Instruments

Chapter
4
Imaging Mass Spectrometry:
Sample Preparation,
Instrumentation, and
Applications
Kamlesh Shrivas , and Mitsutoshi Setou
Contents
1. Introduction
2. Ionization Methods for Imaging Mass Spectrometry
2.1. Desorption Electrospray Ionization
2.2. Secondary Imaging Mass Spectrometry
2.3. Laser Ablation Electrospray Ionization
2.4. Matrix-Assisted Laser Desorption/Ionization
3. MALDI Imaging
3.1. Sample Handling
3.2. Choice of Matrix
3.3. Application of Matrix Solution
4. Instrumentation
4.1. Quadrupole Mass Analyzer
4.2. Time-of-Flight Mass Analyzer
4.3. Sector-Type Mass Analyzer
4.4. Ion Trap Mass Analyzer
4.5. Orbitrap Mass Analyzer
4.6. Ion Cyclotron Resonance Mass Analyzer
5. IMS Measurements
6. Data Analysis
7. Applications of IMS for Direct Analysis of Tissue
7.1. IMS for Lipidomics
7.2. IMS for Proteomics
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Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama,
Higashi-Ku, Hamamatsu, Shizuoka 431-3192, Japan

Department of Chemistry, Guru Ghasidas University, Bilaspur-495009, CG, India
Advances in Imaging and Electron Physics, Volume 171, ISSN 1076-5670, DOI: 10.1016/B978-0-12-394297-5.00004-0.
c 2012 Elsevier Inc. All rights reserved.
Copyright
145
146
Kamlesh Shrivas and Mitsutoshi Setou
7.3. IMS for Pharmacokinetic Studies

7.4. IMS for Metabolomics
8. Summary
Acknowledgments
References
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180
182
183
184
1. INTRODUCTION
The ability to visualize the molecular distribution in biological material
such as tissue samples has helped scientists to provide a better understanding of the principles of life. The study of biomolecule distribution in
organs and its alterations with disease remains one of the most challenging and intriguing scientific issues of recent times. Various techniques are
used in laboratories around the world to visualize molecular systems
techniques such as magnetic resonance imaging (MRI) technology (Hurd
and Freeman, 1989) and positron electron tomography (PET) (Ametamey
et al., 2008). The Nobel Prizewinning MRI and PET technologies are
known as noninvasive techniques for medical diagnosis. Nuclear magnetic resonance spectroscopy (NMRS) is also helpful for imaging and
identification of biomolcules in tissue sample (Hiltunen et al., 2002). The
limitations of these techniques are the relatively poor resolution, sensitivity, and requirement of labeling of molecules for detection (in the case of
the PET method).
Imaging mass spectrometry (IMS) was introduced for spatial distribution analysis of biomolecules without the need for extraction, purification,
separation, or labeling of biological samples. Recent developments in
molecular imaging have created new opportunities to perform molecular
diagnostic and therapeutic procedures. The technique can be exploited to
visualize cellular and molecular processes that occur in two-dimensional
(2D) or three-dimensional (3D) fashion without perturbing the structure
of the system (Caprioli et al., 1997; Setou et al., 2010).
Mass spectrometry (MS) is a technique based on the measurement
of the charged ions in an electric or magnetic field. Generally, a mass
spectrometer contains three distinct parts: (1) an ion source producing
ions from sample molecules; (2) a mass analyzer separating the different molecules with respect to their mass-to-charge ratios (m/z), and
(3) a detector, registering the ion m/z and the intensity at which the
ions were detected. Data are collected and visualized in a mass spectrum where the different m/z ratios are displayed as a function of their
signal intensity (Gross, 2004). MS is a great scientific tool because of the
wide range of molecules that can be accurately detected and identified:
large organic compounds and biomolecules of low molecular weight.
Imaging Mass Spectrometry
147
In the beginning, mass spectrometric analysis was limited to samples

that had undergone excessive preparation procedures, such as purification, separation, and concentration steps. These procedures not only
jeopardize sample integrity, but also lead to the complete loss of any spatial distribution information. MS instruments are equipped with different
ionization methods, including electron ionization and chemical ionization (Fales et al., 1972), fast atomic bombardment (Morris et al., 1981),
electrospray ionization (ESI) (Fenn et al., 1989), and matrix-assisted laser
desorption/ionization (MALDI) (Karas et al., 1985) for the analysis of a
wide range of organic and bio-organic molecules. The introduction of the
soft ionization sources such as ESI and MALDI transfigured MS, as it
offered the capability to analyze large intact biomolecules.
At present, IMS is a well-recognized technique for profiling the distribution of biomolecules in tissue sample at micrometer to nanometer
resolution (Caprioli et al., 1997; Goodwin et al., 2008; McDonnell and
Heeren, 2007; Pol et al., 2010; Shimma et al., 2008). Data acquisition is performed through scanning a tissue section with a laser, thereby obtaining
one mass spectrum for every pixel. The main principle of IMS is based
on desorption and ionization of biomolecules from the surface of the
tissue sample. There are currently four important desorption/ionization
methods: desorption electrospray ionization (DESI) (Takats et al., 2004),
secondary ion mass spectrometry (SIMS) (Benninghoven, 1973), MALDI
(Tanaka et al., 1988) and laser ablation electrospray ionization (LAESI)
(Nemes and Vertes, 2007).
2. IONIZATION METHODS FOR IMAGING MASS

SPECTROMETRY
2.1. Desorption Electrospray Ionization
DESI was introduced by R.G. Cooks in 2004. In DESI, the molecules are
ionized at atmospheric pressure without the use of any organic matrix
(Dill et al., 2009) in a combination of ESI and desorption ionization (DI).
The charged droplets of solvent generated during the electrospray stage
are used to ionize molecules from the surface of the sample and the ions
produced thereby are directed into an atmospheric inlet of the MS. The
components and use of DESI in IMS are presented in Figure 1a. The spatial
resolution obtained by this method is 0.30.5 mm, which is a low resolution of tissue sample in IMS studies. DESI has been successfully applied
to IMS for the identification of lipids, drug metabolites, and antifungal
molecules in seaweeds (Dill et al., 2009; Lane et al., 2009; Wiseman et al.,
2008).
148
Inlet of mass
spectrometer
Solvent
N2
Secondary
droplets
Spray droplets
Sample
(a)
Inlet of mass
spectrometer
Secondary
ions
Primary ions
Sample
(b)
Laser
attenuator
UV or IR laser
Inlet of mass
spectrometer
Analyte/matrix
mixture
(c)
FIGURE 1 Desorption-ionization techniques used in mass spectrometry imaging.

(a) Desorption electrospray ionization (DESI). (b) Secondary ion mass spectrometry
(SIMS). (c) Matrix-assisted laser desorption ionization (MALDI). UV, ultraviolet;
IR, infrared. Reprinted from Pol et al. (2010) with permission from Springer.
149
2.2. Secondary Imaging Mass Spectrometry

SIMS is a sophisticated technique that uses ion beams from metal ions
such as Ar+ , Ga+ , and In+ (here denoted primary ion beams) to produce
secondary ions from molecules on the surface of a sample. Ionization
is performed in high vacuum to avoid a collision with surrounding gas
molecules, and the primary ion beams can be focused down to 50 nm
on the sample surface, with the resolution depending on the current and
charge state of the ions. SIMS coupled with time-of-flight (TOF-SIMS) is a
superior tool for high-spatial, submicron resolution (< 10 nm). Thus SIMS
can be applied for the differentiation of biomolecules that are present
all the way down to the cellular level. However, fragmentation of larger
molecules on the sample surface is observed when strong laser energy
is applied for the primary ion beam. Hence, SIMS is primarily applicable for the analysis of small molecules (< 1000 Da) (Heeren et al., 2006;
Slaveykova et al., 2009). Figure 1b shows the process of SIMS ionization of molecules from the sample surface. SIMS has been applied for
imaging of samples such as single cells, embryos, brain, cocaine, and cinnamoylcocaine in coca (Colliver et al., 1997; Jones et al., 2007; Wu et al.,
2007). The fragmentation of molecules in SIMS can be overcome through
the treatment of an organic MALDI matrix; this approach is known as
matrix-enhanced (ME)-SIMS (Altelaar et al., 2007).
2.3. Laser Ablation Electrospray Ionization

LAESI was developed by Nemes and Vertes (2007) and is a method for MS
analysis of tissue samples without sample preparation under atmospheric
pressure (Nemes and Vertes, 2007). Laser ablation from a mid-infrared
(mid-IR) laser is combined with a secondary ESI process. The spatial resolution for tissue samples using LAESI technique is better than DESI and
can be used for imaging of biomolecules from the surface of tissue sample at a lateral resolution of < 200 m. The technique has been applied for
imaging and identification of plants, tissues, cell pellets, and even single
cells (Nemes et al., 2010; Shrestha et al., 2010; Sripadi et al., 2010). Recently
it has also been used in 3D imaging of molecules from the sample (Nemes
et al., 2009).
2.4. Matrix-Assisted Laser Desorption/Ionization

MALDI was introduced as a soft ionization technique that causes little
or no fragmentation of the target molecules, allowing for the analysis
of molecules at several hundred kilodaltons (i.e., high m/z values). This
allows for mass spectrometric analysis of a wide range of molecules such
as amino acids, peptides and proteins, carbohydrates, and nucleic acids
and drugs and has proven to be one of the most powerful MS technologies
150
to date. In traditional MALDI, an organic matrix is mixed with the sample on the target plate and irradiated by a ultraviolet or IR light generated
by a pulsed and focused laser. The matrix absorbs the light at the wavelength of the laser, leading to a soft desorption/ionization of the intact
compounds of interest (Gross, 2004; Karas et al., 1985; Tanaka et al., 1988).
Figure 1c illustrates the MALDI mechanism.
3. MALDI IMAGING
By scanning a sample surface with the MALDI matrix/laser setup and
registering individual mass spectra for each pixel, a 2D ion density map
can be reconstructed using appropriate software. Direct MALDI-IMS analysis of clinical samples offers a unique approach to reveal the spatial
expression of biomolecules linked with pathological disease and other
clinical information. MALDI imaging is also suitable as a biomarker
discovery tool by comparing the relative quantities and/or spatial distribution patterns of molecules in pathological and normal samples. The
localization and abundance of biomarkers identified in tissue sections
are used to understand disease progression at a molecular level. The
main advantages of a direct biomarker analysis using MALDI imaging
are that it provides spatial distribution patterns and is free from extraction, purification, or separation steps, hence avoiding procedures that are
both time-consuming and jeopardize sample integrity (Chaurand et al.,
2006; Hayasaka et al., 2010; Herring et al., 2007; Schwartz et al., 2003;
Sugiura et al., 2009). With the currently available imaging software packages, we can construct multiplexed imaging maps of selected biomolecules within tissue sections. The laser energy is used in a raster scan
pattern to ionize the molecules, which are present as discrete spots or
pixel. For each pixel the full mass spectrum is represented. The data acquisition time for IMS was shortened by the introduction of N2 (337-nm) or
neodymium-doped yttrium aluminum garnet (Nd:YAG) (355-nm) lasers
with repetition rates of 2001000 Hz with pulse lengths of 3 ns. The laser
spot size of MALDI-MS is decreased from 100150 to 20 m, rendering
higher spatial resolution of biomolecules on the tissue surface. Further,
a higher spatial resolution can be attained with a MALDI instrument
equipped with a highly focused laser. Chaurand et al. (2007) used a laser
beam at 7 m, which is in the order of the diameter of a single cell to
detect protein ions. However, a decrease in sensitivity is observed while
increasing the resolution in this manner. Figure 2 shows an example of
MALDI-IMS analysis of protein from tissue section.
In addition to increased sample integrity, the great advantage of IMS
is that it allows the construction of numerous ion images of molecules
detected in a single run. This technique does not require previous
Slice frozen tissue on

cryostat (~12 m thick)
Profiling
151
Imaging
Thaw slice onto

MALDI plate,
allow to dry
Low
density
droplet
array
High
density
droplet
array
Apply
matrix
Acquire
mass spectra
r
se
La
Molecular
profiles
r
se
La
Molecular
images
FIGURE 2 Scheme presenting the protein profiling and imaging analytical strategies
from thin tissue sections. Reprinted from Chaurand (2006) with permission from
American Chemical Society.
labeling with fluorescent probes or radioactive isotopes. MS analyses may

be performed for imaging of biomolecules at low concentrations; the
detection of 500 attomol has been reported in a single cell (Northen et al.,
2007). Another advantage when using MS is the specific identification of
molecules; tandem MS is used to identify compounds for which no previous knowledge is required. For this, two MS analyzers are used: one for
the selection of the ion of interest before fragmentation, and the second is
used for the analysis of fragmented masses. Thus the use of MS is rapid,
sensitive, and free from complicated sample procedures for the analysis
of unknown biological tissue samples.
3.1. Sample Handling

Sampling handling is a very important concern for imaging and identification of biomolecules in tissue samples. Consideration must be given to
the storage of the tissue sample after surgical removal from the human or
animal body to prevent ex vivo degradation and alteration processes. The
sectioning, washing, and staining of tissue, the choice of matrix, and its
application on the tissue section are other parameters to optimize in order
to obtain better-quality data.
152
3.1.1. Storage of Samples

Tissue storage is the most important part of the protocol for IMS studies
to maintain the integrity of both the molecular composition and the spatial localization of analytes. When sampling is performed through surgical
removal of tissue, molecular processes such as protein degradation continue in the ex vivo state. These processes should be halted immediately,
either through freezing in liquid nitrogen or heat stabilization (Schwartz
et al., 2003). Chaurand et al. (2008) reported a long preservation method
of tissue samples with ethanol for generating high-quality histological
sections that enable high-quality images of biomolecules in tissue sample. Previously archived samples, on the other hand, are often fixed with
paraformaldehyde and embedded in paraffin. Due to the cross-linking
between molecules caused by this preservation method, special methods
for specific tissue digestion have been developed (Wisztorski et al., 2010).
3.1.2. Sectioning of Tissue

The next important part of imaging experiments is the sectioning of tissue sample into thin slices and the subsequent mounting of these tissue
slices onto an appropriate target. Before tissue sectioning, the frozen tissue
samples are transferred from the 80 C freezer to the cryostat chamber
at 20 C for 30 minutes to thermally equilibrate the tissue. The tissue
is usually embedded on an optimal cutting temperature (OCT) polymer, which supports easy handling and precise microtoming of sections.
However, the use of OCT compounds causes a suppression of MALDI
analyte signals in MS and should, if possible, be avoided (Schwartz et al.,
2003). Figure 3 shows the mass spectra of rat liver with suppression of
MALDI-MS signals when OCT is used as a supporting material.
The use of gelatin is an alternative method for embedding the tissue
sample where the mass spectrum is free from background signals compared with the use of OCT (Chen et al., 2009). The embedded tissue is
fixed on a sample stage and the temperature is maintained between 5 C
and 25 C. The optimal temperature is set depending on the type of tissue
to be analyzed and is followed by slicing of tissue with a steel microtome blade. For MALDI-IMS, the tissue sections are usually 520-m thick
(Chaurand et al., 2006; Schwartz et al., 2003).
The next step is the proper transfer of the sliced tissue section onto an
electrically conductive steel plate or a glass slide. Thicker sections of tissue are more suitable when transferring them to the target plate because
thinner sections break more easily. The first method of tissue transfer is
performed by simply placing the plate in the cryostat chamber kept at
15 C while sectioning. An artists brush is used to pick up the tissue
section and gently place it on the cold plate, followed by gentle warming
153
Intensity
(a)
Tissue slice
(b)
OCT
Intensity
OCT
4500
6000
7500
m/z
9000
10500
FIGURE 3 Effect of optimal cutting temperature (OCT) on MALDI signal from rat liver.
(a) Optimal procedure where OCT is used to adhere the tissue to the sample stage but
does not come into contact with the sliced tissue. The resulting spectrum shows many
intense signals between m/z 4500 and 10500. (b) The tissue was embedded in OCT and
attached to the sample stage. The resulting tissue slice is surrounded by OCT on the
MALDI plate, and the resulting spectrum contains of only about half of the signals as
in (a). Reprinted from Schwartz et al. (2003) with permission from John Wiley and Sons.
of the plate by touching the backside of the plate with a fingertip. The
tissue is thereby thaw-mounted on the target plate. In the second method,
the plate is kept at room temperature and placed over the sliced frozen
section, and the tissue is thereby simply thawed on the target plate.
Great care should be taken with both methods to retain the shape of the
tissue. Obviously, folding or stretching caused during the sectioning of
tissue section may affect the molecule distribution analysis and prevents
detection of some of the molecules from the tissue surface.
3.1.3. Washing Tissue Sections

A tissue sample is generally washed to remove contaminants such as
tissue-embedding media as well as lipids or biological salts that may affect
the profiling and identification of peptides and proteins in MALDI-MS
analysis. Washing a tissue section with 70% ethanol can remove salts, followed by a 90%100% ethanol wash to dehydrate and fixate the tissue
(Lemaire et al. 2006b; Schwartz et al., 2003). Lemaire et al. demonstrated a
procedure for washing a tissue section with five different organic solvents
(chloroform, xylene, toluene, hexane, and acetone) for the identification
of proteins in tissue samples and repeated the procedure with fresh
solvents. The detection of protein signals is increased when the tissue
154
Relative intensities
Acetone
Chloroform
Ethanol
Water
Hexane
Isopropanol
Acetic acid
Methanol
t-MBE
Toluene
Xylene
No wash
0
5000
10000
15000
Mass-to-charge (m/z)
(a)
6226
Intensity
2000
7928
900
1000
600
500
300
9912
13777
7936
1200
1500
6000
20000
9980
4000
7885
500
2000
0
6200 6220 6240 6260
m/z
0
7860 7900 7940 7980
m/z
(b)
(c)
1000
0
9850 9900 9950 10000
m/z
(d)
0
13700
13800
m/z
13900
(e)
FIGURE 4 Average MALDI-IMS protein profiles directly acquired from serial mouse
liver tissue sections not washed or washed with different solvent systems. (a) Full mass
range; (b)(e) selected mass signals showing specific behaviors for the different washes.
Reprinted from Seeley et al. (2008) with permission from Springer.
sections are washed with organic solvent compared with untreated samples (Lemaire et al., 2006b). Seeley et al. (2008) reported a new washing
procedure to enhance protein detection in terms of both the number of
observed peaks and the signal intensity. They demonstrated that the use
of 12 different washing solvents established the most effective condition for direct protein analysis from the surface of tissue section. They
also obtained a high detection sensitivity of protein signals, matrix crystal formations, and histological integrity of the tissues by washing with
70% isopropanol for 30 seconds followed by a 90% isopropanol wash for
30 seconds. Figure 4 shows the MALDI-IMS results for protein detection in
mouse liver tissue sections after washing with different organic solvents.
3.1.4. Histological Staining of the Section

Histological staining of the tissue section is necessary to interpret the ion
images obtained from the IMS results with the tissue section used in the
experiments. The optical image obtained by the microscope is also used to
superimpose the images acquired by IMS analysis to see the localization
of molecules in tissue section. Hematoxylin-eosin (H&E) staining is a very
popular histological method for MALDI-IMS results (Walch et al., 2008).
In IMS, two serial sections are sliced from tissue; one is used for imaging
155
m/z 6651
1 mm
(a)
m/z 2897
(b)
m/z 9685
(c)
1 mm
(d)
m/z 2897
Pars distalis
(e)
Pars intermedia
Pars neuralis
m/z 9685
a.u.
12
10
8
6
4
2
m/z 6651
3000
4000
5000
6000
7000
(f)
8000
9000
10000
m/z
FIGURE 5 MALDI-IMS of a tissue section of rat pituitary gland. (a) Optical microscopic
image of an H&E-stained tissues section. The staining was done after the MALDI
measurement of the tissue section. (b)(d) Visualized selected m/z species representing
features to pars distalis (m/z 6,651; green), pars intermedia (m/z 2,897; red), and pars
neuralis (m/z 9,685; yellow). (e) Merge of (ad). (f) MALDI-TOF-MS spectra obtained
from this case from pars distalis (green), pars intermedia (red), and pars neuralis (yellow)
showing the molecular differences between the histological regions. Reprinted from
Walch et al. (2008) with permission from Springer.
and another section is cut for histological staining. They can then be superimposed on each other and provide an absolute value of the molecular
distribution (Figure 5). Recently a new approach for tissue section staining after the MALDI measurement has been reported. The results obtained
from IMS analysis were correlated with the H&E staining of the tissue
section (Schwamborn et al., 2007).
3.2. Choice of Matrix

The choice of a suitable matrix for MALDI-IMS analysis depends on
the mass range and analyte of interest. The main function of the matrix
is to absorb laser energy from the source and transfer it to the analyte
(Dreisewerd, 2003). The matrix thus ensures that efficient desorption
and ionization occur and protects the tissue section from the disruptive energy of the laser. Sinapinic acid is generally used for the analysis of higher-molecular-weight proteins, and -cyano-4-hydroxycinnamic
acid (CHCA) is used for lower-molecular-weight molecules such as
peptides (Schwartz et al., 2003). 2,4-dihydroxybenzoic acid (DHB) and
2,6-dihydroxyacetophenone (DHA) are generally used for analysis of
156
TABLE 1 Commonly used MALDI matrices for imaging of biomolecules in tissue

samples
Matrix
Applications
References
2,5-Dihydroxybenzoic
acid (DHB)
Lipids, Sugars,
peptides, nucleotides,
glycopeptides,
glycoproteins, and
small proteins
Fournier et al. (2003);

Herring et al. (2007);
Tholey and Heinzle
(2006)
-Cyano-4hydroxycinnamic acid
(CHCA)
Peptides, small
proteins and
glycopeptides
Schwartz et al. (2003);

Tholey and Heinzle
(2006)
2,6Dihydroxyacetophenone
(DHA)
Phospholipids
Jackson et al. (2005);

Seeley et al. (2008);
Tholey and Heinzle
(2006)
2,4,6Trihydroxyacetophenone
(THAP)
Lipids
Stuebiger and Belgacem

(2007)
p-nitroaniline (PNA)
Phospholipids
Estrada and Yappert

(2004); Rujoi et al. (2004)
2-mercaptobenzothiazole
(MBT)
Sinapinic acid (SA)
Phospholipids
Astigarraga et al. (2008)
Peptides and large

proteins
Schwartz et al. (2003)
CHCA/aniline, ionic
matrix
Peptides
Lemaire et al. (2006b)
CHCA/n-butylamine,
ionic matrix
Phospholipids
Shrivas et al. (2010)
phospholipids (Herring et al., 2007; Seeley et al., 2008). A great variety of

matrices are used for the analysis of biomolecules, some of which are listed
in Table 1.
3.2.1. Ionic Matrices for IMS

Ionic matrices (IMs) constitute a new class of organic matrices reported
for the analysis of a number of different molecules in MALDI-MS.
IMs are good for MALDI-MS imaging studies due to the fact that the
process solubulizes several analytes, has vacuum stability, and forms
homogenous crystals with analyte molecules. IMs have been used to
obtain enhanced sensitivity and good reproducibility in the analysis
157
of biomolecules (Armstrong et al., 2001; Laremore et al., 2007). IMs

such as 2,5-dihdroxybenzoic acid butylamine (DHBB) and -cyno-4hydroxycinnamic acid butyl amine (CHCAB) render good crystal formation, signal intensity, and reproducibility compared with conventional
matrices such as DHB and CHCA (Shrivas et al., 2010). The results are
6
786
786
45
4
760
760
30
810
810
(a)
Signal intensity 10
15
(b)
731
703
500
1000
800
731
703
500
800
(i)
1000
(k)
786
786
350
120
760
760
90
810
210
810
60
731
703
30
(c)
731
703
70
(d)
500
1000
800
500
(l)
60
703
100%
3
760
0%
8
703
731
760
786
40
Signal intensity 10
731
20
0
1
(g) DHBB (h) CHCAB
2
0
703
731
760
786
0
0
(n)
50
(f) CHCA
703
731
760
786
(m)
100
(e) DHB
0
0
150
786
1000
800
(j)
400
300
200
100
0
703
731
760
786
0
(o)
(p)
Number of sample analyses
FIGURE 6 The crystal formation of (a) DHB, (b) CHCA, (c) DHBB, and (d) CHCAB
matrixes with phospholipids on to a MALDI target plate. The pictures were taken with an
Ultraflex II TOF/TOF. The scale bar (white color line) is 100 m; images (e) to (h) show
the ion image of phospholipids reconstructed obtained by using (e) DHB, (f) CHCA,
(g) DHBB, and (h) CHCAB matrix at m/z 703, 731, 760, and 786. Images (i) to (l) show
the signal enhancement: 3- to 7-fold enhancement of signal intensity when DHBA IM
(image i) is used as a matrix compared with DHB matrix (image j) and 50- to 100-fold
improvement of signal intensity using CHCAB IM (image k) compared with CHCA matrix
(image l). Graphs (m) to (p) show the six replicate analyses of samples with relative
standard deviation, % by using (m) DHB: 20.540.8%, (n) CHCA: 29.545.8%,
(o) DHBB: 14.521.8%, and (p) CHCAB: 7.510.0%. Reprinted from Shrivas et al. (2010)
with permission from American Chemical Society.
158
shown in Figure 6. Direct tissue analyses of peptides in rat brain tissue

sections using IMs improved the ionization efficiency and increased the
signal intensity of ion images of molecules compared with the conventional
matrix (Lemaire et al., 2006a). IMs were also used for imaging and identification of gangliosides in mouse brain (Chana et al., 2009). DHB and CHCAB
IMs in MALDI-IMS were also used for analysis of mouse liver and cerebellum tissues to identify the different species of lipids; results with CHCAB
were better than with conventional matrices (DHB and CHCA).
3.2.2. Nanoparticles as Matrices for IMS

In addition, nanoparticles (NPs) can be used as a matrix instead of organic
matrices for the analysis of low-molecular-weight molecules (< 500 Da).
One problem with the organic matrix ions is that they themselves produce
an intense peak in the mass spectrum and hence suppress detection of the
analyte of interest, which then obviously decreases the sensitivity of the
method. To circumvent this disadvantage, nanomaterials and inorganic
compounds have been introduced. The Tanaka and Sunner groups investigated the application of cobalt powder (NPs) and graphite microparticles,
respectively, suspended in glycerol to analyze proteins and/or peptides
in MALDI-MS analyses. The use of NPs as a matrix in MALDI-MS allows
for efficient absorption of laser energy as well as efficient subsequent
desorption and ionization of molecules from the sample surface (Sunner
et al., 1995; Tanaka et al., 1988). Desorption/ionization on porous silicon (DIOS) is another matrix-free method that is produced by etching
of the silicon surface. Small molecules can be efficiently ionized using
DIOS as an effective surface (Wei et al., 1993). Today nanomaterial surfaces
are also applied for the direct analysis of tissue samples in MALDI-IMS.
Northens group introduced a new nanostructure surface for imaging
of biomolecules in tissue samples known as ionization nanostructureinitiator mass spectrometry (NIMS) (Northen et al., 2007). Several other
sample preparation procedures, such as graphite-assisted laser desorption/ionization (GALDI) (Cha and Yeung, 2007), nano-assisted laser
desorption/ionization (NALDI) (Vidova et al., 2010), and DIOS have been
proposed for imaging of biomolecules in tissue samples. Taira et al. (2008)
developed another matrix-free method called nanoparticle-assisted laser
desorption/ionization imaging mass spectrometry (Nano-PALDI-IMS)
that can be used to visualize peptides, phospholipids, and metabolites
in tissue sections. Recently silver (Hayasaka et al., 2010) and gold (GotoInoue et al., 2010a) NPs were applied for imaging and identification of
fatty acids and glycosphingolipids, respectively, an analysis that could be
difficult to perform by conventional MALDI-MS using DHB as a matrix.
Figure 7 demonstrates imaging and identification of fatty acids from
mouse liver sections using silver NPs as a matrix (Hayasaka et al., 2010).
More recently, TiO2 NPs were applied for the analysis of low-molecular
159
On tissue with AgNps
40
35
30
25
20
15
10
5
200
220
240
260
280
300
320
Signal intensity (a.u.)
340 m/z
On tissue with DHB
40
35
30
25
20
15
10
5
200
220
240
40
35
30
25
20
15
10
5
200
Only AgNPs
220
240
260
280
300
320
340 m/z
280
300
320
340 m/z
Only DHB
40
35
30
25
20
15
10
5
200
220
m/z 255.4
(16:0)
240
260
280
300
320
340
m/z
(d)
(b)
Optical
image
260
(c)
(a)
m/z 279.4
(18:2)
m/z 281.5
(18:1)
m/z 283.4
(18:0)
m/z 301.2
(20:5)
m/z 303.3
(20:4)
100%
AgNPs
DHB
0%
(e)
FIGURE 7 Identification of fatty acids from mouse liver sections in nano-PALDI-IMS.

The serial sections were sliced to a thickness of 10 m. Silver nanoparticles (NPs) or DHB
matrix solution was sprayed on the surface of the mouse liver sections, respectively.
Their sections were measured with a scan pitch of 100 m by nano-PALDI-IMS analysis in
negative-ion mode. The mass spectra were obtained from the sections sprayed with
silver NPs (a) on tissue section, (b) only silver NPs or DHB matrix, (c) on tissue section,
and (d) only DHB solution. The peaks used to reconstruct the ion image are indicated by
arrows. (e) In the analysis using silver NPs and DHB, the ion signals at m/z 255.4 (16:0),
279.4 (18:2), 281.5 (18:1), 283.4 (18:0), 301.2 (20:5), and 303.3 (20:4) were detected. The scale
bars are 500 m. Reprinted from Hayasaka et al. (2010) with permission from Springer.
weight-biomolecules in mouse brain without observing any NP-related

peaks. More individual signals and higher intensity were obtained when
TiO2 NPs were used as a matrix compared with a DHB matrix (Shrivas
et al., 2011). Thus we can conclude that the use of a nanomaterial surface
is efficient and effective for desorption and ionization of molecules; the
process yields images with higher resolution.
3.3. Application of Matrix Solution

The deposition of matrix solution on the surface of a tissue section is
another important step in obtaining homogeneity, reproducibility, and
good resolution of the biomolecule. The matrix solution consists of three
160
componentsorganic solvent, matrix, and trifluoroacetic acid (TFA).

Crystal formation is affected by the concentration of matrix and the ratio
of organic solvent to water; organic solvent is used to dissolve the solid
matrix and extract the molecules from the tissue section. This extraction is followed by crystal formation on the surface of the tissue section.
The addition of TFA provides free protons for effective ionization of the
analytes, and typically, singly charged [M + H]+ ions are formed. A number of devices are useful for the deposition of matrix solution on the
surface of tissue sectionsfor example, chemical inkjet printer spotter
(Baluya et al., 2007), robotic spotting depositors (Aerni et al., 2006), electrospray depositors (Altelaar et al., 2007), and airbrush sprayers (Hayasaka
et al., 2009). The sublimation (Hankin et al., 2007) and stainless steel sieve
(Puolitaival et al., 2008) methods have demonstrated good signal intensity
and sample reproducibility. Figure 8 shows a thin layer chromatography
(TLC) sprayer (image a), sublimation apparatus (b), air brush sprayer (c),
and a chemical inkjet printer (d) used for matrix deposition. The goal of
these matrix deposition approaches is to improve the homogeneity of the
sample surface and enhance the signal intensity for the identification of
biomolecules compared with direct deposition of the matrix.
MALDI
plate with
tissue slice
(a)
(b)
(c)
(d)
FIGURE 8 Apparatus used to deposit matrix on the tissue section. (a) Thin-layer
chromatography sprayer, (b) sublimation apparatus, (c) air brush sprayer, and (d) chemical
inkjet printer. Reprinted from Hankin et al. (2007) with permission from Springer.
161
4. INSTRUMENTATION
4.1. Quadrupole Mass Analyzer
A quadrupole mass analyzer is made from four parallel rods maintained
at fixed direct current (DC) with an alternating radiofrequency (RF). With
this setup molecular ions formed at the source pass through the middle
of the quadrupoles in the electric field region and the ions of a specific
m/z have a stable trajectory path and may pass all the way to the detector, while the remaining ions collide with the electrodes and never reach
the detector (Gross, 2004). Using a continuous and controlled manner to
change the frequency and potential, the quadropole transmits molecules
at certain m/z values. Figure 9a shows a diagram of quadrupole mass analyzer. The sensitivity of the instrument can be enhanced by increasing the
number of qaudupoles from two to three (triple quadrupole) in series. In
triple-quadrupole analyzers, the first (Q1 ) and third (Q3 ) quadrupoles act
as filters, and the second (Q2 ) quadrupole functions as a collision cell. The
third (Q3 ) quadrupole is worked at normal RF/DC or in the linear ion
trap (LIT) mode (Douglas et al., 2005). Hopfgartner et al. (2009) demonstrated the fast imaging of complete rat sections using MALDI coupled
with a triple-quadrupole LIT where the precursor ion mode can be used
to monitor the presence of the parent drug in the tissue section.
4.2. Time-of-Flight Mass Analyzer

The TOF-MS analyzer has become valuable for direct analysis of biomolecules from tissue samples. In TOF-MS, the different masses of ions are
separated based on their differences in travel time through a drift region.
The lighter ions produced from the source travel faster at the end of the
drift region compared with heavier ions in the tube (see Figure 9b). However, TOF-MS has disadvantages in mass accuracy, resolving power, and
its inability to perform tandem MS experiments (Goto-Inoue et al., 2011;
Gross, 2004). This drawback has been overcome by the introduction of an
orthogonal geometry (oTOF)-MS analyzer to extract pulsed ions from a
continuous ion beam. Huang et al. (2011) investigated the use of oTOF-MS
for imaging and simultaneous detection of metal and nonmetal elements
in tissue section with spatial resolution of 50 m.
Ion mobility (IM) spectrometry can also be coupled with the TOF-MS
system for direct analysis of tissue samples. The instrument has oTOF-MS
and is equipped with an IM spectrometer located between the quadrupole
and the TOF-MS analyzer. The IM spectrometer separates ions based on
their IM (i.e., their shape) and TOF-MS separates ions according to their
m/z ratio in the MS (Verbeck et al., 2002). Separation of structurally similar
162
Ring
electrode
Entrance
slit
Quadrupole
rods
Exit
slit
(a)
Flight
tube
Entrance
endcap
electrode
Linear
detector
Exit
endcap
electrode
(d)
Detector
electrode
Detector
electrode
Central
electrode
Reflector Reflector
detector (lon mirror)
(b)
Superconductive
magnet
Excitation
plates
Magnet
Flight
tube
(e)
Exit
slit
Trapping
plates
(c)
Detector
plates
(f)
FIGURE 9 Schematic description of six mass analyzers used in mass spectrometers.

(a) Quadrupole, (b) time-of-flight, (c) magnetic sector, (d) ion trap, (e) orbitrap, (f) ion
cyclotron resonance. Reprinted from Pol et al. (2010) with permission from
Springer.
ions and ions of the same charge state is thus possible through their different mobility in the IM spectrometer. The combined techniques of IM and
TOF-MS were used for imaging and identification of digested proteins.
IM separates isobaric ions that cannot be distinguished by MALDI-TOF
alone, providing mass- and time-selected ion images of biomolecules in
tissue samples (Stauber et al., 2010).
163
In addition, the combination of a quadrupole (Q) mass analyzer with

a TOF-MS is called a Q-TOF-MS system and is used for structural analysis
with tandem MS. The localization of a xenobiotic substance in skin has
been reported by applying a Q-TOF-MS (Bunch et al., 2004). Another
approach for imaging and identification of molecules is the combination
of two TOF mass analyzers; this hybrid is called TOF/TOF. First, TOF-MS
separates precursor ions using a velocity filter; second, TOF-MS analyzes
the fragment ions (Gross, 2004). MALDI-TOF/TOF is a simple, rapid, and
sensitive technique for MALDI imaging of biomolecules in tissue sections
(Hayasaka et al., 2010; Sugiura et al., 2009).
4.3. Sector-Type Mass Analyzer

The sector mass spectrometer consists of large electromagnetic (B
sector) and electrostatic focusing devices (E sector) that, depending
on the different manufacturers use, differ in their geometries (Cottrell
and Greathead, 1986). The motion of the ions in the trajectory pathway
depends on the strength of electric and magnetic field where each ion
(m/z) travels with different speeds (see Figure 9c). Magnetic sectors are
used for high-resolution elemental imaging and identification of samples
in combination with dynamic SIMS. The magnetic sector and several movable detectors allow a simultaneous detection of several elements or small
molecules (within a narrow mass range) with higher sensitivity. Slodzian
et al. (1992) used a SIMS coupled with a magnetic sector double-focusing
mass spectrometer for simultaneous imaging of several elements in tissue
sample.
4.4. Ion Trap Mass Analyzer

A quadrupole ion trap (QIT or 3D-IT) operates in a 3D quadrupole field
maintained at constant DC and RF fields to trap the moving ions of m/z
range. A QIT consists of three hyperbolic-shaped electrodes: the central ring electrode and two adjacent end cap electrodes (entrance and
exit) (see Figure 9d). A 3D-IT is a small, relatively inexpensive instrument for sensitive analysis; it can also be used for MSn analysis of
molecules in the tissue samples (Gross, 2004; Hopfgartner et al., 2004).
Shimma et al. (2008) reported their use of a MALDI-QITTOF-MS instrument for imaging of phospholipids, glycolipids, and tryptic-digested
proteins. MS analyses were performed to confirm their presence. Recently
a mass microscope coupled with a high-resolution atmospheric pressurelaser desorption/ionization (AP-MALDI) and QIT-TOF was used for
imaging and identification of volatile substances in ginger (Harada et al.,
2009). This instrument allows researchers to precisely determine the
164
specific tissue section prior to IMS and has spatial resolution (10 m)
higher than the naked eye.
In a linear quadrupole ion trap (LIT) or 2D traps (2D-IT), the ions are
trapped in a 2D quadrupole field and then pass axially. The 2D-IT ion
trap produces reasonable mass accuracy, mass resolution, and sensitivity
(Schwartz et al., 2002). LIT has a better ion storage capacity and a higher
trapping efficiency compared with 3D-IT. However, the disadvantage of
LIT is the relatively narrow mass range of small molecule analysis. Garrett
et al. (2007) described a new MALDI-LIT-MS for imaging of tissue samples and also used for MSn analyses to confirm the molecules. Enomoto
et al. (2011) demonstrated the visualization of phosphatidylcholine (PC),
lysophosphatidylcholine, and sphingomyelin in mouse tongue using LTQ
(linear trap quadrupole)-MALDI-IMS (Enomoto et al., 2011).
4.5. Orbitrap Mass Analyzer

In an orbitrap mass analyzer, the ions are rotated around a central electrode by applying an appropriate voltage between the outer and central
electrodes. Hence, the ions of a specific m/z ratio cycle in rings that
oscillate around the central spindle and then pass through the detector (Makarov et al., 2006). Figure 9e shows the overview of the orbitrap mass analyzer. LTQ-Orbitrap has been used to analyze compounds
with high resolving power and excellent mass accuracy that appreciably decrease false-positive peptide identifications in the sample (Adachi
et al., 2006; Makarov et al., 2006). Verhaert et al. (2010) demonstrated
the use of LTQ-orbitrap for imaging of neuropeptides in neural tissue
samples. In addition, it has also been used for identification and sequencing of neuoropeptides from neural tissue using MALDI-MS with an ion
traporbitrap hybrid instrument. Landgraf et al. (2009) showed the high
resolution and accurate measurement of ion images of lipids in spinal
cord using MALDI-LITorbitrap-MS. Manicke et al. (2010) demonstrated
imaging of lipids in rat brain tissue section with a high-resolving power
instrument of DESI-LTQorbitrap-MS.
4.6. Ion Cyclotron Resonance Mass Analyzer

In an ion cyclotron resonance (ICR)-MS analyzer, the ions of a particular
m/z ratio are isolated based on the cyclotron frequency of the ions in a
constant magnetic field. The oscillation of ions in ICR induces an alternating current that is equivalent to their m/z ratios. Figure 9f shows the
schematic for an ICR analyzer. Fourier transforms (FT)-ICR-MS continues to deliver the highest mass-resolving power and mass measurement
accuracy (Gross, 2004). The combination of MALDI-TOF-MS with the
FT-ICR-MS technique is useful for highspatial resolution analysis and
165
identification of unknown biomolecules in tissue samples. Thus the high

mass resolution of the FT-ICR-MS can be used to analyze compounds
that cannot be distinguished with lowermass resolution mass spectrometers (Taban et al., 2007; Wang et al., 2011). MALDI-FT-ICR has also been
reported for IMS analysis of drugs and metabolites in tissue. The accurate
mass measurement can be performed using FT-ICR-MS, which provided
a molecular specificity for the ion images obtained from tissue sample
analysis (Cornett et al., 2008).
5. IMS MEASUREMENTS
MALDI-IMS experiments can be performed after the deposition of matrix
on the tissue section and using different types of MS instruments as discussed above. The setting of the laser energy, detector gain, and random
walk function are optimized in order to obtain better signal intensity of the
target molecules during the IMS analysis. Either a particular region of
the tissue or the entire tissue section is selected for analysis, depending on
the particular interest. At present, the commercially available instruments
can perform IMS analyses with the highest spatial resolution of 25 m
(Goto-Inoue et al., 2009a). Recently we developed a mass microscope that
can move a sample stage by 1 m, and the finest size of the laser diameter is approximately 10 m (Harada et al., 2009). The measurement time of
IMS experiments depends on the number data spots, the frequency of the
laser, and the number of shots per spot.
6. DATA ANALYSIS
Due to the large (gigabytes) size of the dataset, high-capacity visualization
software is required to visualize the ion image and distribution pattern of
biomolecules in tissue samples. New computing methods are required for
both rapid, accurate data acquisition and the interpretation of the IMS
analysis results. Therefore, in addition to instrumental improvements,
data acquisition and software development have been important for the
production of reliable data. The databases used are based on algorithms
that perform analysis through statistical evaluation of observed and
theoretical spectra of bimolecules. BioMap (http://www.maldi-msi.org,
Novartis, Basel, Switzerland) and flexImaging (http://www.bdal.com,
Bruker Daltonics GmbH, Bremen, Germany) imaging software are used
to identify biomolecules in various sample types. The intensity of the
different color images obtained by both software packages can relate
the distribution of biomolecules in the tissue section. These software
packages also help in understanding the localization of biomolecules at
166
particular regions of interest (ROIs) for mass spectral comparison and

other statistical analysis.
BioMap imaging software can be used for different instruments such
as PET, nuclear magnetic resonance (NMR), computed tomography (CT),
and near-infrared fluorescence (NIRF) as the result of multiple modifications. Interactive data language Virtual Machine (Research Systems,
Boulder, CO) is required in the system to process the data obtained from
IMS analysis. BioMap software can also be used for baseline correction,
spatial filtering, and averaging of spectra for presentation of the IMS
results.
The flexImaging software is used for the acquisition and evaluation
of MALDI-TOF imaging results. The mass peaks (at m/z) obtained in the
mass spectrum are normalized to total ion current and then the peak
intensity is taken into account to study the molecules distribution on
the tissue section. The imaging MS experiments are performed by collecting spectra at a resolution of 50 to 400 m in the same m/z range as
above. Principal component analysis (PCA) is an unsupervised statistical method used to identify groups of closely correlated variables; for
MS imaging datasets these variables are spatial coordinates and mass.
This approach also reduces the multidimensional datasets to the lower
dimensions (Chou, 1975). PCA analysis is performed using ClinProTools
2.1 software (Bruker Daltonics). Zaima et al. (2009) performed a PCA for
screening of metabolites in the fatty liver. Several differences were found
in identifying the metabolites in fatty and normal liver tissue samples.
PCA was also used in proteomics studies (Deininger et al., 2008; Djidja
et al., 2010; Yao et al., 2008).
7. APPLICATIONS OF IMS FOR DIRECT ANALYSIS OF TISSUE

7.1. IMS for Lipidomics
Lipids are the main constituents of cell membranes; the major functions of
lipids are transportation of ions and signals across the cell membrane. Various types of lipids, such as glycerophospholipids (GPLs), sphingolipids,
sterol lipids, saccharolipids, waxes, and fat-soluble vitamins are found in
biological systems. Membranes act as barriers to separate compartments
within eukaryotic cells and to separate all cells from their surroundings
(Brown, 2007; Fahy et al., 2009; Lee et al., 2003). The identification and
quantification of lipids in tissue sample can help in understanding several
biosynthetic and metabolic pathways that govern human diseases, such
as insulin-resistant diabetes, Alzheimers disease, schizophrenia, cancer,
atherosclerosis, and infectious diseases (Oresic et al., 2008). Thus the analysis of lipids in tissue samples is a very important issue. High-performance
liquid chromatography (HPLC) (McCluer et al., 1986), TLC (Touchstone,
167
1995), and MS have been used to analyze lipids in tissue samples. However, the sample preparation procedures in chromatographic techniques
are lengthy and the localization of biomolecules in tissue sample surface
cannot be established. Therefore, different IMS systems are successfully
used for imaging of lipids. The analysis of glycerophospholipids, sphingolipids, and neutral lipids is discussed in detail in the following
sections.
7.1.1. Glycerophospholipids
GPLs are glycerol-based phospholipids and essential components of cell
membranes. They act as second messengers involved in cell proliferation, apoptosis, and metabolism. The determination of GPL content in
tissue samples is useful for finding potential biomarkers for diseases such
as atherosclerosis or rheumatism (Fuchs et al., 2005; Schmitz and Ruebsaamen, 2010). Altered levels of lipids are found in many pathological
conditions such as Alzheimers disease (Han et al., 2001, 2002), Down syndrome (Murphy et al., 2000), diabetes (Han et al., 2007), and NiemannPick
disease (He et al., 2002). Figure 10 illustrates the basic structures of common classes of GPLs such as PC, phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) (Jackson and Woods,
2009). PC is easily ionized due to its charged quaternary ammonium head
group and has thus become a popular lipid species to investigate (Pulfer
and Murphy, 2003). The ionized molecules observed in the mass spectrum
are usually either protonated [M+H]+ , sodiated [M+Na]+ , or pottasiated
[M+K]+ in positive-ion mode. Phospholipids such as PE, PS, PA, PG, and
PI may generate negative ions due to the presence of the phosphodiester
moiety and display molecular anions [M-H] (Fuchs et al., 2010). The
addition of potassium acetate (Sugiura et al., 2009) or LiCl (Jackson et al.,
O
P
O O
O
+
N
O
O
H O
R1
H 2N
R2
HO
OH
O
OH P
O
O
OH
O
O
H O
R1
R2
O
H2N H
P
HO
O
O
OH
C
O
R2
O
O
H O
O
Phosphatidylinositol
R1
O
H O
O
Phosphatidylethanolamine
O
Phosphatidylcholine
HO
HO
O
P
O
O
OH
Phosphatidylserine
R1
R2
FIGURE 10 Structure of glycerophospholipids. Reprinted from Jackson and Woods

(2009) with permission from Elsevier Science.
168
2005) to the matrix solution has also been reported for effective ionization
of molecules in tissue samples.
The selection of MALDI matrices is an important issue. For MALDIIMS, the matrix should have good vacuum stability and homogenous
crystal formation containing analyte molecules. Various matrices have
been reported for the identification and characterization of lipids in
MALDI-MS, including DHB (Petkovic et al., 2001; Puolitaival et al., 2008;
Schiller et al., 1999), DHA (Jackson et al., 2005; Shimma et al., 2007),
p-nitroaniline (PNA) (Estrada and Yappert, 2004; Rujoi et al., 2004), and
9-aminoacridine (Eibisch and Schiller, 2011; Teuber et al., 2010). However,
PNA and dihydroxyacetone phosphate (DHAP) were unstable under high
vacuum conditions and started to evaporate after their introduction into
the MALDI-MS instrument (Jackson et al., 2005; Rujoi et al., 2004; Shrivas
et al., 2010). DHB matrix exhibited a lower sensitivity for the detection
of PA, PS, PE, PI, and PG in negative-ion mode, possibly due to its acidity (Estrada and Yappert, 2004; Petkovic et al., 2001). DHA can be used
in both positive and negative ionization modes for analysis of phospholipids (Woods et al., 2006). PNA is another good matrix for the analysis of
phospholipids in either positive-ion or negative-ion modes (Estrada and
Yappert, 2004). Recently, 2-mercaptobenzothiazole (MCT) was added as
an alternative to the use of DHB for MALDI-MS analysis of phospholipids in brain and liver tissue samples (Astigarraga et al., 2008). The main
advantages of MCT over the commonly used matrices DHB, DHA. and
PNA are low vapor pressure, low acidity, and homogenous crystal formation, which allowed for detection of more lipid species in negative mode,
with high sensitivity and high detection reproducibility. Ionic matrices
have also been used in MALDI-IMS owing to the good vacuum stability,
homogenous crystal formation, and good solubility of analytes for efficient ionization and desorption of molecules (Chana et al., 2009; Lemaire
et al., 2006a; Shrivas et al., 2010). Shrivas et al. (2010) used an ionic matrix
of CHCAB to image and identify lipids in mouse cerebellum and found
that this ionic matrix yields a higher number of ion images compared
with the use of DHB matrix in MALDI-IMS (Figure 11). Use of NPs is
another good approach for selective and sensitive analysis of lipids and
small metabolites in tissue samples without matrix-oriented peaks in the
low-molecular-mass range (Cha and Yeung, 2007; Goto-Inoue et al., 2010a;
Hayasaka et al., 2010; Shrivas et al., 2011; Taira et al., 2008).
Sugiura et al. (2009) showed the imaging of polyunsaturated fatty acid
containing PC in mouse brain using MALDI-IMS. The results demonstrated that arachidonic acid (AA) and DHA-containing PC were found
in the hippocampal neurons and cerebellar Purkinje cells, respectively.
Figure 12 shows the localization of PC species in different layers of the
mouse brain (Sugiura et al., 2009). The distribution of PC species also has
been reported in the mouse retinal section via MALDI-IMS analysis. The
localization of PC (16:0/18:1) was found in the inner nuclear layer and
169
GL
ML
WM
(a)
ND
m/z 734.5
m/z 772.5
m/z 826.5
m/z 846.5
m/z 734.5
m/z 772.5
m/z 826.5
ND
m/z 756.5
m/z 798.5
m/z 832.5
m/z 850.6
m/z 756.5
m/z 798.5
m/z 832.5
ND
m/z 760.5
m/z 810.5
m/z 834.5
m/z 864.6
m/z 760.5
m/z 810.5
m/z 834.5
ND
m/z 769.5
m/z 822.6
m/z 835.6
m/z 770.5
m/z 824.5
m/z 844.5
(b)
m/z 870.5
m/z 769.5
m/z 822.6
m/z 835.6
m/z 770.5
m/z 824.6
m/z 844.5
m/z 846.5
ND
m/z 850.6
ND
m/z 864.6
ND
m/z 870.5
(c)
FIGURE 11 (a) H&E-stained mouse cerebellum showing three layers with 1.5-mm scale
bar (white color). The ion images of lipids in mouse cerebellum tissue section obtained
by using (b) CHCAB and (c) DHB as a matrix at m/z 734.5 [(PC(16:0/16:0)+H)]+ , 770.5
[PC(16:0/16:1)+K]+ , 772.5 [PC(16:0/16:0)+K]+ , 798.5 [PC(16:0/18:1)+K]+ , 834.5
[PC(18:0/22:6)+H]+ , and 870.5 [PC(18:1/22:6)+K]+ were localized in the molecular layer
of cerebellum; at m/z 760.5 [PC(16:0/18:1)+H]+ , 832.5 [PC(18:0/20:4)+Na]+ , 844.5
[PC(16:0/22:6)+K]+ , and 846.5 [PC(18:1/20:4)+K]+ were specific to the granular layer;
and at m/z 756.50 [PC(16:0/16:0)+Na]+ , 810.5 [PC(18:0/18:1)+Na]+ , 824.5
[PC(18:0/18:2)+K]+ , and 826.5 [PC(18:0/18:1)+K]+ and were found to be concentrated in
the white matter of cerebellum. The ion images at m/z 769.5 [SM(d18:1/18:0)+K]+ and
835.6 [SM(d18:1/24:1)+Na]+ illustrated that the molecules were distributed in the region
of molecular layer of tissue. The ion images at m/z822.6 [GalCer(d18:1/22:0)+K]+ and
850.6 GalCer(d18:1/24:0)+K]+ were localized in the white matter of mouse cerebellum.
ND indicates the molecules were not detected. GL, granular layer; ML, molecular layer;
WM, white matter. Reprinted from Shrivas et al. (2010) with permission from American
Chemical Society.
the outer plexiform layer; PC (16:0/16:0) in the outer nuclear layer and
inner segment; and PC (16:0/22:6) and PC (18:0/22:6) in the outer segment
and pigment epithelium (Hayasaka et al., 2008). Differential localization
of PC (16:0/20:4) species was observed between terminal and stem villi of
170
16:0
PC (diacyl-16:0/16:0)
16:0
CBX
CTX
HPF
TH
m/z 772.4
18:0
PC (diacyl-18:0/18:1)
Structure of PCs
CH3
O
N+ CH3
H2C O P O
CH3
O
HC O R2 (sn2)
18:1 (OA)
PC (diacyl-16:0/18:1)
H2C O
m/z 826.5
m/z 798.4
PC (diacyl-16:0/20:4)
Cp
20:4 (AA)
PC (diacyl-18:0/20:4)
R1 (sn1)
18:1 (OA)
PC (diacyl-18:1/20:4)
m/z 848.5
m/z 820.5
PC (diacyl-16:0/22:6)
PC (diacyl-168:0/22:6)
m/z 846.5
22:6 (DHA)
PC (diacyl-18:1/22:6)
m/z 844.5
0%
m/z 872.6
m/z 870.6
100%
FIGURE 12 Identification of molecular species of PC in sagittal mouse brain sections

by MALDI-IMS. Among the PC, AA-PC showed characteristic localization in the
hippocampal cell layers (arrowheads). Among DHA-containing species, two abundant
species, PC (diacyl-16:0/22:6) and PC (diacyl-18:1/22:6), were commonly enriched in the
granule layer of the cerebellum, while PC (diacyl-18:0/22:6) showed a characteristic
dotted distribution pattern near the cell layer (arrows). CBX, cerebellar cortex;
CP, corpus striatum; CTX, cerebral cortex; HPF, hippocampal formation; TH, thalamus.
Reprinted from Sugiura et al. (2009) with permission from the American Society
for Biochemistry and Molecular Biology.
human placenta, which could be helpful in understanding the pathological involvement of fetal growth restriction and fetal hypoxia (Kobayashi
et al., 2010). The accumulation of lipid molecules, such as LPC (1-acyl 16:0),
PC (1-acyl 36:4), and shingomyelin (SM) (d18:1/16:0) around the damaged
valvular region was investigated and suggested an association of these
molecules with tissue inflammation and resultant valvular incompetence
(Tanaka et al., 2010). PC (diacyl-16:0/20:4) containing an AA was found
at higher concentration in prefrontal cortex tissue compared with occipital cortex tissue in the brains of patients with schizophrenia (Matsumoto
et al., 2011). The specific localization of five PC species in the cochlea
was also examined using the mass microscope. A differential distribution of PC species was observed; (16:0/18:1) in the organ of Corti and the
stria vascularis, (16:0/18:2) in the spiral ligament, and (16:0/16:1) in the
organ of Corti (Takizawa et al., 2010). Recently Goto-Inoue et al. (2009a)
171
investigated use of a TLC-Blot-MALDI-IMS for analysis and characterization of acidic, neutral glycosphingolipids and PC in sample mixtures.
In TLC-Blot, the lipids are separated and transferred to a polyvinylidene
fluoride (PVDF) membrane without any change in the stability of the
molecules. PVDF membranes with the sample may then be placed on the
target plate for MALDI-IMS analysis. This method might be useful for
the detection of minor components that could not be detected by the conventional TLC method. SIMS is also used for imaging of lipids at high
spatial resolution and sensitive detection. The combined approaches of
MALDI-IMS and SIMS-IMS have been used for imaging of PC in cultured
mammalian neuron. The data obtained from MALDI and SIMS supported
that the signals of small molecules in the low molecular region, such as
PC head groups and fatty acids (detected in SIMS) were obtained from
the intact lipids (Yang et al., 2010). DESI-IMS has been used for imaging of
most commonly encountered brain lipid species such as PE and PI in rat
spinal cord cross sections in negative-ion mode. The ion image of PI (38:4)
was shown in grey matter regions such as the cortex and hippocampus.
The ion image of PE (at m/z 888) showed the white specific region in the
brain (Dill et al., 2009).
7.1.2. Sphingolipids
The sphingolipid is a type of lipid obtained from the aliphatic amino alcohol sphingosine. The main functions of sphingolipids are transmission
and cell recognition. The investigation of sphingolipids is very important because they are indicative of aging and may function as a disease
marker. Sphingolipids contain a sphingoid base backbone and include
sphingomyelins (SM), sulfatides (ST), ceramides, cerebrosides, and gangliosides (Merrill et al., 2009) (Figure 13). Changes in the levels of lipids,
HO
OH
HO
HO
C13H27
H HN
HO
OH
O
+
N
O
O P
O O
C13H27
H HN
O
Ceramide
HO H
HO
H
C13H27
H HN
OH
OH O S
O O
HO
OH
O
HO H
C13H27
H HN
O
Sphingomyelin
R
O
Cerebroside
Sulfatide
R
O
FIGURE 13 Structure of sphingolipids. Reprinted from Jackson and Woods (2009) with
permission from Elsevier Science.
m/z 725.5
Relative intensity
Normal
Cancer
616.1
725.5
Relative intensity
172
Normal
Cancer
m/z 616.1
Merged image
Green: m/z 616.1

Red: m/z 725.5
725 727 729
(m/z)
616 618 620

(m/z)
(a)
(b)
(c)
FIGURE 14 Imaging of normal and cancerous cells in human liver sample. (a) The most
prominent signal at m/z 725 showed the higher expression for cancerous cells than
normal cells. (b) The signal at m/z 616 showed the higher distribution of this molecule
in normal cells. (c) Merged images at m/z 725 and 616. Reprinted from Shimma et al.
(2007) with permission from Elsevier Science.
in particular ceramide, also have been observed in apoptosis or cell death

(Fuchs et al., 2007; Thomas et al., 1999). MALDI-MS/MS analyses were
used to image liver tissue samples at a thickness 3 m from a patient with
colon cancer. A higher expression of sphingomyelin (SM, 16:0) at m/z 725
was observed in cancerous tissue than in normal tissue by MS/MS analyses (Figure 14). In contrast, a strong distribution of an ion at m/z 616 was
observed in the normal but not cancerous tissue sample (Shimma et al.,
2007). IMS has also been used to detect seminolipid, a glycolipid synthesized in sperm. Here, seminolipid localization was performed in mice
testis during testicular maturation Goto-Inoue et al. (2009b). In another
study, the distribution pattern of ganglioside molecular species (C-18
and C-20) in mouse hippocampus was demonstrated using MALDI-IMS.
The localization of C-18 species was found in the frontal brain and C-20
species contained in the entorhinal-hippocampus projections of the molecular layer (ML) of the dentate gyrus. Figure 15 shows the distribution
of C-20-sphingosinecontaining gangliosides in the hippocampal formation (Sugiura et al., 2008). In a study using gold NPs in Nano-PALDI-IMS
and comparing it with the use of DHB matrix, the PI, ST, and ganglioside species (GM3, GM2, GM1, GD1, and GD3) were all detected with
higher sensitivity. This is the first report of the visualization of minor
sphingolipids by IMS analyses using gold NPs in tissue sections (GotoInoue et al., 2010). Higher expression of sulphatides in ovarian cancer cells
was reported compared with a normal sample with MALDI-IMS analysis and similar results were obtained by a transcriptomic approach of
lipid analysis (Liu et al., 2010). The high spatial resolution localization
of glucosylceramide in spleen of a mouse model of Gaucher disease was
HPF
(a)
(b) m/z 878.6
(c) m/z 906.6
CTX
MB
TH
Mad/P
Cp
ST (22:0 OH)
(e) m/z 1902
(d) m/z 1874
[GD1(18:0/d18:1) + K - 2H]
(f) Merged
[GD1(18:0/d20:1) + K - 2H]
(g) m/z 1858
(h) m/z 1886
(i) Merged
[GD1(18:0/d18:1) + Na - 2H]
[GD1(18:0/d20:1) + Na - 2H]
(j) m/z 1544
(k) m/z 1572
(l) Merged
GM1
GD1
ST (24:0 OH)
C20
C18
[GM1(18:0/d18:1) - H]
[GM1(18:0/d20:1) - H]
(A)
(a)
(b) m/z 878.6
(c) m/z 906.6
SO
SR
SLM
ML
CA1
ML
DG
ST (22:0 OH)
CA3
C18
[GD1(18:0/d18:1) + K - 2H]
(e) m/z 1902
(g) m/z 1858
(h) m/z 1886
[GD1(18:0/d18:1) + Na - 2H]
[GD1(18:0/d20:1) + Na - 2H]
(j) m/z 1544
(k) m/z 1572
[GM1(18:0/d18:1) - H]
[GM1(18:0/d20:1) - H]
(B)
FIGURE 15
(Continued)
(f) Merged
[GD1(18:0/d20:1) + K - 2H]
GM1
GD1
(d) m/z 1874
ST (24:0 OH)
C20
(i) Merged
(l) Merged
173
174
FIGURE 15 Localization of C20-sphingosinecontaining gangliosides in the

hippocampal formation. IMS at 50-mm raster step size was used to gain an overview of
ganglioside distribution in different brain regions (A), and IMS at 15-mm raster size was
used to study in detail the distribution pattern of gangliosides in the hippocampus (B). In
both panels, schematic diagram of the brain section (a) and ion images of STs (b)(c) are
presented. For ions corresponding to the GD1 molecular species, we observed the ion
distributions of both sodium and potassium complexes; that is, the ions at m/z 1858 (f)
and m/z 1886 (g), which correspond to the [M+Na-H] form of C18- and C20-GD1, and
those at m/z 1874 (h) and m/z 1902 (i), which correspond to the [M+K-H] form of C18and C20-GD1, respectively. The ion distribution patterns corresponding to the GD1-Na
salts and GD1-K salts are fairly uniform for both C18- and C20-species. For GM1, m/z 1544
(d) and m/z 1572 (e), which correspond to C18- and C20-sphingosinescontaining GM1,
respectively, are shown. HPF, hippocampus formation; MadP, -; CTX,
cerebral cortex; MB, -; TH, Thalamus; SO,-; SR, stratum
radiatum; SLM, stratum lacunosum molecular; ML, molecular layer. Reprinted from
Sugiura et al. (2008) with permission from Public Library of Science.
also demonstrated using MALDI-IMS (Snel and Fuller, 2010). Ganglioside

GM2, asialo-GM2 (GA2), and sulfatides in brain from a mouse model of
Tay-Sachs/Sandhoff disease (Chen et al., 2008) and sulfatides in mouse
kidney (Marsching et al., 2011) have been reported.
7.1.3. Nonpolar Lipids

Imaging and identification of nonpolar lipid in tissue sections is not
easy, perhaps because of the difficulty in the ionization of molecules in
MALDI-MS. Thus only a few species of nonpolar lipids have been successfully reported. One example is cholesterol, a highly abundant lipid
in many tissues. It is usually detected at m/z 369 after the loss of a
water molecule using an organic matrix in MALDI-MS. Cholesterol is
a vital constituent of the cell membrane, required for lipid organization and cell signaling. Changes in the quantity of cholesterol in tissue
can cause myocardial infarctions and stroke, as well as other disorders
(Fernandez et al., 2011). SIMS has been used for imaging of cholesterol
with the capability to analyze single cells. In this setup one drawback was
that cholesterol was fragmenting (Piehowski et al., 2008). However, the
use of NIMS could directly analyze the brain cholesterol metabolites in
Smith-Lemli-Opitz syndrome without the fragmentation of molecules in
MS (Patti et al., 2010). The distribution of triglycerides (TAG) in mouse
embryo was also investigated using MALDI-IMS (Hayasaka et al., 2009).
TAG is an ester derived from glycerol and three fatty acids and is the main
constituent of vegetable oil and animal fats. Figure 16 is illustrates the distribution of different molecular species of TAG [(16:0/18:2/18:1)+Na]+ ,
[(16:0/18:1/18:1)+Na]+ , [(16:0/20:3/18:1)+K]+ in mouse embryo. The
ion images of TAG were concentrated mainly around the brown adipose
and liver tissue (Hayasaka et al., 2009).
175
Lung
Brown adipose
tissue
Liver
Brian
Intestine
Hypoglottis
Heart
Thymus
(a)
(TAG (16:0/18:2/18:1) + Na)+
100%
(TAG (16:0/18:1/18:1) + Na)+

100%
0%
0%
(b)
(c)
(TAG (16:0/20:3/18:1) + K)+
Merged
100%
100%
0%
0%
(d)
(e)
FIGURE 16 MALDI-IMS of neutral lipids. Distribution of triglycerides (TAG) in

mouse embryo. (a) H&E staining, (b) ion images of [TAG (16:0/18:2/18:1)+Na]+ ,
[TAG 16:0/18:1/18:1)+Na]+ , and [TAG (16:0/20:3/18:1)+K]+ are shown. (b)(d) The ion
images were merged with the optical image of an H&E-stained section. (e) Three merged
TAG images demonstrate the same distribution. Reprinted from Hayasaka et al. (2009)
with permission from Springer.
7.2. IMS for Proteomics

The study of proteomics is useful for biomarker discovery of a large number of diseases, using tissue samples such as vascular tissue, heart, brain,
lung or bone, with a current major focus on cancer and malignant tissues
(MacAleese et al., 2009). Today MALDI-IMS is increasingly being used
for direct analysis of peptides and proteins from tissue sections; the main
advantage is that it requires no labeling reagents (McDonnell and Heeren,
2007; Stoeckli et al., 2001). Immunohistochemistry (IHC) has long been the
standard technology for imaging of peptide and protein distribution in
tissue. The sensitivity of IHC is usually excellent. However, this method
requires a specific binder, usually an antibody, to detect a previously
defined protein from the sample. Only a very small number of molecules
may be detected in parallel, and these all need to be known beforehand.
176
Antibody availability and specificity are other constraints with IHC that
limit its capacity (Luongo de Matos et al., 2010). Thus the introduction of
IMS has provided complementary resources.
What IMS lacks in terms of sensitivity, it compensates for by enabling
untargeted studies with the possibility to detect hundreds of molecules
in a single run. In the so-called bottom-up approach, the proteins present
in the tissue sample must first be subjected to in situ digestion by a proteolytic enzyme. Usually trypsin is used since it renders peptides that
contain at least one lysine or arginine amino acid and hence are easily
ionized. Setou et al. (2008) investigated whether the addition of detergent into the trypsin solution could improve the digestion efficiency of
proteins for direct analysis of tissue section in MALDI-.IMS. Trypsin solution can be spotted directly onto the tissue sections, rendering spot sizes
150200 m. Considering the possibility for peptide migration within
this spot area, the tryptic spot size can also be said to determine the
resolution of the IMS experiment, although the spatial resolution from
the actual data acquisition is determined by the instrument used and is
usually lower. Organic matrices such as DHB and CHCA are used for
ionization.
For biomarker studies, the tissues available through biobanks around
the world have generally been treated with formalin for increased tissue
stability over time. Formalin fixation and subsequent paraffin embedding
allows for stable histomorphology, but it also causes difficulty in IMS since
it cross-links proteins and hampers protein mining. This problem has been
overcome by deparaffinization methods followed by the same antigenretrieval methods used in IHC experiments (enzymatic or heat-mediated)
(Aoki et al., 2007). Recently formalin-fixed, paraffin-embedded tissue
microarrays were analyzed in MALDI-IMS and MS/MS experiments to
study the gastric carcinoma tissue, thereby identifying the histone (H4)specific signal in poorly differentiated cancer tissue samples (Morita et al.,
2010). Other groups have demonstrated the direct analysis and identification of tryptically digested proteins from tissue samples of lung cancer
(Groseclose et al., 2008), breast cancer (Ronci et al., 2008), prostate cancer
(Cazares et al., 2009), and pancreatic adenocarcinoma (Djidja et al., 2009).
Chaurand et al. (2004) showed the level of the binding protein (S100B)
in tissue samples using MALDI imaging to distinguish a high-grade and
low-grade glioma. In addition, the combined approach of MALDI-IMS
and MS/MS analyses of digested myelin basic protein (MBP) in a coronal section of rat brain has been demonstrated (Figure 17ac) (Groseclose
et al., 2007). After digestion, a total of eight tryptic peptides from MBP
were detected (Figure 17d). This protein is essential for the formation of
myelin in the central nervous system. MALDI-IMS also has been used to
classify a pancreatic cancer tissue microarray where a number of proteins
that appear to discriminate between different tumor classes were detected
(Djidja et al., 2009). Direct proteomic-based imaging was also performed
177
High
Low
(a)
(b)
(c)
High
m/z 699.47
(NIVTPR)
Low
m/z 1131.84
(TTHYGSLPQK)
m/z 726.46
(HGFLPR)
m/z 1067.60
m/z 1909.92
(FSWGGRDSR) (FSWGGRDSRSGSPMARR)
m/z 1336.69
m/z 1460.90
(YLATASTMDHAR) (TQDENPVVHFFK)
m/z 1502.98
(TTHYGSLPQKSQR)
(d)
FIGURE 17 (a) H&E stain of rat brain tissue section serial to the sections used for
digestion and imaging. (b) Tissue section spotted with a sinapinic acid matrix solution.
(c) Image of the 14.2-kDa isoform of myelin basic protein. (d) Images of 8-tryptic
peptides generated from the digestion of the 14.2-kDa isoform of myelin basic protein.
Reprinted from Groseclose et al. (2007) with permission from John Wiley and Sons.
on a gene knockout mice tissue section of rat that could be useful for the
diagnosis of human diseases (Yao et al., 2008). Figure 18 shows the PCA
of mass spectra from Scrapper-knockout (SCR-KO) and WT mouse brains
analyzed by MALDI-IMS.
7.3. IMS for Pharmacokinetic Studies

Imaging of pharmaceuticals samples is performed to examine pharmacokineticsthat is, the absorption, distribution, metabolism, and excretion of drugs in laboratory animals and humans. HPLC combined with
MS/MS is used to analyze and characterizze most drugs. However,
HPLC-MS/MS analyses cannot provide the distribution of drugs in different organs or tissues of laboratory animal experiments (Hsieh et al., 2003).
Whole-body autoradiography (WBA) is normally used for the visualization of drug candidates in all tissues; however, it requires the compound
of interest to be radioactively labeled (Kertesz et al., 2008). This disadvantage of WBA can be overcome by using MALDI-IMS to analyze the
drugs in tissue samples. The drug distribution profile obtained by IMS
tells whether the oral administration of an exogenous compound affects
the endogenous metabolites (Rubakhin et al., 2005). Reyzer et al. (2003)
reported images of two antitumor drugs in mouse tissue samples using
178
Relative intensity
WT
SCR-KO
Cerebral corte
WT
Cerebral corte
SCR-KO
Hypothalamus
WT
Cerebral cortex hypothalamus

Corpus striatum pons/medullary
m/z 2500
5000
(a)
7500
(b)
Pons/medullary
Corpus striatum
0.3
0.4
15
Load 1
1
PC 1
15
m/z 7109
0.8
10
WT
SCR-KO
m/z 7420
Hypothalamus
SCR-KO
10000
1Load 1
PC 1
20
0.6
10
m/z 7420
Load 2
0.8
12 PC 2
100%
Load 2
0.6
15 PC 2
m/z 7109
0%
100%
0%
SCR-KO
WT
Cerebral cortex
Hypothalamus
0.8
10
0.8
10
m/z 5004
PC 1
15
25
Load 1
0.8
PC 2
20
m/z 5004
0.8
15
PC 1
Load 2
0.8
100%
100.8
m/z 4285
m/z 4285
0.4
Load 2
0.6
PC 2
0%
Load 1
100%
0%
(c)
FIGURE 18 In situ proteomics of the SCR-KO mouse brain using IMS and PCA.
(a) H&E-stained images of the WT and SCR-KO mouse brain. The regions focused in
IMS analyses are indicated by colors. (b) Mass spectra obtained from each region of the
WT or SCR-KO mouse brain sections. Specific signals of the regions are indicated by
arrowheads. (c) Distributions of principal component scores of mass spectra from
various brain regions (left spray graphs; WT, blue; KO, red) and the loading factors
plot (right graphs). The signal intensities of mass spectra of the substances with indicated
m/z are shown in the reconstructed images of the mouse brain analyzed by IMS.
Reprinted from Yao et al. (2008) with permission from John Wiley and Sons.
179
MALDI-IMS. The results showed the spatial distributions of drugs in

brain tissue section were elucidated using a Q-TOF instrument operated in
selective reaction monitoring (SRM) mode to provide good sensitivity for
tissue analysis. This work demonstrated the proof of MALDI-IMS in monitoring a drug distribution in different parts of body organs. MALDI-IMS
can provide the spatial information for both drugs and their metabolites. Figure 19ad shows the distributions of the drug olanzapine and its
metabolites (N-desmethyl metabolite and 2-hydroxymethyl) in tissue after
post dosing of 2 hours and 6 hours (Khatib-Shahidi et al., 2006). Further,
(b)
(c)
(d)
Kidney
Fur
(a)
Testis
Bladder
Spleen
Liver Lung
Thoracic
cavitv
Spinal cord Brain
Thymus
100%
100%
100%
FIGURE 19 Detection of drug and metabolite distribution at 2 hours after dosing in a

whole rat sagittal tissue section using IMS analysis. (a) Optical image of a 2-hr post
olanzapine-dosed rat tissue section across four gold MALDI target plates Organs are
outlined in red. A pink dot used as a time point label. (b) MS/MS ion image of
olanzapine (m/z 256). (c) MS/MS ion image of N-desmethyl metabolite (m/z 256).
(d) MS/MS ion image of 2-hydroxymethyl metabolite (m/z 272). Scale bar, 1 cm.
Reprinted from Khatib-Shahidi et al. (2006) with permission from American Chemical
Society.
180
SIMS and NIMS were used for imaging of drugs in tissue samples and the
mass spectrum obtained was free of the matrix-oriented peaks. The direct
analysis of clozapine and its metabolites in dosed rat brains has been illustrated using TOF/TOF mass analyzers (Yanes et al., 2009). NIMS-IMS is
compatible with both ion beam and laser sources available on commercial
SIMS and MALDI instruments. In addition, fewer laser shots are required
per spot compared with the MALDI technique.
7.4. IMS for Metabolomics

Metabolomics is the study of metabolites, including metabolic intermediates such as lipids, amino acids, organic acids, and small signaling
molecules. Concentration changes of metabolites in tissue samples might
reflect a specific physiological or pathological condition of the organism
(Dunn, 2008; Nicholson and Lindon, 2008). Liquid chromatography
mass spectrometry and gas chromatographymass spectrometry are wellknown techniques for metabolite analysis (Griffiths and Wang, 2009;
Novotny et al., 2008). Here, the tissue samples are homogenized before
analysis and thus it is impossible to assess their actual tissue distribution. However, IMS can be directly used to profile a broad range
of small molecules, including nucleotides, amino acids, proteins, lipids,
and carboxylic acids, in tissue samples with their unique distributions.
MALDI-IMS has been used for imaging and identification of 13 primary metabolites, such as adenosine monophosphate (AMP), adenosine
diphosphate (ADP), adenosine triphosphate (ATP), uridine diphosphate
(UDP), or N-acetyl-D-glucosamine (GlcNAc) in rat brain sections (Benabdellah et al., 2009). The distribution pattern of lipids such as cholesterol,
cholesterol sulfate, vitamin E, and glycosphingolipids in skin and kidney
sections of patients with Fabry disease using the combined approaches of
MALDI-TOF and cluster-TOF-SIMS was demonstrated by Touboul et al.
(2007). The MALDI-based imaging technique was also used to visualize
energy metabolism in the mouse hippocampus via imaging of energyrelated metabolites. Cellular metabolic processes use ATP as an energy
source and converting it into ADP or AMP. Thus the imaging of these
molecules in tissue samples can provide useful information about energy
production and how it can be used in the function of tissue (Sugiura et al.,
2011). The phenomenon of energy metabolism is shown in Figure 20.
Metabolomics studies of plants have also been performed to elucidate
the structure, function, and biosynthetic pathways (Lisec et al., 2006). Carbohydrates, amino acids, vitamins, hormones, flavonoids, phenolics, and
glucosinolates are the main metabolites found in plants and are needed
for growth, stress adaptation, and defense (Hounsome et al., 2008). In
combination with soft ionization methods such as ESI and MALDI, MS
proved useful for direct analysis of plant tissue sections. The spatial
distribution of sugars, metabolites, and lipids in plant tissue samples was
investigated using MALDI-IMS. Cha et al. (2009) exploited the use of
ADP
AMP
Seizure
(30 min)
Control
ATP
nmol/g tissue
(a)
ATP
120
ADP
**
400
80
**
40
200
AMP
2000
Sham
Seizure (30 min)
1000
0
**
**
**
Increase
ADP
AMP
0
1
*
*
2
Decrease
Decrease
Increase
Increase
ATP
Decrease
Log ((int.(sham/KA))
(b)
Entire region
CA1 cell layer
DG cell layer
CA3 cell layer
(c)
Energy charge
High
CA1
CA3
Control
Seizure
Low
(d)
ATP
Succinyl AMP
IMP
AMP
ADP
Inosine
Adenine
Adenosine
(e)
FIGURE 20 (Continued)
Hypoxanthine
182
FIGURE 20 CA3 cell-selective consumption of adenosine triphosphate (ATP) and

adenosine diphosphate (ADP) during a kainate-induced seizure. (a) MALDI imaging of
adenosine nucleotides in a mouse hippocampus. (b) Absolute quantification of ATP, ADP,
and adenosine monophosphate (AMP) in a mouse cerebrum using CE-MS. Massive
reductions in the levels of ATP and ADP, but not AMP, were observed during
kainate-induced seizures. (c) Results of the relative quantification of ion intensity for
ATP, ADP, and AMP calculated from the averaged mass spectra of each hippocampal
subregion obtained using MALDI imaging. The values shown are logarithmic ratios of ion
intensities between sham-operated (sham) and kainate-treated mice (KA). (d) Mapping of
energy-charge index values on tissue sections. The region-specific reduction of these
values in the CA3 region (arrows) suggests massive energy metabolism in CA3 neurons.
(e) Relative quantitative comparison of adenosine nucleotides and related metabolites
using CE-MS. Each result is mapped on the metabolic pathway and clearly shows the
depletion of ATP and ADP due tp their conversion into downstream metabolites. The
colored graphs indicate significant increases (orange) and decreases (blue). IMP, inosine
50 -monophosphate. Reprinted from Sugiura et al. (2011) with permission from Public
Library of Science.
colloidal silver NPs for direct profiling of an epicuticular wax on leaves

and flowers from Arabidopsis thaliana in LDI-IMS. Recently, Goto-Inoue
et al. (2010b) illustrated the spatial distribution of gamma-aminobutyric
acid (GABA) in the seed of eggplant and the presence of GABA was
confirmed by MS/MS analysis. The localization of GABA in eggplant is
shown in Figure 21. Zhang et al. (2007) showed imaging and identification
of fatty acids, sugars, and other small metabolites using colloidal graphite
NPs in GALDI-IMS, which was free from matrix background noise in the
low molecular region. The distribution of lysophosphatidylcholine and
PC in rice endosperm and bran and alpha-tocopherol in the germ has also
been reported (Zaima et al., 2010).
8. SUMMARY
Several advances in sample preparation, ionization, and MS instrumentation have been achieved, steadily improving sensitivity, spatial resolution,
and identification capabilities for MALDI-IMS. These improvements are
broadening the MS imaging applications for lipid, peptide, and protein
biomarker identification, as well as drug and metabolite imaging. NanoPALDI, the use of ionic matrices, and the mass sicroscope techniques
are new developments that could be powerful tools in obtaining highresolution images for biomolecular distribution in biological samples. In
the future, MALDI-IMS has the potential to become a routine tool for
imaging of tissues, helping us to understand the link between the localization of certain molecules and their function during pathogenesis, disease
progression, or treatment.
183
5 cm
3 mm
(a)
Optical image of
eggplant section
m/z 104.07
2.5 mm
100%
Optical image of
eggplant section
0%
High-power field of
red rectangle area
m/z 104.07
0.5 mm
0%
(b)
(c)
m/z 104.0 on tissue
104.0
58.1
100
30 40 50 60 70 80 90 100 110 120 130 140

(m/z)
(d)
Relative intensity (%)
Relative intensity (%)
GABA (standard)
100
100%
104.0
100
100
58.0
0
30 40 50 60 70 80 90 100 110 120 130 140

(m/z)
(e)
FIGURE 21 Optical images of eggplant, the results of IMS and tandem mass analyses.
(a) Optical images of eggplant, vertically cut eggplant, and round-cut eggplant. A grey
rectangle in a round-cut image shows the region of analyses by IMS. (b) Optical image of
eggplant section and ion image of the m/z values at 104.07. The red arrows in the optical
image show seed locations. Scale bar: 2.5 mm. Reproducibility was confirmed (n = 3).
(c) Optical image of eggplant section and ion image of the m/z values at 104.07 with
higher spatial resolution at 25 m on a seed. Scale bar: 0.5 mm. (d) The tandem mass
spectrum of standard gamma-aminobutyric acid (GABA) and (e) m/z 104.0 on eggplant
tissue. Reprinted from Goto-Inoue et al. (2010b) with permission from The Japan Society
for Analytical Chemistry.
ACKNOWLEDGMENTS
We thank the Japanese Society for the Promotion of Science, Japan,
for a postdoctoral fellowship (to K.S.). This work was also supported
by a grant-in-aid for SENTAN from the Japan Science and Technology
184
Agency (to M.S.). Cecilia Eriksson (Medical Mass Spectrometry, Uppsala

University) is acknowledged for assistance in developing this chapter.
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Chapter

Higashi-Ku, Hamamatsu, Shizuoka 431-3192, Japan

Kamlesh Shrivas and Mitsutoshi Setou

7.3. IMS for Pharmacokinetic Studies

Imaging Mass Spectrometry

In the beginning, mass spectrometric analysis was limited to samples

2. IONIZATION METHODS FOR IMAGING MASS

Kamlesh Shrivas and Mitsutoshi Setou

FIGURE 1 Desorption-ionization techniques used in mass spectrometry imaging.

Imaging Mass Spectrometry

2.2. Secondary Imaging Mass Spectrometry

2.3. Laser Ablation Electrospray Ionization

2.4. Matrix-Assisted Laser Desorption/Ionization

Kamlesh Shrivas and Mitsutoshi Setou

Imaging Mass Spectrometry

Slice frozen tissue on

Thaw slice onto

labeling with fluorescent probes or radioactive isotopes. MS analyses may

3.1. Sample Handling

Kamlesh Shrivas and Mitsutoshi Setou

3.1.1. Storage of Samples

3.1.2. Sectioning of Tissue

Imaging Mass Spectrometry

3.1.3. Washing Tissue Sections

Kamlesh Shrivas and Mitsutoshi Setou

3.1.4. Histological Staining of the Section

Imaging Mass Spectrometry

3.2. Choice of Matrix

Kamlesh Shrivas and Mitsutoshi Setou

TABLE 1 Commonly used MALDI matrices for imaging of biomolecules in tissue

Fournier et al. (2003);

Schwartz et al. (2003);

Jackson et al. (2005);

Stuebiger and Belgacem

Estrada and Yappert

Astigarraga et al. (2008)

Peptides and large

Schwartz et al. (2003)

Lemaire et al. (2006b)

Shrivas et al. (2010)

phospholipids (Herring et al., 2007; Seeley et al., 2008). A great variety of

3.2.1. Ionic Matrices for IMS

Imaging Mass Spectrometry

of biomolecules (Armstrong et al., 2001; Laremore et al., 2007). IMs

(g) DHBB (h) CHCAB

Kamlesh Shrivas and Mitsutoshi Setou

shown in Figure 6. Direct tissue analyses of peptides in rat brain tissue

3.2.2. Nanoparticles as Matrices for IMS

On tissue with AgNps

Signal intensity (a.u.)

Signal intensity (a.u.)

Imaging Mass Spectrometry

On tissue with DHB

Signal intensity (a.u.)

FIGURE 7 Identification of fatty acids from mouse liver sections in nano-PALDI-IMS.

weight-biomolecules in mouse brain without observing any NP-related

3.3. Application of Matrix Solution

Kamlesh Shrivas and Mitsutoshi Setou

componentsorganic solvent, matrix, and trifluoroacetic acid (TFA).

Imaging Mass Spectrometry

4.2. Time-of-Flight Mass Analyzer

Kamlesh Shrivas and Mitsutoshi Setou

FIGURE 9 Schematic description of six mass analyzers used in mass spectrometers.