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Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging

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Cardiovascular diseases are the world’s number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na+, K+, and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging. Figure Workflow of the 3D reconstruction. A Tissue section, B gold deposition is done by sputter coating, C, C1 SIMS-ToF mass analyzer, C, C2 mass spectral peaks, C, C3 datacube images; D, E Reconstruction of the heart showing 3D-spatial distributions of three different ions 145 m/z (red), 23 m/z (green), and 39 m/z (blue); F coregistration of 40 individual MS imaging
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ORIGINAL PAPER
Three-dimensional molecular reconstruction of rat
heart with mass spectrometry imaging
Lara Fornai &Annalisa Angelini &Ivo Klinkert &
Frans Giskes &Andras Kiss &Gert Eijkel &
Erika A. Amstalden-van Hove &Leendert A. Klerk &
Marny Fedrigo &Giuseppe Pieraccini &
Gloriano Moneti &Marialuisa Valente &Gaetano Thiene &
Ron M. A. Heeren
Received: 20 June 2012 /Revised: 17 September 2012 / Accepted: 21 September 2012
#Springer-Verlag Berlin Heidelberg 2012
Abstract Cardiovascular diseases are the worlds number
one cause of death, accounting for 17.1 million deaths a
year. New high-resolution molecular and structural imag-
ing strategies are needed to understand underlying path-
ophysiological mechanism. The aim of our study is (1) to
provide a molecular basis of the heart animal model
through the local identification of biomolecules by mass
spectrometry imaging (MSI) (three-dimensional (3D) mo-
lecular reconstruction), (2) to perform a cross-species
validation of secondary ion mass spectrometry (SIMS)-
based cardiovascular molecular imaging, and (3) to
demonstrate potential clinical relevance by the application
of this innovative methodology to human heart speci-
mens. We investigated a MSI approach using SIMS on
the major areas of a rat and mouse heart: the pericardi-
um, the myocardium, the endocardium, valves, and the
great vessels. While several structures of the heart can be
observed in individual two-dimensional sections analyzed
by metal-assisted SIMS imaging, a full view of these
structures in the total heart volume can be achieved only
through the construction of the 3D heart model. The
images of 3D reconstruction of the rat heart show a
Please address reprint requests to: Prof. Dr. R.M.A. Heeren, FOM-AMOLF,
Science Park 104, 1098 XG Amsterdam, The Netherlands
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-012-6451-3) contains supplementary material,
which is available to authorized users.
L. Fornai (*):A. Angelini :M. Fedrigo :M. Valente :G. Thiene
Cardiovascular Pathology, Department of Cardiac,
Thoracic and Vascular Sciences, University of Padua,
Via Gabelli 61,
35121 Padua, Italy
e-mail: lara.fornai.1@unipd.it
L. Fornai :I. Klinkert :F. Giskes :A. Kiss :G. Eijkel :
E. A. A.-v. Hove :L. A. Klerk :R. M. A. Heeren
FOM-AMOLF,
Science Park 104,
1098 XG Amsterdam, The Netherlands
L. Fornai
e-mail: fornai@amolf.nl
R. M. A. Heeren
e-mail: heeren@amolf.nl
G. Pieraccini :G. Moneti
CISM Mass Spectrometry Centre,
Viale Pieraccini 6 University of Florence,
50139 Florence, Italy
R. M. A. Heeren
The Netherlands Proteomics Centre, Utrecht University,
H.R. Kruytgebouw, Padualaan 8,
3584 CH Utrecht, The Netherlands
Anal Bioanal Chem
DOI 10.1007/s00216-012-6451-3
highly complementary localization between Na
+
,K
+
, and
two ions at m/z 145 and 667. Principal component analysis of
the MSI data clearly identified different morphology of the
heart by their distinct correlated molecular signatures. The
results reported here represent the first 3D molecular recon-
struction of rat heart by SIMS imaging.
Keywords 3D molecular reconstruction .Secondary ion
mass spectrometry .Heart .Imaging
Non-standard abbreviations and acronyms
DAG Diacylglycerols
FFA Free fatty acids
MALDI-
MSI
Matrix-assisted laser desorption/ionization-
mass spectrometry imaging
MetA-
SIMS
Metal-assisted-secondary ion mass
spectrometry
MS Mass spectrometry
MSI Mass spectrometry imaging
PCA Principal component analysis
SIMS Secondary ion mass spectrometry
TAG Triacylglycerols
ToF Time-of-flight
VARIMAX VARIance MAXimization
Introduction
Heart failure is among the leading causes of morbidity
and mortality and can result from either primary or
secondary heart muscle disease [1]. The causes of car-
diac dysfunction in most heart diseases are still largely
unknown but are likely to result from underlying alter-
ations in gene and protein expressions or downstream
metabolic processes. The functional complexity of an
organism far exceeds that indicated by its genome se-
quence alone, and this is dependent on the products of
gene expression, including transcriptomics, proteomics,
lipidomics, and metabolomics [25]. Proteome databases
containing two-dimensional (2D) gel electrophoresis, 2D
gel images, and protein spot identifications have been
compiled for canine and rat myocardial tissues [6,7].
An animal model is crucial to evaluate new basic mo-
lecular insights prior to their application in human stud-
ies. Rats exhibit physiological characteristics similar to
those of humans and have been a key experimental
model in biomedicine for over a century [8]. However,
to date, there is no comprehensive molecular image
database for the rat heart. The construction of such
databases in animal model is important for the identifi-
cation of the molecular basis of pathological substrates
caused by a cardiovascular disease. This requires a
molecular imaging method that provides detailed insight
into the spatial distribution of a broad range of elements
and molecules. A mass spectrometer is described as the
smallest weighing scale ever used in the world [9].
Mass spectrometry (MS) is an analytical technique that
is used to determine the molecular weight of a variety
of chemical compounds. Because the mass of a chemi-
cal compound is dependent on its elemental composi-
tion, it is an important determinant of its identity. Mass
spectrometry imaging (MSI) is a technique that enables
the localization of molecules directly from biological
surfaces. The advantage of MSI is its ability to detect
the distribution of hundreds of unknown compounds in
a single measurement without the use of chemical or
immunological labels [10,11]: It is a true label-free
molecular imaging technique. The aims of the present
study were: (a) the establishment of a molecular atlas of
the heart through a direct and large-scale, local analysis
of lipid and elemental ions in a healthy rat heart tissue;
(b) to perform a cross-species validation through the
analysis of mouse and human heart tissues and compare
and contrast our molecular findings; (c) to test this
methodology on clinical human samples as the last
element of our translational research.
Ethics statement
The animals used in this study were purchased from Harlan
Laboratories (Boxmeer, The Netherlands). The Center for
Cardiovascular Pathology of the University of Padova spe-
cifically approved the use of human tissues. Written in-
formed consent was acquired from human subject involved
in the study.
Methods and materials
Rat heart from adult rats (type WU) and mouse heart (type
9CFW-1) were frozen and stored at 80 °C until sectioning.
The tissue sections were stained with hematoxylin (Sigma
Diagnostics, Zwijndrecht, The Netherlands) and eosin
(Merck Diagnostica, Darmstadt, Germany) after secondary
ion mass spectrometry (SIMS) analysis to correlate the
observed molecular profiles with morphological features.
Several successive heart sections from three different rats
and one mouse heart were analyzed using standardized and
optimized workflows.
Rat and mouse hearts were sectioned at 12 μmthick-
ness and sliced into sagittal sections at 20 °C on a
cryomicrotome (Microm International, Waldorf, Ger-
many) and then mounted on indiumtinoxide-coated
glass slides (ITO, 48Ωresistance; Delta Technologies,
Stillwater, MN, USA). All samples slices were stored at
L. Fornai et al.
80 °C prior to use and dried in a vacuum desiccator
for 30 min prior to MS analysis. The 12 μm thickness
ensures that enough analyte molecules are available for
ionization and no problems occur with the insulating
properties of tissue [12]. Sample metallization as a
means to improve molecular ion yields was performed
by sputter deposition of a 1-nm layer of gold [13].
Samples were analyzed by high-resolution time-of-
flight secondary ion mass spectrometry (ToF-SIMS) im-
aging. This procedure is often referred to as metal-
assisted-secondary ion mass spectrometry (MetA-SIMS)
when sample metallization is used. ToF-SIMS analysis
of the cross-sectioned heart was done on a Physical
Electronics (Eden Prairie, MN) TRIFT-II (triple focusing
time-of-flight) ToF-SIMS system, equipped with a
22 keV gold liquid metal ion source. The analysis was
performed in positive ion mode. The instrument was
calibrated in positive ion mode on high occurrence
elements and fragments such as H
+
,C
n
H
m
+
,Na
+
,and
K
+
. Since the sample size is approximately 2 × 2 cm,
the resulting image fidelity of such ToF-SIMS experi-
mentsiscloseto0.6μm per pixel. The raster size (or
fieldofview(FOV))usedwas150μm per tile, with
resolution of 256×256 pixel per tile. The FOV is auto-
matically calculated by WinCadence 4.4.0.17 software
(ULVAC-PHI Inc., Kanagawa, Japan) based on the max-
imum raster size defined by the user and the total
measurement area. The heart tissue was analyzed using
a mosaic mode of 128×128 tiles with a spectral reso-
lution of 22-bit mass channels, for a total of about one
billion pixels. The acquisition time was 3 s for each
tile. Both FOV and acquisition time were constant
throughout the experiment, which covered the entire
sample surface. SIMS is an extremely surface sensitive
technique in which ions are exclusively generated from
a depth of no more than 50 nm from the tissue surface.
We used the static SIMS mode, where the primary ion
dose is so low that each incoming ion hits a unique
spot on the surface, less than 1 % of the surface area is
analyzed [14,15]. In our work, a list is created from
the recorded data containing the position as a two-
dimensional coordinate, the channel number, c(which
is linearly related to the time-of-flight and hence the m/z
value), and the number of counts (n) for that respective
ion. This dataset, which represents a so-called datacube,
can subsequently be converted into an x×yby cunfold-
ed datacube containing the number of counts for each
spectral and spatial combination. These datacubes can
be visualized with the DataCube Explorer (http://
www.maldi-msi.org) developed at the FOM Institute
AMOLF, a lightweight visualization tool providing a
platform to share and explore MSI datasets. Several
useful features are available to dynamically scroll
through the data, analyze selected regions, and process
and classify the image [16]. Here, we will focus on a
description of the high-resolution metA-SIMS molecular
atlas that was recently completed from us.
PCA analysis and variance maximization (VARIMAX)
rotation
Principal component analysis (PCA) was employed to
extract meaningful information out of the complex and
extremely large datasets generated by high-resolution
imaging mass spectrometry (IMS). PCA is a statistical
technique to find patterns in data of high dimensional-
ity. The data are described in such a way that corre-
lated similarities and differences are highlighted. This
is realized by transforming a number of data features
(variables) into a smaller number of orthogonal varia-
bles called principal components (PCs), which de facto
consist of correlated spectral features. The first princi-
pal component accounts for the largest amount of var-
iance in the data. Each successive component describes
a smaller part of the remaining variance. PCA is there-
fore well-suited to be applied on hyperspectral datasets
as used to construct the molecular atlas described in
this paper. The hyperspectral SIMS data have both a
large spatial as well as a large spectral dimension.
Additional optimizations can be done after completion
of the PCA to enhance the spectral contrast in the data.
One method is an additional fitting of the principal
components to maximize the variance expressed in
each component. There is a number of maximization
criteria, but the VARIance MAXimization (VARIMAX)
is the most common. It can be used as a post-
processing step after PCA, as previously described
[17]. By rotation of the orthogonal axis, components
with a higher contrast are created. To highlight the
spectral correlations in the three-dimensional (3D) at-
las, PCA was used with VARIMAX optimization.
Rat heart high-resolution imaging mass spectrometry data
Imaging mass spectrometry data are often displayed as
a total-ion-count image; the output of a mass spectrom-
eter consists of a set of mass-to-charge ratios (m/z)of
detected ions. IMS produces such a set of mass-to-
charge ratios for each pixel in a 2D grid, allowing
analysis of the chemical structure of the sample. The
resulting grid of m/z histograms is commonly visual-
ized in the form of a total-ion-count image. For each
pixel, a total-ion-count image maps the number of
items in the corresponding set of m/z values to an
intensity value. Other methods of visualization are
based on various forms of multi-variate analysis on
Three-dimensional molecular reconstruction of rat heart with MSI
the m/z data, producing maps of chemically similar
regions as show from Smit et al. [18].
Human heart Human failing left-ventricular free-wall
heart explants were obtained from the heart transplant
collection at Padua University. Samples (left ventricle)
from explanted heart (heart failure) were frozen and
stored at 80 °C until sectioning. Heart tissue transverse
sections (12 μm thickness) were cut using a cryomicro-
tome and thaw-mounted onto an ITO slide. The same
SIMS-ToF method and statistical analysis described for
rat and mouse heart were applied to the human sample.
A sample of the left ventricle was further divided into
manageable blocks for paraffin embedding. From each
block, three serial sections of 4 μm thickness were
taken. Adjacent (serial) sections were then stained with
one of the three following techniques to correlate hearts
molecular profile with morphological features: (a) hema-
toxylin and eosin (H&E) staining, (b) Sirius red-
collagen-staining for connective tissue, and (c) Heiden-
hain's Azan stained for connective tissue. Comparison
was made with a serial staining section to show the
anatomical structure and confirm the distribution of
connective tissue in that location.
Results
2D MSI-based molecular imaging of rat heart sections
Surface rastering of heart tissue sections generated a
plethora of secondary ions with a molecular weight up
to m/z 1,500 (Fig. 1AC). Several distinctive MS peaks
and correlated image patterns were observed in the
positive-ion mode for the heart sections analyzed. The
selected ion images are highly sensitive to the specific
anatomical tissue types within the sections (Fig. 1A, B).
Several peaks are often visible within a single m/z
range, and a single peak can be used to create descrip-
tive molecular ion images. The resulting intensity map
is usually visualized using a pseudo-color map. Because
of the lack of an efficient MS/MS method that could be
associated with ToF-SIMS imaging, structure attributions
or assignments of ion peaks were made according to the
instrument resolution and accuracy, the valence rule, and
the biological characteristics of the tissue. All the mass
assignments have been done from data taken from the
literature [1922].
The high-spatial-resolution images shown in Fig. 2dem-
onstrate that the spatial resolution obtained in these SIMS
experiments is more than sufficient to reveal in detail all
major anatomical substructures in the rat heart. Note that
conventional matrix-assisted laser desorption/ionization-
mass spectrometry imaging (MALDI-MSI) with a pixel size
of 100 μm is not capable of revealing the detailed structures
shown in Fig. 3where the distribution of a wide variety of
secondary ions imaged in the heart is observed. The ions at
m/z 667 and m/z 840 localized very precisely within the
aorta wall (Fig. 3c, d). For instance, m/z 83 is localized in
aorta wall, semilunar valve, and endocardium, whereas it is
hardly visible in both ventricles and atria (Fig. 3a). The
image in Fig. 3f shows the ion at 145 m/z as very much
localized in pulmonary artery, right atrium, and atrioventric-
ular valve. Interestingly, m/z 175 and m/z 213 are observed
only in ventricles. The bottom image Fig. 3m, in contrast,
shows m/z 334 localized only in the pericardium. The image
in Fig. 3q shows the high spatial distribution at 969 m/z in
both atria, aorta wall, left atrioventricular valve, and right
coronary artery. Significant peaks in our data are seen at m/z,
representing cholesterol ion [MH
2
O+H]
+
whose distribu-
tion was imaged. Important tissue differences are distin-
guished upon examination of the cholesterol distribution.
Cholesterol shows higher intensity in both atria, aorta wall,
atrioventricular valves, and the coronary artery but is ob-
served with lower intensity in ventricles (Fig. 3i). VARI-
MAX rotation was used to enhance the spectral contrast of
the PCs. This axis rotation results in a higher molecular
contrast not only in the spectra, but also in a higher molec-
ular image contrast. Several signals of fatty acids show a
variation in their spatial distribution that corresponds direct-
ly to the degree of lipid unsaturation, and hence energy
catabolism. Diacylglycerol (DAG) species that were identi-
fied as [M+HOH]
+
(C
35
H
65
O
4
)atm/z 549, [M+HOH]
+
(C
37
H
69
O
4
)atm/z 577, [M+HOH]
+
(C
39
H
71
O
4
)atm/z 603
and ceramide at m/z 604 could be detected. Substantial
differences are also seen in the amount of free choline
present in the various tissue parts. The distributions based
on the PCA results revealed a clear image of the different
areas where m/z 104 (choline) displayed a high intensity
signal. The m/z 104 signal strongly localizes in atria,
aorta, pulmonary artery, and the atrioventricular and
semilunar valves but has lower intensity in ventricles,
asshowninFig.4B. The phosphocholine headgroup
at m/z 184 (C
5
H
15
PNO
4
) was used to localize the
phosphocholine-containing phospholipids, i.e., sphin-
gomyelins and phosphatidylcholines [23]. The phos-
phocholine headgroup was additionally localized by
imaging a specific fragment (m/z 86). This signal
was localized in the left and right ventricles with
low intensity. In right and left atria, aortic wall, and
aorta valve, the intensity of m/z 86 signal was higher
(Fig. 3a). The localization of chemical components in
the tissue reveals structural information that can be
used for creating an atlas (Fig. 3). Separated ion images
of relevant molecules provide this image information. PCA is
used to find spatially correlated molecules; the resulting PCA
L. Fornai et al.
score images greatly enhance image contrast in comparison
with the separate ion images (Fig. 4).
3D molecular reconstruction of the rat heart
The generation of a three-dimensional dataset requires
an additional z-dimension. In our experiments, this was
achieved by successive tissue sectioning with well-
defined and measured spatial intervals [24]. Using the
micrometer scale of a cryomicrotome, an entire cryo-
preserved rat heart was sectioned. Forty sections at
irregularly spaced but well-documented intervals were
taken through the heart as shown in Figure 1A(Elec-
tronic supplementary material). The workflow of the 3D
reconstruction is show in Fig. 5.Thez-position was
recorded for each of the sections. High-resolution
metA-SIMS datasets were acquired from each section
with a 22 keV Au
+
primary ion beam. Each dataset
was acquired in 12 h. Forty datasets were acquired
resulting in a total of 42,949,672,960 spectra in the
raw data files. These data were subsequently combined
and processed to reveal the three-dimensional molecular
features. The processing protocol included spectral and
spatial binning to reduce the total dataset size prior to
molecular feature visualization using our datacube ex-
plorer. The 3D data volumes can be explored using the
Volume explorer software that is used to reconstruct a
3D-data grid out of the 40 individual 2D-datasets. The
molecular images of specific m/z range of interest areas
are put together into a three-dimensional volume in
which the pixels are turned into voxels. The Volume
explorer then uses volume-extraction by combining vox-
els above a certain threshold into a volume. The co-
registration of individual molecular images was obtained
by manual alignment in the Volume explorer of MSI-
section on the base of anatomical structures. This is, to
Fig. 1 A,B1,CMetal-assisted SIMS images of a sagittal section of
three different rat hearts; A1 anatomic differences from rat heart visu-
alized with H&E staining after SIMS analysis. B,B1 SIMS-MSI
images of ions detected from representative rat heart valves and
H&E-stained tissue sections. DMetal-assisted SIMS images of a
sagittal section of mouse hearts (datacube images), D1 H&E-stained
tissue sections and D2 spectrum of SIMS
Three-dimensional molecular reconstruction of rat heart with MSI
the knowledge of the authors, the first time three-
dimensional SIMS-based molecular volumes were con-
structed with a 1 μm lateral resolution and a 100 μm
depth resolution. The consistent observation of identical
correlated anatomical and molecular structures within
each of the technical replicates is shown in Figure 1B
of the Electronic supplementary material.
The images of the 3D reconstruction additionally
show a highly complementary localization between
Na
+
,K
+
,andanionatm/z 145. Na
+
is localized in
the atria, while K
+
is strongly localized in the ventricles
as previously reported [25](Fig.5D). Three-dimensional
reconstructions of three selected individual mass spectral
peaks can be simultaneously displayed using a Red
GreenBlue color scheme. Each color represents a spe-
cific molecular feature (Fig. 5E). The overlaid 3D mo-
lecular model obtained is a representation of the whole
heart. The result of the combination and co-registration
of 40 individual MS imaging data cubes is demonstrated
in Fig. 5F. The data volumes can be explored using
software which allows a close molecular look inside
the heart. The molecular visualization of the different
valvular structures, the pericardium, the atria, the coro-
nary arteries, and various other anatomical features pro-
vides a new tool in molecular pathology. While several
structures of the heart can be observed in individual 2D
sections analyzed by Meta-SIMS imaging, a full view of
these structures in the total heart volume can be
achieved only through the construction of the 3D heart
model. The data show significant differences in the ions
distribution in the various heart structures and reveal
distinctive molecular localization in this organ.
Mouse heart
A cross-species validation was performed through the
analysis of mouse heart sections (Fig. 1D,D1andD2).
We observed similar molecular patterns when the
mouse results were compared with the rat results. This
demonstrates, among others, the diversity in biological
systems in which this technology can be applied.
Cholesterol-related ions again demonstrated to have
specific higher intensity in atria, aorta, and pulmonary
artery and lower intensity in ventricles (Fig. 1D). The
ion at m/z 369 localized very precisely within the
pulmonary artery, tricuspid with high intensity in both
atria, aorta, pulmonary artery, and in the atrioventricu-
lar and semilunar valves but is detected in ventricles
with lower intensity. The similarity of these molecular
findings with the results obtained in the rat heart fur-
ther corroborates the across-species molecular consis-
tency at the lipid level.
Human heart
The true relevance of a new molecular imaging technol-
ogy as described here is demonstrated through the ap-
plication of high-resolution imaging MS on explanted
human heart samples taken from patients suffering from
ventricular failure. The application of MSI in the field
Fig. 2 High-resolution
(8,192× 8,192 pixels) SIMS
total ion images in black and
white and complementary
H&E-stained images showing
the different morphological
structures of the heart observed
in both molecular imaging
modalities
L. Fornai et al.
of human cardiovascular pathology has a direct potential
for clinical diagnoses and treatments. The larger size of
the human heart combined with the time-consuming
experimental procedure is prohibitively limitative for
the generation of a full three-dimensional molecular
model of the human heart. Instead, we have opted for
the analysis of smaller subsets of the human heart, in
this case, segments of the left ventricle.
In all the experiments, the several distinctive structures in
the ventricle can be clearly seen, originating from different
lipid composition and signal intensities. An abundant signal
of cholesterol (m/z 369) [MH
2
O+H]
+
can be observed
(Fig. 6). Cholesterol ion exhibit a high intensity in the
myocardium. The observed intensities in the pericardium
and endocardium are substantially lower. The m/z 104 cho-
line signal co-localizes with high intensity in myocardium
and endocardium (Fig. 6). The oleic (47 %) and palmitic
(19 %) acids are known to be the dominant fatty acids in the
human heart. Results from human and animal models of
heart failure generally support the concept of decreased fatty
acid β-oxidation in heart failure [26]. These findings are
consistent with our molecular images presented below that
show DAG species tentatively identified as [M+HOH]
+
(C
35
H
65
O
4
)atm/z 549, [M+HOH]
+
(C
37
H
69
O
4
)atm/z
577, [M+HOH]
+
, and ceramide at m/z 604. As expected,
they could be detected with high intensity in the pericardium
and low intensity in the myocardium. The SIMS imaging
data demonstrated that several important catabolic mole-
cules and their spatial features can be readily identified
and localized in a single MS imaging experiment (Fig. 6).
This in turn leads to a better fundamental understanding of
the pathological pathways whose molecular constituents are
visualized.
Adjacent human left ventricle sections were stained
with H&E, Sirius red, and trichrome and compared
with the SIMS imaging datasets obtained (Fig. 6A
D). Figure 6BDillustrates a transverse-section of left
ventricular muscle that contains a vein structure. The
different tissue types in this section can only be dis-
tinguished by a combination of three different staining
Fig. 3 Metal-assisted SIMS images of a sagittal section of a rat heart.
Top image shows the spatial distribution of ions (a,b,c,d) in the aorta
wall. For instance, the images in eand ishow distribution for the main
cholesterol ions (m/z 369 and m/z 385) that are localized to the aorta
wall, aorta valve, right coronary artery, and right and left atria but are
not observed in both ventricles. The image in fshows the ion at 145 m/
zvery localized in pulmonary artery, right atrium, and atrioventricular
valve. Interestingly, m/z 175 and m/z 213 are observed only in ven-
tricles. The bottom image m, in contrast, shows m/z 334 localized only
in the pericardium The image in qshows the high spatial distribution of
at 969 m/z in both atria, aorta wall, left atrioventricular valve, and right
coronary artery. All ion image scale bars0100 μm
Three-dimensional molecular reconstruction of rat heart with MSI
procedures, revealing the morphology and the presence
of connective tissue. The molecular images obtained
with SIMS provide all of this information (and more)
in a single experiment. Statistical analysis of human
heart (three technical replicates) yields correlation coef-
ficients of overall spectra of A versus B 0.9934, A
versus C 0.9858, B versus C 0.9925, respectively. This
again confirms the reproducibility and selectivity of
this innovative method for molecular histology (see
Figure 2, Electronic supplementary material). The tis-
sue sections were stained with H&E after SIMS anal-
ysis to correlate the hearts molecular profile with the
observed morphological features.
Discussion
SIMS imaging was able to consistently detect sodium
(23 m/z), potassium (39 m/z), choline (104 m/z),
phosphocholine (184 m/z), cholesterol (369 and 385 m/z),
DAG species (549 m/z,577m/z,603m/z), ceramide (604 m/
z), the ions at 145 m/z,175m/z,334m/z, 213 m/z,969m/z,
and several other molecules in rat, mouse, and human heart
structures. The assignments of molecules were made based
on the unique masses of single elements (e.g., Na
+
m/z
22.989, K
+
m/z 39.098), the calculated molecular weight
of more complex substances, and by comparison with
chemical standards. The lipid database was employed to
correlate specific molecules found by SIMS imaging within
the heart structures. The ability to identify specific biomole-
cules is crucial for biological, and especially pathological,
investigations. Molecular biology thrives on molecular imag-
ing techniques that aim at the investigation of the relationship
between spatial organization, structure, and function of mole-
cules in biological systems. Although impairment in calcium
homeostasis, abnormal myocyte energetics, and myocardial
remodeling have been described to be associated with cardiac
dysfunction, the underlying molecular mechanisms involved
Fig. 4 A PCA spectral results after VARIMAX optimization show a
strong contribution for among others cholesterol ([MH
2
OH]
+
at m/z
369.1 and [MH]
+
at m/z 385) and choline (m/z 104) showing corre-
lation with aorta wall, left and right atria, semilunar valve, atrioven-
tricular valve, left ventricle, right ventricle, and coronary artery. The
distributions based on the PCA results (B) resulted in a molecular
underpinning of the different areas that morphologically identified with
H&E staining (C). The intensity of each ion is indicated in the color
chart on the left from white (high) to dark (absent)
L. Fornai et al.
in the transition from normal cardiac function to heart failure
remain poorly understood [27]. Insight into these processes
and pathways will be important in the development of new
therapeutic strategies for treatment and prevention of heart
failure. Under normal physiologic conditions, the heart uti-
lizes fatty acids as its chief energy substrate. Because there is
limited capacity for triglyceride storage in the cardiomyocyte,
the uptake and oxidation of fatty acids is tightly coupled. The
accumulation of triglycerides in the heart, caused by a mis-
match between the uptake and the oxidation of fatty acids, is
associated with a number of pathophysiological conditions. In
animal models (rats) of obesity and diabetes, triglyceride
accumulation within cardiomyocytes is associated with im-
paired contractile function. Although it is unclear how lipids
induce cardiac dysfunction, accumulation of intramyocardial
triglycerides is associated with altered gene expression [28].
The lipid droplet is endured by a core of lipids, which mostly
consists of TAG (9099 %) and to a lesser amount of DAG,
FFA, phospholipids, and monoacylglycerols. Several distinc-
tive MS peaks and correlated image patterns consistent with
the expected oleic/palmitic acid ratio were observed in
consecutive SIMS experiments used for the 3D reconstruction
of rat heart.
Each molecular pattern in these sections was ana-
lyzed in a search for molecular classifiers. For exam-
ple, cholesterol was found to distinguish directly atria
from ventricles. The difference in the relative choles-
terol content in atria and ventricles was observed in a
direct comparison between rat and mouse results
(Fig. 1A, C, D). These results corroborate and expand
earlier studies performed on heart substructure homo-
genates that were analyzed with liquid chromatography
[29]. Here, for the first time, we visualize the heart
cholesterol distribution directly on histological tissue
sections without the use of any labeling approach to-
gether with a plethora of other molecules. We are not
able to assign a molecular structure to the ions at
145 m/z, 175 m/z, 334 m/z,213m/z, and 969 m/z
(Fig. 3f, h, m, n, q) showing a different distribution
in the tissue sections. In summary, SIMS is shown to
provide extensive local molecular information that
complements the conventional histological approaches
Fig. 5 Workflow of the 3D reconstruction. ATissue section, Bgold
deposition is done by sputter coating, C,C1 SIMS-ToF mass analyzer,
C,C2 mass spectral peaks, C,C3 datacube images; D,E
Reconstruction of the heart showing 3D-spatial distributions of three
different ions 145 m/z (red), 23 m/z (green), and 39 m/z (blue); Fco-
registration of 40 individual MS imaging
Three-dimensional molecular reconstruction of rat heart with MSI
for the determination of the chemical properties of
specific, known anatomical structures inside the heart.
As any analytical technique, SIMS has limitations and
advantages for molecular histology. In the next section,
we briefly discuss the most important aspects of SIMS
in cardiovascular research.
Limitations of SIMS The extreme surface sensitivity
requires careful sample treatment. Sample contamina-
tion and molecular diffusion, which can affect the
reproducibility of the data, complicate their analysis,
or affect the quality of the image, are major consider-
ations in the sample preparation protocols. The multi-
ple molecules present in a tissue section can negatively
influence each othersdesorption and ionization effi-
ciency and prevent optimal detection. This phenomenon
is called ion suppression and can limit the number of
detected molecules. The SIMS imaging technique is
only capable of a semi-quantitative measure of the
distribution of elements and molecules in tissue.
Absolute quantitation requires the use of isotopically
labeled standards. Identification of species is based on
single m/z values exclusively; as in most SIMS instru-
ments, no tandem mass spectrometry can be performed.
New instruments will overcome this limitation but are
not yet commercially available.
Advantages of SIMS
SIMS allows for label free molecular imaging of mul-
tiple species in parallel directly on tissue. SIMS instru-
ments are used for imaging unknown compounds
present in the biological sample without any a priori
knowledge or labels in a single experiment. It is a true-
discovery methodology. It has been used for imaging
of elemental species in cells at high spatial resolution
(50 nm) and can typically analyze ions up to
1,500 m/z. In the last decade, there have been a grow-
ing number of studies that show the capacity of SIMS
for analyzing biological materials, including different
Fig. 6 A Metal-assisted SIMS images of human heart. All the images
obtained are from the same section. All ion images scale bar0100 μm. B
H&E staining. CSection stained with Sirius red. Connective tissue stains
red. In this trichrome-stained specimen (image D), collagen is colored
blue and smooth muscle is red. The images in B,C,andDillustrating a
transverse-section of left ventricle muscle, clearly include an intra-
myocardial blood vessel to illustrate the staining pattern in gross sheets
of cardiomyocytes that can be seen towards the periphery of the figures
L. Fornai et al.
tissues such as mouse brain, human adipose tissue,
muscle, kidney, and single cultured cells [3035]. More
information on different MS imaging technologies for
biomedical purposes can be found in current review of
the various imaging MS technologies for molecular
pathology [11].
A major advantage is the reduced amount of sample used;
often, after SIMS analysis, the tissue surface can be ana-
lyzed or stained as if the tissue was pristine. This, in turn,
allows the direct comparison with other ex vivo techniques
(e.g., fluorescence and immunostaining) for orthogonal val-
idation of the findings. These techniques are highly specific
within the class but allow the visualization of labeled mol-
ecules only with a limited number of detectable analytes per
section [36].
Future and perspective
The application of this method in future studies can be used to
identify changing tissue regions that are indicative of human
cardiac disease. SIMS improves tissue classification necessary
to perform retrospective studies, will assist clinical studies
from the bench to bedside, and can guide therapeutic choices.
The direct application of SIMS on the same heart tissues as
used by pathologists improves and accelerates molecular di-
agnoses. Molecular tissue classification after SIMS based on
known biomarkers or using unsupervised multivariate analy-
ses can positively affect patient treatment. The evolution of a
heart disease during treatment can be monitored based on the
tissue biomarkers identified by MSI. In cases where traditional
biomarkers cannot be clearly detected in biopsies, SIMS could
become critical to the outcome. At this point, it is obvious that
further clinical studies using SIMS technology are required to
fully validate this method. Nonetheless, SIMS as well as
MALDI-MSI have opened the door to molecular tissue clas-
sification, not only for diagnostic and prognostic purposes but
also for treatment development. MS-based molecular imaging
is becoming one of the basic information providers for per-
sonalized medicine, especially when used in complement with
magnetic resonance imaging (MRI). A major advantage for
SIMS will be its coupling with positron emission tomography,
X-ray, computed tomography instrumentation, and MRI for
both preclinical and clinical research. The complementarities
between non-invasive techniques and molecular data obtained
from SIMS-MSI will result in a more precise diagnosis of the
molecular stateof a living system.
In clinical studies, the need for information on the spatial
localization of pathologically gene-encoded products has
become more pressing. The three-dimensional volume
reconstructions generated by SIMS-MSI data now offer
the possibility of comparing the molecular data with
data obtained using positron emission tomography or
MRI [37]. These multi-modal molecular imaging
approaches will strengthen the fundaments of molecu-
lar imaging research.
Conclusions
The SIMS imaging approach can be used to detect and
probe the molecular content of tissues in an anatomical
context. Anatomical atlases based on optical images are
widely used for anatomical and physiological reference.
A series of secondary ion images obtained from succes-
sive tissue sections of rat heart can be used to produce
a 3D molecular reconstruction that contains both pieces
of information. SIMS provides detailed high-resolution
molecular images of tissue surfaces. The results reported
here represent the first 3D molecular reconstruction of
rat heart by SIMS imaging. The measurements were
extended on mouse and human heart samples. Human
tissue analysis is demonstrated to benefit from the po-
tential of SIMS imaging for the investigation of the
distribution of elements and biomolecules directly on
the surface of cardiovascular tissues.
Acknowledgments We gratefully acknowledge the assistance and In-
formatics (CWI) in the generation of the full-resolution SIMS images.
Sources of funding This work is supported by the Cardiovascular
Pathology, Department of Cardiac, Thoracic, and Vascular Sciences,
University of Padua Medical School, and by a grant from the Italian
Society of Cardiology. This work is also part of the research program
of the Stichting voor Fundamenteel Onderzoek der Materie (FOM) and
is financially supported by the Nederlandse organisatie voor Weten-
schappelijk Onderzoek (NWO).
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Supplementary resource (1)

... These methods generate layers of matrix crystals of different sizes and thickness. In the case of metal-assisted SIMS, a thin layer of gold can be deposited on the sample to improve molecular ion yield [31]. The attainable spatial resolution in matrix-based MSI strategies is determined by the matrix Fig. 1 Schematic overview of the workflow for MSI. ...
... Other optional mass analysers are a quadrupole TOF [35], a LTQ XL linear ion trap [36], and an orbitrap Fourier transform MS (FTMS) [37]. For SIMS MSI, a TOF-based mass spectrometer is most often used [19,25,31,[38][39][40]]. An ion source, for instance, gold or bismuth liquid metal ion gun, generates a pulse of primary ions that are accelerated towards the surface. ...
... Cardiovascular lipid imaging was mostly performed with a (Q)TOF or TOF-TOF instrument, coupled with MALDI [23,27,34,46,47] or SIMS [19,25,31,[38][39][40]. Structural information, high mass accuracy, and identification were done using an FR-ICR, LTQ XL linear ion trap, or orbitrap coupled with MALDI [17,34,36,37,47] or DESI [20,32]. ...
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Mass spectrometry imaging (MSI) is a widely established technology; however, in the cardiovascular research field, its use is still emerging. The technique has the advantage of analyzing multiple molecules without prior knowledge while maintaining the relation with tissue morphology. Particularly, MALDI-based approaches have been applied to obtain in-depth knowledge of cardiac (dys)function. Here, we discuss the different aspects of the MSI protocols, from sample handling to instrumentation used in cardiovascular research, and critically evaluate these methods. The trend towards structural lipid analysis, identification, and Btop-down^ protein MSI shows the potential for implementation in (pre)clinical research and complementing the diagnostic tests. Moreover, new insights into disease progression are expected and thereby contribute to the understanding of underlying mechanisms related to cardiovascular diseases.
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