT results are
very well reflected in the histological slide where the structures V
appears very pink indicating a high concentration of the contrast
agent 9c, while II and III being white highlighting the absence of
contrast agent. Thus the staining results and image quality
compare very well with the respective results obtained with the
eosin Y disodium salt (2a).[5a] A clear advantage is seen here in
the enormous reduction of the contrast agent used to stain the
biopsy samples from 300 mg/mL for 2a to 25 mg/mL for 9c. Even
though the color of the dibromo fluorescein barium salt (9c)
shifted to orange-pink when compared to the eosin Y disodium
salt (2a), the pathologists observed no problems to perform their
histological analysis. Thus, the new X-ray stain dibromo
fluorescein barium salt (9c) proved superior and is thus suitable
for µCT even at low concentrations.
To conclude, the improved solubility of 9c in water was crucial to
obtain this new X-ray staining agent that allows to
non-destructively and selectively visualize the cell cytoplasm of
biological and medical soft-tissue samples in three dimensions.
The application of the staining protocol to turkey liver and mouse
kidney tissue pieces underlines the reliability of the protocol and
emphasizes the use for different tissue types. The ability to
counterstain the biopsy samples using standard histological
methods paves the way for establishing a convenient 3D X-ray
histology approach as a complementary tool for future histological
analysis. This will offer access to additional information and
support histologists where 2D imaging is facing its boundaries and
thus meeting the demands to provide answers to advanced
medical questions, which will benefit from targeted staining of
specific biological structures as well as non-destructive 3D
imaging techniques.
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Acknowledgements
This work was funded by the Emmy-Noether (DFG, GU 1134-3)
and Heisenberg (DFG, GU 1134-4) program of the German
Research Foundation (DFG) to T.G. M.B. thanks the European
Union Horizon 2020 research and innovation program under the
Marie Skłodowska-Curie Grant Agreement No. H2020-MSCA-IF-
2015-703745-CONSALT.
[9]
For the synthesis and characterization of the Pb(II) eosin y salt (2e) see
C. Anselmi, D. Capitani, A. Tintaru, B. Doherty, A. Sgamellotti, C. Miliani,
Dyes Pigm. 2017, 140, 297-311.
Keywords: microCT • eosin y • fluorescein • stain
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10.1002/chem.202005203
Chemistry - A European Journal
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The dibromo fluorescein barium salt proved to be suitable staining agent enabling the diagnostic screening of tissue samples by µCT
without impending further diagnostics through histological methods.
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