A comparison of sample preparation strategies for biological tissues and subsequent trace element analysis using LA-ICP-MS
A comparison of sample preparation strategies for biological tissues and subsequent trace element analysis using LA-ICP-MS
Maximilian Bonta 0 1 2
Szilvia Török 0 1 2
Balazs Hegedus 0 1 2
Balazs Döme 0 1 2
Andreas Limbeck 0 1 2
Andreas Limbeck 0 1 2
0 Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center Vienna, Medical University of Vienna , 1090 Vienna , Austria
1 Department of Tumor Biology, National Koranyi Institute of Pulmonology , Budapest 1121 , Hungary
2 Institute of Chemical Technologies and Analytics, TU Wien , Getreidemarkt 9/164-IAC, 1060 Vienna , Austria
Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is one of the most commonly applied methods for lateral trace element distribution analysis in medical studies. Many improvements of the technique regarding quantification and achievable lateral resolution have been achieved in the last years. Nevertheless, sample preparation is also of major importance and the optimal sample preparation strategy still has not been defined. While conventional histology knows a number of sample pre-treatment strategies, little is known about the effect of these approaches on the lateral distributions of elements and/or their quantities in tissues. The technique of formalin fixation and paraffin embedding (FFPE) has emerged as the gold standard in tissue preparation. However, the potential use for elemental distribution studies is questionable due to a large number of sample preparation steps. In this work, LA-ICP-MS was used to examine the applicability of the FFPE sample preparation approach for elemental distribution studies. Qualitative elemental distributions as well as quantitative concentrations in cryo-cut tissues as well as FFPE samples were compared. Results showed that some metals (especially Na and K) are severely affected by the FFPE process, whereas others (e.g., Mn, Ni) are less influenced. Based on these results, a general recommendation can be given: FFPE samples are completely unsuitable for the analysis of alkaline metals. When analyzing transition metals, FFPE samples can give comparable results to snapfrozen tissues.
Biological samples; Laser ablation; Mass spectrometry ICP-MS
Laser ablation-inductively coupled plasma-mass spectrometry
(LA-ICP-MS) is a tool nowadays widely used for the laterally
resolved assessment of trace elements in biological tissues .
Exceptional limits of detection for a wide range of metallic
analytes and good lateral resolutions combined with
reasonable acquisition times make this technique a valuable tool for
such imaging applications [2, 3]. During the last decades, a
variety of works on bio-imaging using LA-ICP-MS have been
presented and the technique has already been included in
clinical research in some examples. The tissue type investigations
have been performed on (e.g., liver , brain [5, 6], kidney
, or tumor tissues [8, 9]) are as numerous as the elements
that have been analyzed already; here, naturally occurring
minor (e.g., Na, K) and trace elements (e.g., Ni, Cu, Zn), as
well as elements artificially introduced during chemotherapy
(e.g., Pt [8, 9]) or being used as contrasting agents (e.g., Gd
), have to be named in this context. LA-ICP-MS does not
only give the opportunity of acquiring qualitative distribution
images but also quantification is possible [11, 12]. With regard
to the quantification strategy, a wide variety of approaches has
been presented in the past, as LA-ICP-MS suffers from severe
matrix effects and slight variations in tissue composition may
already induce unwanted signal changes that do not reflect the
actual composition of the sample. Thus, the use of appropriate
standard materials, as well as employing a suitable internal
standard, is imperative . As certified reference materials
are scarcely available for biological tissues, alternative
approaches have to be used ranging from the preparation of gel
standards , over printed patterns on paper , to the use of
polymeric layers , or the online addition of aqueous
standards . Also concerning internal standardization,
possibilities are various, including not only the sample inherent
carbon but also additionally applied materials/reagents such as
thin gold layers [17, 18], protein-metal tags , or polymer
thin films . Each of the named approaches has their
advantages and weaknesses. Their description is, however, not
within the scope of this work.
However, an important part of accurate analysis is sample
preparation and pretreatment. Here, some research is required
to evaluate appropriate strategies for obtaining reliable results
from bio-imaging experiments. Typical histology knows a
variety of sample preparation techniques, each one fit for
different purposes, whether performing conventional histological
staining, immunohistochemistry, or other, more specialized,
techniques. Despite the variety of sample fixation,
preparation, and cutting strategies, one method has emerged as the
gold standard: formalin fixation and paraffin embedding
(FFPE) . Main advantages over other techniques are that
the samples can be stored under ambient conditions for
decades and thin cutting is fairly simple and can also be carried
out at room temperature, i.e., no cryo-microtome is required.
Due to the exceptional storage properties, large tissue archives
of FFPE tissues are available, even from very rare diseases.
However, the sample preparation process involves a number
steps which might alter the actual elemental distribution.
During formalin fixation, the tissue specimen is immersed in
a solution of formaldehyde in water (known as formalin),
resulting in cross-linking of proteins within the tissue.
Subsequently, water in the tissue is substituted by an organic
solvent (often xylene) in several solvent exchange steps.
Finally, the sample is embedded in paraffin; this sample block
can further be used to prepare thin cuts of the sample. Thin
cuts are deposited on suitable sample carrier material (e.g.,
microscopic slides, silicon wafers) before the paraffin is
removed. This involves an additional washing step using an
organic solvent. In contrast, elemental distributions and
quantities in snap-frozen and unfixed tissue sections were expected
to represent the actual in vivo conditions in the best way. This
sample preparation strategy involves the least sample
processing of all techniques; tissue specimen are snap frozen in liquid
nitrogen and thin cuts of some micrometer thickness are
prepared in a cryotome without a fixation step. Major
disadvantage is that unfixed tissues are scarcely available for clinical
studies mainly due to reasons of storage and handling.
Cryosamples need to be stored at −80 °C to avoid proteolytic
reactions, which is an important fact hampering general
applicability of this sample preparation approach. The suitability of
FFPE tissues for molecular analysis techniques has already
been investigated earlier . Also, bulk element
concentrations of selected metals in specific tissues have already been
compared between FFPE sample preparation and
cryosectioning [22, 23]. However, to the best of the authors’
knowledge, no study including multiple organs and
physiologically relevant metals ranging from alkalines over earth
alkalines to transition metals has been presented where
elemental distributions as well as bulk concentrations have been
In this work, the suitability of FFPE sample preparation for
trace elemental distribution analysis using LA-ICP-MS is
examined. Organs from one single mouse were partly used for
FFPE sample preparation and for cryo-preparation, which is
considered being the reference method. By using the same
organ of one mouse for both preparation techniques, possible
biological variations are sought to be reduced to a minimum.
While the tissue has to undergo several washing steps where
ionic species may potentially be washed out or their spatial
distribution may be altered, the preparation for cryo-cutting is
far more straightforward, leading to the probably most
accurate picture of the physiological conditions. Elemental
concentrations of nine elements (Na, Mg, K, Ca, Mn, Fe, Ni,
Cu, and Zn) as well as their distributions were examined and
compared leading to a comprehensive picture of the suitability
of FFPE sample preparation for elemental imaging
Xylene (isomer mixture, histological grade), aqueous
formaldehyde solution (≥36.0%, for molecular histology), and
paraffin wax (for histology) were all purchased from Sigma
Aldrich, Buchs, Switzerland. Ultra-pure water (resistivity
18.2 MΩ cm) was dispensed from a Barnstead EASYPURE
II water system (ThermoFisher Scientific, Marietta, OH).
Ethanol, toluene, (3-aminopropyl)-triethoxysilane (APES),
acetone, and nitrate salts of Na, K, Mg, Mn, Fe, Cu, and Zn
were all of p.a. quality and purchased from Sigma Aldrich,
Buchs, Switzerland. Conc. HNO3 was of p.a. quality and
obtained from Merck, Darmstadt, Germany.
All LA-ICP-MS measurements were performed using an
NWR213 LA system (ESI, Fremont, CA) equipped with fast
washout cell and a 213-nm frequency quintupled Nd:YAG
l a s e r c o u p l e d t o a n i C A P Q c I C P - M S i n s t r u m e n t
(ThermoFisher Scientific, Bremen, Germany). Coupling of
the two instruments was established using a 1.0-m-long
PTFE tubing with an inner diameter of 1.0 mm. Laser ablation
parameters were optimized in preliminary experiments to
ablate the complete sample material in one run of analysis while
maintaining the integrity of the sample material surrounding
the area of the incident laser beam. A lateral resolution of
40 μm was selected to be appropriate for the performed
investigations; this value provided a good trade-off between
timeefficient measurement and good image quality. While all
instrumental settings for the LA device were kept constant
throughout all measurements, the ICP-MS had to be tuned
on a daily basis to obtain best sensitivity for the
measurements. Optimization was performed using the very same dried
droplet standards used for signal quantification. A dried
droplet standard prepared using the highest analyte concentration
(10.0 mg L−1 Na, K, and Mg; 4.0 mg L−1 Ca and Fe;
2.0 mg L−1 Zn; 1.0 mg L−1 Mn; and 0.40 mg L−1 Ni and
Cu) was used. Performance was optimized to reach maximum
signal intensities for 23Na, 56Fe, 63Cu, and 64Zn; typical
measurement parameters are summarized in Table 1.
Gold coating of standards and samples was performed
using an Agar B7340 sputter coater (Agar Scientific
Limited, Essex, UK) equipped with a gold target. Sputter
parameters were kept constant over all experiments, as reported
in a previous study . Microscopic images of the samples
were acquired using a light microscope operated in reflective
light mode at 50× magnification (Leica DM2500M, Leica
Microsystems, Wetzlar, Germany).
Sample preparation and ICP-MS measurement
The animal-model protocol was developed and conducted in
accordance with the ARRIVE guidelines and the animal
welfare regulations of the Department of Tumor Biology,
National Koranyi Institute of Pulmonology (permission
number: 22.1/1268/3/2010). Mice were kept on a daily 12-h light/
12-h dark cycle and held in conventional animal house in
microisolator cages with water and laboratory chow ad
libitum. After sacrification, five organs (heart, lung, kidney,
liver, and brain) as well as pieces of muscle tissue were
removed from the animals and each divided into two halves.
One half underwent the complete FFPE procedure, whereas
the other half was snap frozen in liquid nitrogen directly after
sampling and stored at −80 °C until further use. This
procedure was found appropriate to obtain optimal comparability
between FFPE and cryo-cut samples. During the sample
preparation process and sample cutting, care was taken to keep
both samples in a similar orientation, which would then
facilitate inter-sample comparison. Even though this procedure
showed to be more complicated than sampling the organs
from two individual mice, it offered the opportunity to exclude
the influence of biological variation between two specimens.
In the preparation of the cryo-cuts, the tissue specimen was
attached to a sample holder using the Shandon Cryomatrix
(Thermo Scientific, Cat. No: 6769006). At −20 °C,
cryocuts with 10 μm thickness were prepared using a cryotome
(Leica CM3050 S, Leica Microsystems, Wetzlar, Germany).
Slices were deposited onto squared 1 × 1 cm surface-modified
silicon wafers for minimization of background signals
originating from the carrier material—a necessity for high
sensitivity trace element measurements with optimal quantification
accuracy. Silicon wafers (Infineon Technologies Austria AG,
Villach, Austria) were surface coated before deposition of the
tissue samples to ensure optimal adhesion of the thin sections.
The surface coating was performed as reported previously
. Samples were allowed to dry at room temperature before
further analysis. FFPE samples were prepared according to
standard procedures used in clinical laboratories. In analogy
to the cryo-cuts, they were also cut to 10 μm thickness and
deposited on silicon wafers; the thin cuts were washed with
toluene to remove excess paraffin.
Before LA-ICP-MS analysis, all samples were coated with
a thin gold layer to be utilized as a pseudo-internal standard.
Previous experiments showed that deposition of the gold layer
is very reproducible with relative standard deviations of the
amount of deposited material below 4%. This metal layer with
a thickness in the nanometer range is ablated simultaneously
with the sample material. It has been shown in earlier studies
Summary of instrumental parameters used for the LA-ICP-MS imaging experiments
Mass resolution at m/z 238
that such signal normalization approach allows the
compensation of matrix effects (i.e., material ablation and transport),
instrumental drifts during measurement time, and day-to-day
signal variations .
For quantification of trace elements in the tissue samples, a
dried droplet approach reported earlier to deliver valid results
for tissue investigations  has been employed. In this
simple method, defined amounts of liquid standards are deposited
onto pre-cut filter pieces and allowed to dry. A stock solution
containing 10.0 mg L−1 Na, K, and Mg; 4.0 mg L−1 Ca and Fe;
2.0 mg L−1 Zn; 1.0 mg L−1 Mn; and 0.40 mg L−1 Ni and Cu in
1% nitric acid (v/v) was used for preparation of the dried
droplet standards. A standard series was generated by dilution
of this stock by factors 2.0, 3.0, 5.0, 10.0, and 20.0. The stock
itself and a blank solution without analyte addition were also
used for preparation of the dried droplets. Every liquid
standard, a concentration of 1.0 mg L−1 indium was added to serve
as internal standard for correction of inaccuracies during
pipetting. Ten microliters of standard solution were applied to
each filter piece, six replicates of each concentration level
were prepared. For the standards, gold normalization is
applied in the same manner as for the samples: After evaporation
of the liquid, a pattern of dried droplets is coated with a thin
layer of gold. The same instrumental parameters as for the
tissue samples are used, ensuring comparable thickness of
the gold layers on samples and standards. Measurement of
the standards was performed using the very same laser
parameters than for the images; just the stage scan speed has been
reduced to 40 μm s−1 in order to ablate the complete filter
material. Using radial line scans, one line across the diameter
of the filter was ablated, derived signals were normalized and
averaged, and a calibration function to be used for signal
quantification was calculated. Using this calibration function,
area concentrations of each analyte in the tissue can be
calculated (e.g., ng cm−2) which can then be further transformed
into mass concentrations knowing the tissue thickness and the
density of the wet tissue .
Results and discussion
With six tissue types, each measured in three replicates, and
two different sample preparation strategies, a total of 36 tissue
samples were investigated in this comparative study between
FFPE and snap-frozen tissue preparation. Dimensions of the
tissues ranged from approx. 3 × 3 to 8 × 8 mm2. LA-ICP-MS
measurements provided signals above background level for
all selected elements on all samples. Normalized signal
intensities for each pixel were within the signal range obtained for
the dried droplet calibrations. Quantification of the obtained
signal intensities was performed by transforming the data
matrix of each element using the calibration functions from the
dried droplet calibration. Derived element concentrations
ranged between 0.1 μg g−1 (Mn) to 3000 μg g−1 (K).
Whenever possible, two isotopes of each element were
monitored and quantification was performed. The isotope with the
higher natural abundance was always used for data evaluation,
the other one for quality control purposes. In all cases, the
distribution images of two isotopes were well correlated;
calculated analyte concentrations did not differ significantly.
Repeatability of the measurements was validated using
consecutive tissue slices.
Exemplary elemental distribution images from the
LAICP-MS analyses are shown in Fig. 1. The distributions of
Mg in heart tissue (FFPE; Fig. 1a, d), Zn in muscle tissue
(cryo-cut; Fig. 1b, e), and Cu in brain tissue (cryo-cut;
Fig. 1c, f) are shown.
Figure 1 demonstrates that the elemental distributions in
the samples are inhomogeneous and vary with morphological
structures of the tissue. Such findings were also reported
earlier in other laterally resolved elemental distribution studies
[26, 27]. Found distributions are in good accordance with
previously reported data. Given that the analyzed sections
are representative for the complete tissue/organ, these average
concentrations across the complete section would correspond
well with bulk metal concentrations. For calculation of
average concentrations over the tissue material, the 13C signal was
used as indicator for the presence of tissue material; a region
of interest was defined for each tissue thin cut including all
pixels where 13C signal was above background level.
Additionally, visual interpretation was used to verify that the
13C signal did not originate from possibly remaining
embedding medium. For each element, average concentrations over
these regions of interest were calculated to yield an average
concentration. Thereby calculated concentrations from the
measurement of three consecutive tissue thin cuts were
averaged to obtain a mean concentration and a standard deviation,
respectively. The calculated bulk metal concentrations for all
elements and all tissue types are shown in Table 2.
The average concentrations calculated from the elemental
distribution images vary between 1.0 μg g−1 for nickel in brain
tissue and 2500 μg g−1 for potassium also in brain tissue. The
relative standard deviation of the calculated averages (n = 3)
was usually below 15%, a value which is acceptable with
regard to possible biological variations even within the same
organ. To the best of the authors’ knowledge, this is the first
study reporting average data as well as laterally resolved trace
metal images from multiple elements in a large range of
organs from one individual. Such extensive data set allows
convenient investigations on the inter-relations of different
elements in various tissue types. Found concentrations for Na
and K are usually very high (lowest values of 238 μg g−1 for
Na and 591 μg g−1 for K) in the analyzed tissues. This is
explained by the fact that the alkaline elements are responsible
for maintaining the physiological conditions and the osmotic
pressure in cellular systems by creating ionic potentials .
Fig. 1 Elemental distributions of
Mg (d) in a FFPE-treated heart
section cut along the transverse
plane, Zn distribution in a
cryocut muscle sample (e), as well as
Cu distribution in a cryo-cut
coronal brain sample (f) with the
corresponding micrographs of the
heart (a), muscle (b), and brain (c)
The absolute concentrations as well as the ratios between the
element concentrations of Na and K change with the
respective tissue type but the K concentration is in all cases higher
than the Na concentrations. Less variation in the absolute
amounts and overall lower concentrations can be detected
for the earth alkaline elements Mg and Ca. High
concentrations and overall presence of these four elements can be
explained by their general and wide purpose. As already
mentioned, Na and K are part of every tissue to keep up
physiologically suitable conditions. Besides also occurring in free
form, Mg and Ca are also important cofactors in protein
complexes, for example, in enzymes used for DNA replication,
which takes place in every tissue type. All other elements
(transition metals) are related to rather specific functions as
Table 2 Average concentrations
of all measured elements in all
analyzed tissues with a
cryosamples and FFPE preparation;
all units are given in μg g−1, and
errors are given as a single
standard deviation (n = 3)
cofactors in proteins and other macromolecules. Thus, their
concentrations in different tissue types can change widely. As
an example, the iron concentration is higher in tissues where
blood is processed: liver, kidney, and heart. While the highest
found concentration is in the liver (256 μg g−1), the lowest
calculated average concentration is more than a factor of 10
below (18.6 μg g−1 in the muscle). The average Mn
concentrations follow the trend of Fe, which correlates well with the
similar function of those two metals . Brain tissue exhibits
higher amounts of Cu and Zn, while having rather low Fe
content compared to other tissues. This fact underlines the role
of Cu and Zn in neurological functions .
The concentrations found in the snap-frozen tissue samples
are regarded to be most representative for the in vivo
conditions. This is supported by the element concentrations, for
example, found in lung tissue; the determined values correlate
well with those reported earlier by Carvalho et al.  in a
study using x-ray-based analysis techniques. Lopez-Alonso
et al.  recently analyzed trace elements in commercial
beef; liver and muscle tissue were examined in their study.
Even though another species, the approximate trace metal
concentrations should be comparable within the class of
mammals. The found concentrations for transition metals correlate
well with those found in the present study. Detected average
concentrations found in heart tissue are in accordance with
those reported earlier by Becker et al. in the same tissue type
. Similar findings were also reported for brain [5, 34]. All
reported studies have used unfixed tissue. Thus, the obtained
results are in good accordance with literature data and the
comparison with the concentrations obtained for FFPE tissues
should allow valid conclusions.
Average trace element contents
Due to the large number of sample preparation steps, the
absolute element concentrations are expected to be influenced by
the FFPE treatment. To demonstrate such possible wash-out
effects in detail, the average concentrations derived from the
elemental distribution images were considered. A full
compilation of all values calculated from the LA-ICP-MS
measurements is presented in Table 2. For better visualization, the data
for lung and brain tissue were extracted to be represented in a
graph, which is shown in Fig. 2, respectively. For better
visualization, the concentrations are shown in logarithmic scale.
The largest difference between snap-frozen and FFPE
samples can be found for the alkaline metal concentrations. With
concentrations of some hundreds of micrograms per gram for
Na and over 1000 μg g−1 for K, large amounts can be detected
in the snap-frozen tissue samples. In comparison, values
found in the FFPE samples are much lower. For K less than
1% recovery can be calculated in all investigated sample
types; for Na, the recovery is in no case above 5%. This
finding can be easily explained by the high mobility of alkaline
metals in tissues: They occur completely in the form of free
ions, unbound to larger molecular structures . As a result,
any aqueous solvents as used in parts of the FFPE process will
readily wash these metals out from the tissue structure.
Less severe analyte loss could be found for Mg. Its
concentration is generally lower in the FFPE samples, recoveries
compared to the snap-frozen reference vary between 21%
(lung) and 83% (muscle). This weaker wash-out compared
to Na and K can be explained by looking at the speciation of
Mg in biological systems: On average, about 30% of the total
Mg occurs in free unbound form in a complete organism,
while the rest is bound in complexes ; the exact ratio
between free ionic and bound Mg depends on the tissue type.
Similar to alkaline metals, free Mg can also easily be washed
out from the tissue, whereas chelated Mg ions are more tightly
bound to the macromolecular tissue matrix and are therefore
less likely to be affected by the fixation and embedding
process. A similar finding can be stated for iron. Average amounts
found in snap-frozen brain, muscle, and liver tissue do not
differ significantly from the respective FFPE samples. The
other tissue types show significant analyte loss when
comparing cryo-samples to FFPE. As for Mg, a portion of Fe is not
bound to larger molecules . This fraction can again be
Fig. 2 Comparison of the average metal concentrations between a snap-frozen and a FFPE sample of mouse lung (a) and mouse brain (b)
easily removed from the sample material by a solvent
compared to the chelated fraction of Fe. The ratio between these
two groups of species depends on the tissue type, explaining
the different grades of wash-out.
The trace metals Mn, Ni, and Cu do not exhibit any
significant concentration difference between the snap-frozen and
the FFPE samples. Those metals are toxic to mammalian
organisms when occurring in free, unbound form . Thus,
they appear in the organism almost completely bound to
chelate complexes with different proteins or other
macromolecules. High binding affinity makes it hard to break the chelate
bonds by aqueous solvents. Thus, the concentration is barely
affected by the FFPE sample preparation. The finding could
be confirmed for all investigated tissue types.
In comparison to the metals having lower or similar
concentration in the FFPE samples compared to the snap-frozen
reference, Ca and Zn exhibit a higher average concentration in
the FFPE samples of all investigated tissue types. Such
increase is likely to be caused by contaminations introduced
during any of the sample processing steps—another weakness
of the FFPE sample preparation: Due to the vast number of
process steps, a contamination of metals at the trace level can
be easily introduced. Especially as the sample preparation is
usually performed in clinical laboratories which might not be
equipped according to the requirements for trace element
investigations. Problems might be reagents with trace impurities
as well as a workflow that introduces trace contaminations
(e.g., the use of glassware). In the present case, the source of
Ca and Zn could be easily identified. LA-ICP-MS
measurements of thin cuts without having the embedding medium
removed showed high Ca and Zn contents also in areas
Fig. 3 FFPE (a) and snap-frozen
(e) samples of a mouse kidney
with the elemental distributions of
Na (b, f), Fe (c, g), and Mn (d, h)
without tissue. Such Ca and Zn contamination originating
from the embedding medium will also result in elevated metal
contents in the sample, even after removing the paraffin.
Comparison of element distributions
In order to evaluate if apart from the average contents also the
lateral element distributions in different tissues are
compromised by the FFPE process, tissue samples from one organ
were compared. As indicated by the calculated average
concentrations, FFPE sample preparation affects trace metals in
the tissue. If bulk concentrations were altered, also some
variations in element distributions were expected. In this part of
the study, hematoxylin/eosin stain was used to identify
relevant substructures in the respective tissues. Due to practical
reasons, only selected tissue types and elemental distributions
A kidney sample is shown in Fig. 3. Microscopic scans
with indication of the important substructures renal cortex
and renal medulla are shown in Fig. 3a for the FFPE sample
and in Fig. 3e for the corresponding snap-frozen sample from
the very same kidney. It has to be pointed out that an
incomplete section of the kidney is shown for the cryo-cut sample.
This can be explained by difficulties which occurred during
the sample preparation procedure where the organ had to be
divided (cutting the organ correctly into two equal pieces).
Additionally, correct positioning of the cryo-section on the
sample carrier always poses a source for ruptures or other
damages to the tissue. The position of the section relative to
the complete kidney is indicated by the blue overlays. It has to
be noted that the scale bars for Na are different, while the other
elements have the same color scale for FFPE and cryo-cut
sample. In general, the distribution of all three selected
elements seems much more defined in the snap-frozen sample
(Fig. 3f–h). The concentration of Na is slightly higher in the
area of the medulla, whereas Fe and Mn are rather enriched
towards the cortical structures. Similar distributions of Na and
Mn were reported in another study . Mn even shows a very
distinct feature with a band of higher concentration near the
renal pyramids, located at the corticomedullary junction. The
increased concentration of Fe in the cortex can be explained
by the higher density of blood vessels in contrast to the
medulla. In comparison, the elemental distributions in the FFPE
sample show a rather uniform distribution across the whole
section, with Na and Fe showing slightly lower concentrations
in the medulla. Even though less pronounced, the distributions
of Fe in both sample preparation types are similar. The
maximum concentration of Na decreases significantly from
around 1500 to 60 μg g−1. Concentrations maxima of Fe
and Mn in both samples are in a comparable range, supporting
the finding from the investigations on the mean metal
concentrations in the tissues. However, even if they are not removed
from the tissue structure, also these elements seem to be
influenced by the FFPE process, creating a slightly more blurred
A comparison of the trace element distributions in a
snapfrozen and a FFPE liver sample is shown in Fig. 4. Both thin
cuts represent the right lateral lobe of the liver, the similar
shape can easily be recognized; no macroscopic substructures
can be identified. Color scale bars for Mg and Ni are equally
scaled for FFPE and cryo-cut samples, while K distribution
images had to be scaled differently due to the large
concentration differences between the sample preparation strategies.
The elemental distributions are in general rather uniform in the
snap-frozen section as well as in the FFPE sample. The
distribution of Ni is slightly more structured in both samples;
similar distribution could be found, for example, for Fe. This
corresponds well with findings reported earlier for trace
elements in the liver . One semicircular area of higher
potassium concentration in the center of the FFPE sample indicates
the position of a large blood vessel. This feature is also
represented by other metal distributions such as Mn and Cu (not
shown). However, it does not appear in the snap-frozen
sample, which makes a direct comparison of the elemental
distributions in this specific area impossible. Besides that, the
potassium distribution in the FFPE sample also shows high
concentrations in the outer sample areas. This appearance is not
represented in the snap-frozen sample. Therefore, it is very
likely that the distribution obtained from the FFPE sample is
influenced by the pre-treatment of the sample, leading to
wash-out of analytes. Samples with the remaining higher
concentrations might have higher density or hydrophobicity
which hampers the release of analyte into the aqueous solvent.
These findings were observed for all replicate sample
All other investigated organs show similar results:
Elemental distributions are usually more homogeneous in
Fig. 4 FFPE (a) and snap-frozen (e) samples of a mouse liver with the elemental distributions of Mg (b, f), K (c, g), and Ni (d, h)
the FFPE samples, a fact that can be well explained by
diffusional processes occurring during fixation and embedding.
However, the analyte distortion is less pronounced for
transition metals than for alkaline elements; the strength of the
effect of the embedding process on Mg lies between those two
groups. Results of this comparative distribution study indicate
that FFPE samples can be used only to a limited extent for
elemental distribution studies. The qualitative distributions of
many elements seem to be biased towards more homogeneous
distributions compared to the snap-frozen samples, which are
believed to represent the actual in vivo conditions to a good
FFPE is a widely used preparation strategy for tissue specimen.
Due to the large availability of samples in archives, it would be
interesting if these samples could also be used for elemental
distribution studies. In the comparison with snap-frozen tissues
which were used as reference samples, elemental distributions
as well as average metal concentrations showed to be partly
altered in the FFPE samples, which restricts the general
applicability of FFPE sample preparation in metallomics studies.
FFPE samples showed to be completely unsuitable, if the
investigation of alkaline metals is required; distributions as well
as absolute amounts were heavily influenced. Especially the
relative distributions of some transition metals as well as their
bulk concentrations showed to be altered to a lesser extent in
the FFPE samples. These results indicate that if only the
analysis of transition metals is required, also FFPE samples can be
used; even if finer structures appeared to be blurred, the general
elemental distributions were comparable. For Ca and Zn,
contaminations introduced during the embedding process could be
identified, highlighting that metal analysis in FFPE samples still
has to be carefully considered in order not to obtain results
which are biased by the sample preparation process.
However, even if snap-frozen samples without fixation
definitely provide the most accurate way for analysis of metals in
tissue samples, FFPE samples can also deliver valid
quantitative elemental distribution results.
Acknowledgments Open access funding provided by TU Wien (TUW).
M.B. wants to acknowledge the MEIBio PhD program of the TU Wien
for providing a scholarship in the period 2013–2016.
Compliance with ethical standards The animal experiments were
conducted in accordance with the ARRIVE guidelines, as well as the
animal welfare regulations of the Department of Tumor Biology,
National Koranyi Institute of Pulmonology, under the following
permission number: 22.1/1268/3/2010.
Open Access This article is distributed under the terms of the Creative
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creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
1. Becker JS , Zoriy M , Matusch A , Wu B , Salber D , Palm C. Bioimaging of metals by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) . Mass Spectrom Rev . 2010 ; 29 ( 1 ): 156 - 75 . doi:10.1002/mas.20239.
2. Russo RE , Mao X , Gonzalez JJ , Zorba V , Yoo J. Laser ablation in analytical chemistry . Anal Chem . 2013 ; 85 ( 13 ): 6162 - 77 . doi:10.1021/ac4005327.
3. Günther D , Hattendorf B. Solid sample analysis using laser ablation inductively coupled plasma mass spectrometry . TrAC Trend Anal Chem . 2005 ; 24 ( 3 ): 255 - 65 . doi:10.1016/j.trac. 2004 .11.017.
4. M-M P , Weiskirchen R , Gassler N , Bosserhoff AK , Becker JS . Novel bioimaging techniques of metals by laser ablation inductively coupled plasma mass spectrometry for diagnosis of fibrotic and cirrhotic liver disorders . PLoS One . 2013 ; 8 ( 3 ) :e58702 . doi:10.1371 /journal.pone.0058702.
5. Becker JS , Matusch A , Palm C , Salber D , Morton KA . Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics . Metallomics . 2010 ; 2 ( 2 ): 104 - 11 . doi:10.1039/b916722f.
6. Hare DJ , Lee JK , Beavis AD , van Gramberg A , George J , Adlard PA , et al. Three-dimensional atlas of iron, copper, and zinc in the mouse cerebrum and brainstem . Anal Chem . 2012 ; 84 ( 9 ): 3990 - 7 . doi:10.1021/ac300374x.
7. Shariatgorji M , Nilsson A , Bonta M , Gan J , Marklund N , Clausen F , et al. Direct imaging of elemental distributions in tissue sections by laser ablation mass spectrometry . Methods . 2016 ; 104 : 86 - 92 . doi:10.1016/j.ymeth. 2016 .05.021.
8. Gholap D , Verhulst J , Ceelen W , Vanhaecke F. Use of pneumatic nebulization and laser ablation-inductively coupled plasma-mass spectrometry to study the distribution and bioavailability of an intraperitoneally administered Pt-containing chemotherapeutic drug . Anal Bioanal Chem . 2012 ; 402 ( 6 ): 2121 - 9 . doi:10.1007/s00216- 011 - 5654 -3.
9. Bonta M , Lohninger H , Laszlo V , Hegedus B , Limbeck A. Quantitative LA-ICP-MS imaging of platinum in chemotherapy treated human malignant pleural mesothelioma samples using printed patterns as standard . J Anal At Spectrom . 2014 ; 29 ( 11 ): 2159 - 67 . doi:10.1039/C4JA00245H.
10. Birka M , Wentker KS , Lusmöller E , Arheilger B , Wehe CA , Sperling M , et al. Diagnosis of nephrogenic systemic fibrosis by means of elemental bioimaging and speciation analysis . Anal Chem . 2015 ; 87 ( 6 ): 3321 - 8 . doi:10.1021/ac504488k.
11. Hare D , Austin C , Doble P. Quantification strategies for elemental imaging of biological samples using laser ablation-inductively coupled plasma-mass spectrometry . Analyst . 2012 ; 137 ( 7 ): 1527 . doi:10.1039/c2an15792f.
12. Limbeck A , Galler P , Bonta M , Bauer G , Nischkauer W , Vanhaecke F. Recent advances in quantitative LA-ICP-MS analysis: challenges and solutions in the life sciences and environmental chemistry ABC highlights: authored by rising stars and top experts . Anal Bioanal Chem . 2015 ; 407 ( 22 ): 6593 - 617 . doi:10.1007/s00216- 015 - 8858 -0.
13. Austin C , Fryer F , Lear J , Bishop D , Hare D , Rawling T , et al. Factors affecting internal standard selection for quantitative elemental bio-imaging of soft tissues by LA-ICP-MS . J Anal At Spectrom . 2011 ; 26 ( 7 ): 1494 - 501 . doi:10.1039/c0ja00267d.
14. Stärk H-J , Wennrich R. A new approach for calibration of laser ablation inductively coupled plasma mass spectrometry using thin layers of spiked agarose gels as references . Anal Bioanal Chem . 2011 ; 399 ( 6 ): 2211 - 7 . doi:10.1007/s00216- 010 - 4413 -1.
15. Austin C , Hare D , Rawling T , McDonagh AM , Doble P. Quantification method for elemental bio-imaging by LA-ICP-MS using metal spiked PMMA films . J Anal At Spectrom . 2010 ; 25 ( 5 ): 722 - 5 . doi:10.1039/b911316a.
16. Pozebon D , Dressler VL , Mesko MF , Matusch A , Becker JS . Bioimaging of metals in thin mouse brain section by laser ablation inductively coupled plasma mass spectrometry: novel online quantification strategy using aqueous standards . J Anal At Spectrom . 2010 ; 25 ( 11 ): 1739 - 44 .
17. Konz I , Fernandez B , Fernandez ML , Pereiro R , Gonzalez H , Alvarez L , et al. Gold internal standard correction for elemental imaging of soft tissue sections by LA-ICP-MS: element distribution in eye microstructures . Anal Bioanal Chem . 2013 ; 405 ( 10 ): 3091 - 6 . doi:10.1007/s00216- 013 - 6778 -4.
18. Bonta M , Lohninger H , Marchetti-Deschmann M , Limbeck A. Application of gold thin-films for internal standardization in LAICP-MS imaging experiments . Analyst . 2014 ; 139 ( 6 ): 1521 - 31 . doi:10.1039/c3an01511d.
19. Frick DA , Giesen C , Hemmerle T , Bodenmiller B , Günther D. An internal standardisation strategy for quantitative immunoassay tissue imaging using laser ablation inductively coupled plasma mass spectrometry . J Anal At Spectrom . 2015 ; 30 ( 1 ): 254 - 9 . doi:10.1039 /c4ja00293h.
20. Berg D , Malinowsky K , Reischauer B , Wolff C , Becker KF . Use of formalin-fixed and paraffin-embedded tissues for diagnosis and therapy in routine clinical settings . Methods Mol Biol . 2011 ; 785 : 109 - 22 . doi:10.1007/978- 1 - 61779 - 286 -1_ 8 .
21. Wisztorski M , Franck J , Salzet M , Fournier I. MALDI direct analysis and imaging of frozen versus FFPE tissues: what strategy for which sample? Methods Mol Biol . 2010 ; 656 : 303 - 22 . doi:10.1007 /978- 1 - 60761 - 746 -4_ 18 .
22. Bischoff K , Lamm C , Erb HN , Hillebrandt JR . The effects of formalin fixation and tissue embedding of bovine liver on copper, iron, and zinc analysis . J Vet Diagn Invest . 2008 ; 20 ( 2 ): 220 - 4 .
23. Olynyk JK , O'Neill R , Britton RS , Bacon BR . Determination of hepatic iron concentration in fresh and paraffin-embedded tissue: diagnostic implications . Gastroenterology . 1994 ; 106 ( 3 ): 674 - 7 .
24. Bonta M , Gonzalez JJ , Derrick Quarles C , Russo RE , Hegedus B , Limbeck A. Elemental mapping of biological samples by the combined use of LIBS and LA-ICP-MS . J Anal At Spectrom . 2016 ; 31 ( 1 ): 252 - 8 . doi:10.1039/c5ja00287g.
25. Bonta M , Hegedus B , Limbeck A. Application of dried-droplets deposited on pre-cut filter paper disks for quantitative LA-ICPMS imaging of biologically relevant minor and trace elements in tissue samples . Anal Chim Acta . 2016 ; 908 : 54 - 62 . doi:10.1016/j. aca. 2015 .12.048.
26. Becker JS . Imaging of metals, metalloids, and non-metals by laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) in biological tissues . Methods Mol Biol . 2010 ; 656 : 51 - 82 . doi:10.1007/978- 1 - 60761 - 746 -4_ 3 .
27. Hare D , Burger F , Austin C , Fryer F , Grimm R , Reedy B , et al. Elemental bio-imaging of melanoma in lymph node biopsies . Analyst . 2009 ; 134 ( 3 ): 450 - 3 . doi:10.1039/b812745j.
28. Ussing HH , Kruhøffer P , Thaysen JH , Thorn NA . The alkali metal ions in biology . I. The alkali metal ions in isolated systems and tissues. II. The alkali metal ions in the organism . Berlin: Springer Berlin Heidelberg; 1960 . doi:10.1007/978- 3 - 642 - 49246 -4.
29. Beyer Jr WF , Fridovich I. In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli . J Biol Chem . 1991 ; 266 ( 1 ): 303 - 8 .
30. Linert W , Kozlowski H. Metal ions in neurological systems . 2012 .
31. Carvalho ML , Magalhães T , Becker M , von Bohlen A. Trace elements in human cancerous and healthy tissues: a comparative study by EDXRF, TXRF, synchrotron radiation and PIXE . Spectrochim Acta B . 2007 ; 62 ( 9 ): 1004 - 11 . doi:10.1016/j.sab. 2007 .03.030.
32. López-Alonso M , Miranda M , Benedito JL , Pereira V , GarcíaVaquero M. Essential and toxic trace element concentrations in different commercial veal cuts in Spain . Meat Sci . 2016 ; 121 : 47 - 52 . doi:10.1016/j.meatsci. 2016 .05.013.
33. Becker JS , Breuer U , Hsieh H-F , Osterholt T , Kumtabtim U , Wu B , et al. Bioimaging of metals and biomolecules in mouse heart by laser ablation inductively coupled plasma mass spectrometry and secondary ion mass spectrometry . Anal Chem . 2010 ; 82 ( 22 ): 9528 - 33 . doi:10.1021/ac102256q.
34. Hare D , Reedy B , Grimm R , Wilkins S , Volitakis I , George JL , et al. Quantitative elemental bio-imaging of Mn, Fe, Cu and Zn in 6- hydroxydopamine induced parkinsonism mouse models . Metallomics . 2009 ; 1 ( 1 ): 53 - 8 . doi:10.1039/b816188g.
35. Durlach J. Magnesium in clinical practice . London: J. Libbey; 1988 .
36. Yehuda S , Mostofsky DI. Iron deficiency and overload: from basic biology to clinical medicine . New York : Humana Press ; 2010 .
37. Brewer GJ . The risks of free copper in the body and the development of useful anticopper drugs . Curr Opin Clin Nutr Metab Care . 2008 ; 11 ( 6 ): 727 - 32 . doi:10.1097/MCO.0b013e328314b678.