Chapter 11

Electrospray Laser Desorption Ionization

(ELDI) Mass Spectrometry for Molecular Imaging
of Small Molecules on Tissues
Min-Zong Huang, Siou-Sian Jhang, and Jentaie Shiea

Abstract
The use of an ambient ionization mass spectrometry technique known as electrospray laser desorption
ionization mass spectrometry (ELDI/MS) for molecular imaging is described in this section. The tech-
nique requires little or no sample pretreatment and the application of matrix on sample surfaces is unneces-
sary. In addition, the technique is highly suitable for the analysis of hard and thick tissues compared to
other molecular imaging methods because it does not require production of thin tissue slices via micro-
tomes, which greatly simplifies the overall sample preparation procedure and prevents the redistribution of
analytes during matrix desorption. In this section, the ELDI/MS technique was applied to the profiling
and imaging of chemical compounds on the surfaces of dry plant slices. Analyte distribution on plant slices
was obtained by moving the sample relative to a pulsed laser and an ESI capillary for analyte desorption
and post-ionization, respectively. Images of specific ions on sample surfaces with resolutions of 250 μm
were typically created within 4.2 h for tissues with sizes of approximately 57 mm × 10 mm.

Key words ELDI, Ambient ionization, Molecular imaging, Plant slice

1 Introduction

Ambient mass spectrometry, an extension to MS, is a set of useful

techniques for the analysis of samples under open-air conditions.
The feature of ambient MS is its capacity for direct, rapid, real-time,
and high-throughput analyses with little or no sample pretreat-
ment. It allows for the analyses of a wide range of substances from
various surfaces and matrices [1]. With the development of new
variants, combinations, and hybrids, several different ambient ion-
ization techniques have been developed and described [2–4].
Examples of these methods are desorption electrospray ionization
(DESI) [5], direct analysis in real time (DART) [6], and electro-
spray laser desorption ionization (ELDI) [7]. These techniques use a
variety of sampling, desorption, and ionization processes including
bombardment of charged droplets and metastable atoms, thermal

Lin He (ed.), Mass Spectrometry Imaging of Small Molecules, Methods in Molecular Biology, vol. 1203,
DOI 10.1007/978-1-4939-1357-2_11, © Springer Science+Business Media New York 2015

107
108 Min-Zong Huang et al.

and laser desorption, and post-ionization in electrospray ionization

(ESI) and atmospheric pressure chemical ionization (APCI)
plumes, respectively. Several novel techniques have been developed
over the last few years with many applications, in which one of the
most important applications is imaging mass spectrometry with
ambient ionization techniques because it is able to determine the
spatial distributions of chemical constituents on sample surfaces.
Desorption electrospray ionization (DESI) has been used to con-
struct molecular images of several biological tissues, such as mouse
pancreatic tissues, rat brain tissues, metastatic human liver adeno-
carcinoma tissues, human breast tissues, and canine abdominal
tumor tissues [8–11]. The capability of electrospray laser desorp-
tion ionization (ELDI) for profiling and imaging several biological
tissue slices and painting has been demonstrated [3, 12]. A similar
technique known as laser ablation electrospray ionization (LAESI)
has been used for chemical molecular imaging and depth profiling
of water-rich leaf tissues for usage in metabolic studies [13]; the
most recent use of this technique is for the simultaneous imaging
of small metabolites and lipids in rat brain tissues in situ cell-by-cell
imaging of plant tissues [14]. A technique using a low-temperature
plasma probe (LTP) is also used for molecular imaging and has been
used to analyze works of art including paintings and calligraphy
[15]. Probe electrospray ionization (PESI), an ESI-based ambient
ionization, has been shown to have potential for direct mouse brain
imaging analysis in an atmospheric pressure environment [16].
In short, the variety of these techniques with respect to sampling,
desorption, and ionization capabilities allows for the analysis of a
broad range of samples using imaging mass spectrometry coupled
with ambient ionization.
Here, the 2D molecular imaging of sample surfaces using
electrospray laser desorption ionization mass spectrometry
(ELDI/MS) is discussed in detail. The technique has been demon-
strated to be useful in detecting proteins and small organic com-
pounds on solids under ambient conditions [7, 17, 18]. Analyte
molecules in the solid were desorbed using a pulsed laser and then
post-ionized in an ESI plume. It is possible to obtain data on pre-
dominant chemical compounds on a particular area of the sample
surface with the assistance of a stepper motor and laser desorption
(LD) at a high spatial resolution. This procedure was extended to
illustrate the application of ELDI/MS to the imaging of dry plant
slices from Oldham Elaeagnus, an important traditional Chinese
herb, with an emphasis on small-molecule detection. Line scans
were obtained by continuously moving the sample between each
predefined point. These line scans are combined into an array to
produce a 2D image. Using the protocol provided here, it is possi-
ble to obtain point analyses for qualitative chemical analysis and line
scans for quantitative analysis, after which images can be produced
by combining the individual line scans.
 Electrospray Laser Desorption Ionization (ELDI) MS Imaging 109

2 Materials

1. Dry Oldham Elaeagnus was purchased from a local market.

2. Electrospray solution: Methanol and water (50 %, v/v) with
0.1 % acetic acid (see Note 1).
3. Fused silica capillary (100 μm i.d.).
4. Syringe pump (e.g., model 100 KD Scientific).
5. Double-sided tape.
6. A Bruker Esquire 3000 Plus ion trap mass spectrometer con-
trolled by the EsquireControl 5.2 data processing software was
used; however, a mass spectrometer with a similar atmospheric
pressure interface can be used.
7. 266 nm pulsed Nd:YAG laser (e.g., MINILITE I, Continuum
Electro-Optics Inc., USA).
8. A laser power and energy meter (e.g., SOLO 2, Gentec-EO).
9. A three-axis precision automatic stage (e.g., DMVTEKS Co.
Ltd, Taiwan) with a travel range of 10 cm.
10. Because the moving stage, laser system, and the mass spectrom-
eter work independently, an IMS_Control software was created
to control the three components; a contact signal received from
the laser system and automatic XY moving stage controller trig-
gers the start of data acquisition in EsquireControl 5.2 at the
beginning of each line scan. The data acquisition time per line
established in EsquireControl 5.2 must be equal to the time that
is required to perform one line scan. Control software and inte-
gration of moving stage, laser system, and the mass spectrom-
eter provided for customization are available upon request
from Torbis Technology CO., LTD. (http://www.icpdas.com/
distributors/country/torbis.htm).
11. Home-developed ImagAnalysis v2.1 software, available upon
request.

3 Methods

3.1 ELDI Imaging 1. An ESI emitter continuously sprayed solvent through a fused
Experimental Setup silica capillary at a flow rate of 150 μL/h using a syringe pump
(Fig. 1a) and a 2.5 mL syringe (see Notes 2 and 3). A nebulizing gas,
commonly used in conventional ESI, was not used during
ELDI processes. The ESI plume was directed toward the ion
sampling orifice of the mass spectrometer (i.e., the ESI plume
was parallel to the sample plate). The resulting analyte ions
formed in the ESI plume were sampled into the mass analyzer
through the ion sampling capillary. The electrospray needle,
110 Min-Zong Huang et al.

Fig. 1 (a) Graphic representation of the ELDI setup. The sample deposited on the stainless steel plate was
positioned on the mobile sample stage and irradiated with a pulsed laser beam, where the laser beam was set
behind the plane of the figure at an incident angle of 45°. The laser-ablated material was ionized in an elec-
trospray solvent plume delivered through an electrospray capillary, where the resulting ions entered the mass
spectrometer through the MS inlet tube. The distance between the ESI tip and the MS inlet tube was set as
8 mm, while the distance between the electrospray capillary and sample surface was set as 3 mm; the opti-
mum location of the laser spot on the sample surface was positioned approximately 1 mm below the tip of the
ESI capillary. (b) Schematic representation of the imaging experiment conducted using ELDI/MS. Each scan
line on the sample resulted in a unique spectrum

the sample plate, and the sampling tube voltages were maintained
at +4.5 kV, ground, and −0.5 kV, respectively (see Note 4).
2. A 266 nm pulsed Nd:YAG laser operating at a frequency of
10 Hz, a pulse energy of approximately 250 μJ (measured off-
line using a laser power and energy meter, SOLO 2, Gentec-EO),
a pulse duration of 4 ns, and a spot size of approximately
250 μm was used for profiling and imaging analysis. The stron-
gest ion signal was obtained at an incident laser angle of approx-
imately 45° (see Note 5).
3. The geometry of the source was optimized to achieve an efficient
mixing of ablated analytes with the ESI plume for maximum
signal strength. The distance between the ESI tip and the MS
 Electrospray Laser Desorption Ionization (ELDI) MS Imaging 111

inlet tube was set as 8 mm, while the distance between the
electrospray capillary and sample surface was set as 3 mm; the
optimum location of the laser spot on the sample surface was
positioned approximately 1 mm below the tip of the ESI
capillary (see Note 4).
4. A three-axis precision automatic stage with a travel range of
10 cm was computer controlled when scanning the sample sur-
face. While the laser beam irradiates the tissue slices, the sam-
ple stage is moved according to the laser beam at the speed of
200 μm/s in the longitudinal direction (X). The sample stage
is further moved in the transverse direction (Y) upon computer-
controlled positioning mechanism. Each scan line on the sam-
ple results in a unique spectrum (see Note 6).
5. The data acquisition programs rendered analysis times to the
corresponding XY coordinates and converted the data sets into
two-dimensional distributions. In-house software was used to
produce contour plot images of the distribution of selected
ions (see Note 7).

3.2 Preparation 1. The dry plant was cut into 2–5-mm-thick slices using a razor
of Tissue Sections blade at room temperature. For Oldham Elaeagnus, the sample
was sliced into thin sections of approximately 50 × 60 × 5 mm
(L × W × H) (see Note 8).
2. Place and fix the tissue sections onto sample plate using double-
sided tape (see Note 9).
3. Dry tissue sections can be stored in the freezer for a few months

3.3 ELDI Imaging 1. The plant slice set on the sample plate was positioned on a
Experiments homemade automated XY stage in front of the sampling capil-
lary of a Bruker Esquire 3000 Plus ion trap mass spectrometer.
For small-molecule analysis, the acquisition mass range was set
from 50 to 250 m/z.
2. Use the IMS_Control software to set the moving speed of the
stage and define the scanning area, and line scan number on
the automatic XY stage software controller (see Note 10). For
example, the sample stage is moved according to the laser
beam at the speed of 200 μm/s along the x-axis within a
defined area of 57 × 10 mm (L × W), each time at an increment
of 250 μm in the transverse direction (Y) (see Note 11).

3.4 Data Acquisition 1. Create a sample list in the mass spectrometer acquisition soft-
ware (Bruker EsquireControl 5.2). The total number of samples
in the list is equal to the number of lines in the image. The last
two characters of the file name should index the sequence of
files (e.g., OE_01.yep, OE_02.yep, OE_03.yep, …, and
OE_40.yep).
112 Min-Zong Huang et al.

2. Make sure that the acquisition method contains the correct

acquisition time for each line, e.g., 40 lines with an acquisition
time of 285 s for each experiment.
3. Start the acquisition

3.5 Data Analysis 1. Before data analysis, convert the Bruker EsquireControl 5.2
mass spectra files (.yep extensions) into format files (.ascii)
using Bruker DataAnalysis software, in which the ascii files are
required by home-developed ImagAnalysis v2.1 software,
which is available upon request.
2. The following instructions describe how to generate chemical
images of Oldham Elaeagnus via ImagAnalysis v2.1 software.
(a) Open ImagAnalysis v2.1; (b) click on the LOAD DATA
menu bar to load the converted file (.ascii extensions); (c)
select the rainbow-colored scale and adjust the contrast of the
image by selecting minimum and maximum values on the slide
bars; (d) key in the m/z value (i.e., m/z 60.7, m/z 86.5, m/z
97.5, m/z 111.4, and m/z 137.5) displayed in the mass spec-
trum window; (e) click on the CREATE IMAGE menu bar to
see an image of the distribution of small organic compounds
from Oldham Elaeagnus surface; (f) copy the image and paste
onto the organic photo of Oldham Elaeagnus using PowerPoint;
and (g) repeat steps and the overlay chemical images from
Oldham Elaeagnus surface should be observed (see Note 7).

4 Notes

1. The composition of the electrospray solution can influence the

stability of the electrospray generated during ELDI analysis
and the ability of the technique to detect particular analytes
from the tissue matrix depending on the solubility of the ana-
lyte in the solvent system. The solvent composition is usually
50 % MeOH + 0.1 % acetic acid for most cases.
2. Ensure that the tip of the fused silica capillary is square and not
burred or cracked. A burred or cracked tip will result in elec-
trospray instability leading to irreproducible data.
3. Turn on the syringe pump and the high voltage for the electro-
spray. Make sure that the syringe contains enough solvent to
acquire an image of the desired size. A 2.5 mL syringe filled
with ESI solution is suggested and will typically last for 6 h
with a flow rate of 150 μL/h. Let the electrospray stabilize for
10 min before starting analysis.
4. The voltage will depend on the composition of ESI solution,
geometry of MS inlet, and the distances between the ESI tip,
the MS inlet tube, and the sample plate. The high voltage is
typically set from +4 kV to +6 kV for positive ion scan mode.
 Electrospray Laser Desorption Ionization (ELDI) MS Imaging 113

5. The laser energy will depend on the sample materials. The laser
energy is typically set from 250 μJ to 1 mJ (Warning). Avoid
eye or skin exposure to direct or scattered radiation. Safety
goggles should be worn when performing ELDI experiments.
6. As shown in Fig. 2, molecular imaging analysis of a dry plant
slice was completed in a few hours under a full scan in positive
ion mode of mass spectrometry. The extracted ion chromato-
grams corresponding to m/z 86.5, m/z 111.4, m/z 97.5, and
m/z 60.7 were acquired from a single scan line of different
surfaces of an Oldham Elaeagnus slice.
7. As shown in Fig. 3, a typical set of molecular images on dry
Oldham Elaeagnus slice were obtained by using ELDI/
MS. The images were recorded at m/z 97.5, m/z 137.5, m/z
60.7, m/z 111.4, and m/z 86.5. The experimental procedures
are extremely simple when compared to other MS imaging
techniques because little or no sample pretreatment is required
and matrix application of matrix on the sample surface is
unnecessary. In addition, the technique is highly suitable for
the analysis of hard and thick tissues without microtome pro-
duction of tissue slices. In general, this protocol can be applied
when an ELDI ion source and an automatic XY stage are cou-
pled with a mass analyzer. The analytical steps described here are
general and can be used in other applications involving ELDI
(see Note 12).
8. Perform molecular imaging on a relatively hard and thick plant
surface where a micrometer-scale sample slice cannot be
obtained for the high degree of texture and fragile of dry plant
tissue. The best thickness for this kind of tissue would be
2–5 mm. Please note that the surface of tissue should be smooth
and crackles.
9. Adhere plant tissue samples to metal, plastic, or glass plates
using double-sided tape.
10. To obtain the correct setup for the required image resolution,
divide the sample area of the tissue by the different scan num-
bers according to sample size (see Fig. 1b). For example,
Oldham Elaeagnus with a defined scanning width of 10 mm
will result in 40 scan number (each time at an increment of
250 μm in the transverse direction).
11. Calculate the acquisition time based on the distance of each
line scan and the moving speed of the automatic XY stage. For
example, Oldham Elaeagnus with a defined scanning length of
57 mm and a moving speed of 200 μm/s along the x-axis will
result in a duration of 285 s for each line scan. About 1,425
data points (mass spectra) will be obtained for each line scan
while a Bruker Esquire 3000 plus ion trap mass spectrometer
with a scan rate of 200 ms.
Fig. 2 (a) Photograph of an Oldham Elaeagnus slice, where locations of scanning lanes are indicated by the
dotted lines; (b–e) extracted ion chromatograms from a full scan in positive ion mode corresponding to m/z
86.5, m/z 111.4, m/z 97.5, and m/z 60.7, respectively; (f–i) ELDI mass spectra from the surface of an Oldham
Elaeagnus slice. Analytes were detected on the surface of a slice of Oldham Elaeagnus tree bark using ELDI/MS
under ambient conditions without sample pretreatment. The location of different sampling spots was indicated
by the arrow shown in panel (a)
 Electrospray Laser Desorption Ionization (ELDI) MS Imaging 115

Fig. 3 (a) Photograph of a slice of Oldham Elaeagnus bark and the scanning area with a size of 57 × 10 × 4 mm
(L × W × H); (b–f) molecular images corresponding to m/z 97.5, m/z 137.5, m/z 60.7, m/z 111.4, and m/z 86.5
from the Oldham Elaeagnus sample were recorded using ELDI/MS under ambient conditions

12. All operations should be conducted under appropriate safety

protocols especially with respect to exposure at the operator
and decontamination of laboratory equipment used in these
studies.

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