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\begin{document}

\title{MALDI Mass Spectrometry Imaging sample preparation with wet-interface
matrix deposition for lipid analysis.}



\author[1]{Przemyslaw Mielczarek}%
\author[2]{Piotr Suder}%
\author[2]{Paulina Kret}%
\author[3]{Tymoteusz Słowik}%
\author[3]{Ewa Ewa Gibuła-Tarłowska}%
\author[4]{Jolanta Kotlinska}%
\author[2]{Igor Kotsan}%
\author[2]{Anna Bodzoń-Kułakowska}%
\affil[1]{Maj Institute of Pharmacology Polish Academy of Sciences}%
\affil[2]{AGH University of Science and Technology}%
\affil[3]{Medical University of Lublin}%
\affil[4]{Medical University}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
RATIONALE: Sample preparation is one of the most crucial steps for MALDI
mass spectrometry imaging (MALDI-MSI). The scientists beginning their
adventure with this technique may be overwhelmed by the variety of
matrices, solvents, concentrations, the ways of their applications, and
the lack of widely available knowledge about the influence of these
parameters on the results. Here we would like to present our experiences
with detailed aspects of matrices deposition, hopefully helpful for the
scientific community. METHODS: In our article, we have tested several
MALDI matrices applied by the SunCollect\selectlanguage{ngerman}® system: wet-interface matrix
deposition in the context of lipids analysis. Among them:
2,5-dihydroxybenzoic acid (DHB), norharmane, N-(1-naphthyl)
ethylenediamine dihydrochloride (NEDC), 9-aminoacridine (9AA). We have
optimized the number of matrix layers and nozzle settings in terms of
spectra intensity and the overall quality of obtained ion maps. RESULTS:
Our research presents the influence of the number of matrix layers and
nozzle settings on the results and allows for choosing the optimal
parameters for the analyses. In positive ionization mode, DHB matrix
could be chosen as the first selection. In the negative ionization mode,
DAN matrix produces higher peak intensity in a lower mass range and
seems to provide more information than 9AA. We recommended NEDC for
particular tasks such as glucose analysis. Comparably to remaining
matrices, norharmane significantly changes received ion maps.
CONCLUSIONS: Dealing with such a great amount of data allows us to
notice an interesting conclusion: the obtained ion picture for a
particular ion could differ dramatically with changing the matrix, the
solvent composition, or even the number of matrix layers. This must be
considered when interpreting the result and forces us to compare the
results obtained with different matrices with extreme caution.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

Sample preparation is one of the most crucial steps in every analytical
procedure. Optimization of this part of the experiment is usually the
most time-consuming task. Nevertheless, once successfully optimized, the
procedure allows for certain and repetitive analyses. Sample preparation
for MALDI-MSI is connected to the application of a special matrix before
the analysis. The scientists beginning their adventure with this complex
and powerful technique may be overwhelmed by the variety of matrices,
solvents, concentrations, the ways of their applications, and the lack
of widely available knowledge about the influence of these parameters on
the final results. Some data can be found in the research articles,
technical reports, or protocols provided by the equipment manufacturers,
but usually, it is not easy to find a comprehensive recommendation on
which matrix and sample preparation strategy to select.

In our study, we decided to use common matrices like
2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9AA),
1.5-diaminonaphtalene (DAN), and not as well-known matrices as
norharmane and N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) to
compare their ability to visualize lipids and in some cases (NEDC
matrix) small molecules.

To apply matrices, we used SunCollect\selectlanguage{ngerman}® system, which utilizes a
high-performance pneumatic sprayer. It is recognized as a wet-interface
system, and it is worth to mention that its performance may be different
form the systems based for example on sublimation
process\textsuperscript{1} (see: supplementary materials from mentioned
article). In this device, the matrix solution could be sprayed in
several layers over the tissue sample and from different heights over
the sample. The higher position (e.g., topmost position Z=1) produces
the driest vapor resulting in the finest matrix crystals size. At the
lowest position (Z = 25 mm), the wetter, atomized spray will attain the
target. It means that the matrix solution could penetrate the tissue
sample and allows to observe the substances that must be extracted from
the tissue. In our study, four different positions of the spraying
nozzle above the sample surface and the influence of the multiple matrix
layers on the quality of obtained MS spectra were examined for each
matrix.

DHB matrix is the most common one for the analysis in the positive
ionization mode. In the literature different matrix concentration (from
7 mg/ml\textsuperscript{2}, to even 50 mg/ml\textsuperscript{3} could be
applied. Different solvents are used as well. Mainly methanol
(MeOH)\textsuperscript{4} and acetonitrile (ACN)
\textsuperscript{5,6}but also ethanol\textsuperscript{7} and mixtures of
chloroform and methanol\textsuperscript{8} are used. 9AA is a popular
matrix for the negative ionization mode. In this case, concentration
between 2 mg/ml\textsuperscript{9} and 10 mg/ml\textsuperscript{10} are
used, but the most commonly selected is 7 mg/ml (like
in\textsuperscript{11--13}). Regarding the solvents, mainly aqueous
solutions of methanol and ethanol are used in different concentrations.
Norharmane and NEDC are still gaining their popularity so, there is no
such diversity in their use. In both cases, 7mg/ml is the most popular
concentration\textsuperscript{13}. Regarding norharmane for SunCollect®
application, the concentration of 6 mg/ml and the solution of
chloroform: MeOH:H\textsubscript{2}O, 1:2:0.8 (v/v/v) is used since in
given volumes ratios, the solution creates a homogenous mixture, and
separation of water and chloroform is not observed. Additionally,
1.5-DAN (25 mg/ml, in ACN:H\textsubscript{2}O, 1:1,
v/v)\textsuperscript{14} and 1.5 DAN hydrochloride proposed by Liu et
al.\textsuperscript{15} were tested.

The cross-section of a rat spinal cord was chosen as a tissue model,
representing the features of the central nervous system. Particularly,
its simple structural division into a gray and white matter should be
easily recognizable on MSI tissue scans. Additionally, the slices
obtained from this tissue are small and thus convenient for the fast MSI
measurements. Thickness of the slides was typical for most MALDI-MSI
experiments and was set to 12 \selectlanguage{greek}µ\selectlanguage{english}m. To compare different matrices, we
decided to choose several different peaks representing lipids from the
spinal cord's gray and white matter, well known in the
literature\textsuperscript{16--18}. Such choice allows for visualization
of the structures and comparing the quality of obtained results between
different matrices. Additionally, the care was taken to choose
intensive, as well as relatively small, peaks to show their behavior in
the broad intensity range (see Fig.1). Finally, the parameters of chosen
peaks, especially their intensities, were used for the comparison.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/594870/articles/629130-maldi-mass-spectrometry-imaging-sample-preparation-with-wet-interface-matrix-deposition-for-lipid-analysis}

\textbf{Fig. 1} Examples of averaged mass spectra from the rat spinal
cord slices measured in positive (A) and negative (B) ionization modes.
The peaks used in the analysis and comparison of different matrices have
been signed in the figure.

\textbf{Materials and Methods}

\textbf{Tissue preparation}

All experiments on animals were performed according to the respective
Polish and European Communities Council Directives (86/609/EEC) and were
approved by the local Ethics Committee (approval number 137/2018). The
tissues were obtained as spared from control animals used during other
experiments, according to the so-called 3R (replacement/reduction/
refinement) rule. Immediately after isolation, the rat spinal cords were
flash frozen and kept in a liquid nitrogen storage dewar (LS750,
Worthington Industries, Columbus, OH, USA) until assayed. Approximately
1 h before cutting, the tissue was transferred to the cryomicrotome
chamber (Cryotome FSE, Thermo Fisher Scientific, Chesire, UK). Both, the
chamber, and the specimen temperatures were set to -15\selectlanguage{ngerman}°C. Slices were
cut at 12 \selectlanguage{greek}µ\selectlanguage{english}m slices, and immediately thaw-mounted on the indium-tin
oxide (ITO) glasses (Bruker-Daltonics, Bremen, Germany). The tissue
slices were then vaccum-dried for about 45 min. Prepared samples were
stored in a laboratory freezer at -80\selectlanguage{ngerman}°C (NU9483, NuAire Inc., Plymouth,
MN, USA) in the hermetically sealed boxes. Just before analysis, the
samples were warmed up to an ambient temperature in a vacuum desiccator
to prevent moisture condensation on the sample's surface and dried again
for about 45 min. Before each matrix deposition, an optical image (600
dpi) of the tissue cross\selectlanguage{english}-sections with marked fiducials was recorded.

Additionally, as suggested by Angel et al.\textsuperscript{19}, before
analysis in both modes (pos./neg.), tissue washing by 50 mM ammonium
acetate pH = 6.5 by 10 s has been tested. This procedure is suggested,
especially in the negative ionization mode, as a way to improve the
sensitivity of the measurements.

\textbf{MALDI matrices:}

During the experiments, the following chemicals were used: solvents: ACN
and MeOH (J.T. Baker, Amsterdam, Netherlands), both at the HPLC gradient
grade, ethanol (Avantor, Poland) chloroform, ultrapure water
(Sigma-Aldrich/Merck, Germany). Matrices: 2,5-dihydroxybenzoic acid
(DHB), trifluoroacetic acid (TFA), norharmane, N-(1-naphthyl)
ethylenediamine dihydrochloride (NEDC) all from Sigma-Aldrich/Merck
(Darmstadt, Germany).

\textbf{Matrix application:}

The matices were applied by a SunCollect(r) device (SunChrom GmbH,
Friedrichsdorf, Germany). The flow rate of the matrix solution changed
among layers, according to the manufacturer recommendations: 10 \selectlanguage{greek}µ\selectlanguage{english}L/min
for the first layer, 20 \selectlanguage{greek}µ\selectlanguage{english}L/min for the second layer, 30 \selectlanguage{greek}µ\selectlanguage{english}L/min for the
third layer, 40 \selectlanguage{greek}µ\selectlanguage{english}L/min for the fourth layer, and 60 \selectlanguage{greek}µ\selectlanguage{english}L/min for all
following layers. The nozzle applied the matrix solution with a line
distance of 2 mm, and the speed 600 mm/min. For each matrix, a different
number of layers was applied. The Z value -- the position of the
spraying nozzle above the tissue surface was also tested. Z=1 means the
highest possible (about 50 mm) position above the tissue, whereas Z=25
means about 25mm above the tissue and is determined as the ``extraction
mode''. Four different numbers of layers and four different nozzle
heights were tested for each matrix.

\textbf{MALDI measurements}

Matrix-coated sections were subjected to imaging experiments using the
MALDI--TOF/TOF UltrafleXtreme MS (Bruker-Daltonics, Bremen, Germany)
with a Smartbeam II laser operating at 2 kHz. All following MS
parameters underwent initial, multistep optimization. Ions were
accelerated at 25 kV with a pulsed ion extraction of 120 ns and ion
suppression up to 100 Da. Spectra were recorded in positive and negative
ion modes with reflectron, within an 200--3000 m/z range. They were
externally calibrated with Peptide Calibration Standard II
(Bruker-Daltonics, Bremen, Germany) and known matrix ions, e.g. 273.0399
m/z for DHB matrix
({[}2DHB-2H\textsubscript{2}O+H{]}\textsuperscript{+}ion) and 229.0533
m/z for 9AA matrix ({[}9AA+Cl{]}\textsuperscript{-}ion). A raster width
of 200 \selectlanguage{greek}μ\selectlanguage{english}m was applied to all samples. In total, 400 shots were collected
from each ablation point with 20 shots at the raster spot, and the laser
focus diameter was set to ``3\_medium''. FlexControl version 3.4
(Bruker-Daltonics, Bremen, Germany) was employed for spectra
acquisition, and FlexImaging, version 4.0 was used for data processing
and the creation of molecular images. Mmass software (version 5.5.0,
Open-Source software developed by Martin Strohalm, Academy of Sciences,
Prague, Czech Republic) was used for the spectra
analysis\textsuperscript{20}.

\textbf{Results}

To keep this chapter as clear as possible, we divided it into three
subchapters: tissue washing (1), matrix spraying nozzle height over the
sample, and the number of matrix layers in positive (2) and negative (3)
ionization modes. DHB, norharmane, and DAN matrices were tested in the
positive ionization mode. In the negative ionization mode, norharmane,
NEDC, and 9AA matrices were used. Below there is a brief description of
the optimization procedures.

Tissue washing

Some Authors recommend applying the tissue washing procedure before
analysis to remove substances interfering with the ionization
process\textsuperscript{19,21}. The procedure is easy: the sample on the
ITO glass should be immersed into a solvent a few times and dried. To
test this approach, we used the procedure described by Angel et
al.\textsuperscript{19}: two slices were washed with 50 mM ammonium
acetate by immersing them for 10 s and subsequent drying in the
desiccator for the next 15 minutes.

After washing, we observed four types of effects influencing the final
results:

(1) some substances were completely washed away: their ions, typically
present in non-washed tissue, were not observed,

(2) the final picture was significantly blurred (see Fig. 2: the
difference between both gray and white matters has vanished),

(3) all peak intensities were elevated (probably due to the removal of
substances responsible for the ion suppression effect). However, spectra
enhancement did not significantly influence the interpretation of the
results.

(4) The picture for specific m/z values remains the same before and
after washing.

Summing up, washing procedures, if not thoroughly tested to visualize
the peaks of interest, usually could not lead to the desired results. It
is also important to keep in mind the possibility of delocalization of
the molecules and increase the sample variability generated by the
washing procedure. So, we decided to perform the measurements in the
conditions as unchanged as possible.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2} Blurring effect after tissue washing - the difference
between white and gray matter has vanished. Washing: 50 mM ammonium
acetate for 10 s, matrix: 25 mg/ml DHB (MetOH:H\textsubscript{2}O 1:1,
0,2\% TFA), m/z 718.

The number of matrix layers and the height of the spraying nozzle during
matrix application -- positive ionization mode (DHB, norharmane, and DAN
matrices)

To compare the influence of the different matrix layers and the position
of the spraying nozzle on the spectra quality, we decided to select two
parameters: the peak intensity (int.) and the signal-to-noise ratio
(s/n). Peak intensity alone could be misleading since the increase of
the overall spectra intensity may occur simultaneously with the
background signal increase, which could, in turn, decrease the s/n value
and, in effect, worsen the analysis quality.

\textbf{25 mg/ml DHB matrix (MeOH:H\textsubscript{2}O 1:1, 0.2\% TFA) --
positive ionization mode}

25 mg/ml DHB matrix (50\% MeOH, 0.2\% TFA) could be treated as a
reference matrix in the positive ionization mode. To check the influence
of the parameters of matrix application by the SunCollect\selectlanguage{ngerman}® system, we
have chosen four different numbers of matrix layers (L= 8, 10, 12, 14)
and four different heights of the spraying nozzle (Z=25, 15, 5, 1,).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3} The ion map for m/z 844 and m/z 813 obtained from the
slices of the spinal cord for DHB matrix with the different number of
matrix layers (A) and four different heights of spraying nozzle (B) for
the positive ionization mode (matrix: 25 mg/ml DHB (50\% MeOH, 0.2\%
TFA))\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4} The relationship between the chosen peaks' intensities
and signal-to-noise ratio (s/n) in terms of the number of matrix layers
(A) and the spraying nozzle height (B) for the positive ionization mode,
rat spinal cord tissue slices (matrix: 25 mg/ml DHB (50\% MeOH, 0.2\%
TFA))

Considering the ions intensities, the highest increase in those
parameters was observed between 10 and 12 layers (see: Fig. 3,4). The
parameters for the 12 and 14 layers were similar and did not lead to the
further spectra enhancement. The same trend is observed with the s/n
value. Thus, we decided that 12 layers would be optimal for the
measurements since it allows to save the chemical reagents and time for
tissue preparation\textbf{.} The spraying nozzle height of 5 mm gives
the highest intensity for observed peaks. For s/n ratio the 5 mm and 10
mm positions give the maximum value, but the intensity itself is higher
for 5 mm. Thus, considering both parameters, the 5 mm position was
recommended for the DHB matrix in the positive ionization mode.

\textbf{Norharmane (6 mg/ml matrix (chloroform:
MeOH:H\textsubscript{2}O) -- positive ionization mode)}

Norharmane works differently than DHB matrix. For s/n ratio, 12 and 14
layers show high value, but 14 matrix layers are optimal considering the
peaks intensities only (see: Fig. 5,6). Regarding the position of the
spraying nozzle above the measured sample, the most intensive peaks are
obtained both at the highest (z=1) and the lowest position of the
spraying nozzle (Z=25), preferably with the lowest position. The same is
valid for the s/n ratio.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5} The ion map for m/z 772, m/z 734 and m/z 744 obtained
from the slices of the spinal cord for norharmane matrix with the
different number of matrix layers (A) and four different heights of
spraying nozzle (B) for the positive ionization mode (matrix: 6 mg/ml
norharmane (chloroform:MeOH:H\textsubscript{2}O))\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig. 6} The relationship between the chosen peaks' intensities
and signal-to-noise ratio (s/n) in terms of the number of matrix layers
(A) and the spraying nozzle height (B) for the positive ionization mode
rat spinal cord tissue slices (matrix: 6 mg/ml norharmane
(chloroform:MeOH:H\textsubscript{2}O))

The problem with that matrix lies in the fact that, for several m/z
values, we obtained different ion maps in comparison with DHB matrix
(see discussion chapter).

\textbf{DAN (2.5 mg/ml 50\% ACN -- positive ionization mode)}

For this matrix, the highest position of the nozzle (z=1) and 16 layers
seem to be the best setting when the peaks intensities and the s/n
ratios are considered (see: Fig. 8). But looking at different ion maps
(see: Fig. 7), 14 matrix layers seem to give better results. For several
ion maps, additional matrix layers blurred the picture. The same is
observed for z=1 and z=5; thus, z=5 seems the best.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Fig. 7} The ion map for m/z 734, m/z 766, m/z 798, and m/z 744
obtained from the slices of the spinal cord for the DAN matrix with the
different number of matrix layers (A) and four different heights of
spraying nozzle (B) for the positive ionization mode (matrix: 2.5 mg/ml
DAN 50\% ACN). Consider different ion maps for different spraying nozzle
heights (Z) and the number of layers (L) in the case of m/z 798.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Fig. 8} The relationship between the chosen peaks' intensities
and signal-to-noise ratio (s/n) in terms of the number of matrix layers
(A) and the spraying nozzle height (B) for the positive ionization mode
rat spinal cord tissue slices (matrix: 2.5 mg/ml DAN 50\% ACN)

Here we also observe discrepancies between ion maps produced by
different matrices (see: discussion).

\textbf{The number of layers and the spraying nozzle height -- negative
ionization mode (9AA, norharmane, NEDC and DAN matrices)}

\textbf{9-Aminoacridine (9AA 7 mg/ml 70\% EtOH)) -- negative ionization
mode}

Four different numbers of matrix layers (l= 10, 12, 14, 16) and four
different positions of spraying nozzle height (Z=25, 15, 5, 1) were
tested for 9AA as a reference matrix in the negative ionization mode. As
previously, the peaks intensities and the s/n ratios were compared.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Fig. 9} The ion map for m/z 835, m/z 255, m/z 718, and m/z 747
obtained from the slices of the spinal cord for 9AA matrix with the
different number of matrix layers (A) and four different heights of the
spraying nozzle (B) for the negative ionization mode (9AA 7 mg/ml 70\%
EtOH)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

\textbf{Fig. 10} The relationship between the chosen peaks' intensities
and signal-to-noise ratio (s/n) in terms of the number of matrix layers
(A) and the spraying nozzle height (B) for the negative ionization mode
rat spinal cord tissue slices (matrix: 9AA 7 mg/ml 70\% EtOH)

Nozzle height does not significantly affect the quality of the analysis,
but the peaks on the spectra are more intensive with the higher position
of the nozzle. S/n ratio is definitely higher for a higher nozzle
position. Thus z=5 was chosen as the best one. In the case of matrix
layers, we observe the increase in the peak's intensity with increasing
number of layers (see: Fig. 10). Nevertheless, assessing the quality of
the picture, 14 layers seems to be a good choice. With the 16 layers,
the picture becomes a little bit blurred (see: Fig. 9). Thus, it is not
recommended when we are interested in looking for different brain
structures. Additionally, in the case of \textbf{14 layers,} stable
picture is obtained with different normalization methods, which proves
the good quality of the measurement (see: Fig. 11).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

\textbf{Fig. 11} The reduced quality of the ion map with increasing
number of matrix layers without and with normalization (TIC -- total ion
current and RMS - root mean squared) -- negative ionization mode (9AA 7
mg/ml 70\% EtOH).

\textbf{Norharmane (6 mg/ml matrix
(chloroform:MeOH:H\textsubscript{2}O)) -- negative ionization mode}

In the case of norharmane (6 mg/ml in
chloroform:MeOH:H\textsubscript{2}O, 1:1:1, v/v/v solution) the
intensity of the peaks on the spectrum increased with the number of the
matrix layers but starting from the 12 layers, further increase was
minor. Conversely, the s/n ratio decreases starting from the twelfth
layer. Thus, up to 12 matrix layers seem to be optimal. For the spraying
nozzle position, 15 mm is performing the best since the intensity, and
s/n ratio is relatively high. Lower nozzle position does not change the
intensity but might be responsible for the molecule's delocalization
(see: Fig 12, 13).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}

\textbf{Fig. 12} The ion map for m/z 762 and m/z 847 obtained from the
slices of the spinal cord for norharmane matrix with the different
number of matrix layers (A) and four different heights of spraying
nozzle (B) for the negative ionization mode (matrix: 6 mg/ml norharmane
(chloroform:MeOH:H\textsubscript{2}O))\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}

\textbf{Fig. 13} The relationship between the chosen peaks' intensities
and signal-to-noise ratio (s/n) in terms of the number of matrix layers
(A) and the spraying nozzle height (B) for the negative ionization mode
rat spinal cord tissue slices (matrix: 6 mg/ml norharmane
(chloroform:MeOH:H2O))

Generally, for the negative ionization mode, norharmane gives the
spectra of higher intensity for the lipids around m/z = 728 compared to
9AA and then with the higher m/z range their intensities are going lower
(see: Fig. 21). Additionally, there is no signal observed for low m/z
ions like m/z = 255, or m/z = 283. It seems, that only the lipids of
higher molecular weights are able to ionize with this matrix.

\textbf{N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) --
negative ionization mode}

NEDC (7 mg/ml, 70\% MeOH) is quite destructive when sprayed from the low
position of the spraying nozzle. This effect is seen in Fig. 14.
Spraying nozzle heights 25 and 15 gave quite intensive spectra, but the
problem with matrix crystallization could be clearly observed in the
picture. Therefore, spraying nozzle height at a value \textbf{5 seems
to} be optimal. It gives quite good peak intensity for small molecules
and lipids and the s/n ratio is also acceptable (Fig. 15).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

\textbf{Fig. 14} The ion map for m/z 885, m/z 215 and m/z 744 and m/z
734 obtained from the slices of the spinal cord for NEDC matrix with the
different number of layers (A) and four different heights of spraying
nozzle (B) for the negative ionization mode (matrix: 7 mg/ml NEDC, 70\%
MeOH), (m/z 215 -- {[}glucose +Cl\textsuperscript{-}{]}). Additionally,
a picture of the glass covered with NEDC matrix.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}

\textbf{Fig. 15} The relationship between the chosen peaks' intensities
and signal-to-noise ratio (s/n) in terms of the number of matrix layers
(A) and the spraying nozzle height (B) for the negative ionization mode
rat spinal cord tissue slices (matrix: 7 mg/ml NEDC (70\% MeOH) -- (m/z
215 -- {[}glucose +Cl-{]}).

NEDC is the only matrix able to decrease peaks intensities with the
increasing number of the matrix layers, which is observed for low MW
compounds that could be observed with this matrix (see: m/z 215 for
glucose and 174 for glucose-3-phosphate (G3P), Fig. 15). Here 10 to 12
matrix layers seem to be optimal. For lipids, the situation depends on a
compound. For some of them, the trend is similar, while for others is
the opposite. To effectively observe lipids and small
molecules,\textbf{12} layers seem to be the most promising. It has to be
mentioned that NEDC matrix has the lowest intensity of the three tested
matrices in the negative ionization mode with a comparable s/n ratio
(Fig. 21).

DAN (2,5 mg/ml 50\% ACN -- negative ionization mode)

For DAN matrix Z =1 and L = 16 layers gives the highest intensity, and
the trend for s/n ratio is the same. Spraying nozzle height at the
highest position gives the best results (Fig. 16,17).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image16/image16}

\end{center}
\end{figure}

\textbf{Fig. 16} The ion map for m/z 762 and m/z 821 obtained from the
slices of the spinal cord for DAN matrix with the different number of
layers (A) and four different heights of spraying nozzle (B) for the
negative ionization mode (matrix: 2.5 mg/ml DAN, 50\% ACN)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image17/image17}

\end{center}
\end{figure}

\textbf{Fig. 17} The relationship between the chosen peaks' intensities
and signal-to-noise ratio (s/n) in terms of the number of matrix layers
(A) and the spraying nozzle height (B) for the negative ionization mode
rat spinal cord tissue slices (matrix: DAN 2.5 mg/ml 50\% ACN).

\textbf{Discussion}

\textbf{1. Different matrices, different pictures}

Collecting such a great amount of analysis allows us to notice an
interesting observation: the obtained ion map of a particular ion could
differ dramatically regardless of the used matrix, the solvent
composition, or even the number of matrix layers.

In the positive ionization mode, we have observed this effect for the
norharmane and DAN matrices in comparison with the classical DHB matrix.
Norharmane produces a strong signal from lipids. Nevertheless, the
lipids seem to be located in a whole tissue area, and the discrimination
between the white and the gray matter is lost. For example, the ion map
for m/z 810 and 766 for the DHB matrix suggests that those substances
are present mainly in the white matter, but in the case of norharmane,
these peaks were present all over the tissue slice (see Fig. 18). This
effect occurs probably because the chloroform is used as a solvent.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image18/image18}

\end{center}
\end{figure}

\textbf{Fig. 18} The difference in the ion map between norharmane and
DHB matrix. The structures visible with the DHB matrix are not present
when the norharmane matrix is used. Cresyl violet staining show white
and gray matter in the spinal cord slices.

From 11 peaks taken for the analysis, three behaved in that way: in DHB,
they can be seen in the white or gray matter, whereas in norharmane
their distribution is over the whole tissue (m/z = 766, 810, and 872).
Surprisingly the ion at m/z=798 in the norharmane matrix shows its
distribution in a gray region only. Because of that, the norharmane
matrix seems sufficient for the lipids when the differences among the
structures are not crucial.

The study of the DAN matrix revealed that the way of sample preparation
(the number of matrix layers and the spraying nozzle height) -- may also
influence the overall picture. In the case of this matrix, three ions:
m/z 760, 798, 868 were detected from the gray matter - not from the
whole tissue as in the case of the ``classical'' DHB matrix (see Fig.
19).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image19/image19}

\end{center}
\end{figure}

\textbf{Fig. 19} Discrepancies between ion maps produced by DHB and DAN
matrices in the positive ionization mode.

Similar effects are observed during solvent changes for the same matrix.
Exemplary, for the 9AA in 100\% methanol, we saw the ion map for m/z 281
on a tissue slice in the white matter only, whereas for the 9AA matrix
with 70\% ethanol/water solution, the same signal is distributed all
over the sample (see: Fig.20).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image20/image20}

\end{center}
\end{figure}

\textbf{Fig. 20} The ion map for m/z 281 obtained from the slices of the
spinal cord for 9AA matrix with different concentrations of matrix (7
mg/ml 9AA and 5 mg/ml 9AA) and solvent: A - 70\% EtOH, 50\% EtOH; B -
70\% MeOH and 100\% MeOH).

Such solvent-related, matrix-related effects must be taken into
consideration during the interpretation of the results. If a single ion
seems to be desorbed from a single structure with the aid of exact
solvent/matrix mixture, it does not mean that using the other solvent,
or matrix will give us the same result. Thus, it should be clearly
stated that comparing the ion maps obtained from different matrices and
solvents, must be done with extreme caution.

\textbf{2. Technical considerations about wet interface matrix
deposition.}

Wet-interface matrix deposition devices allow to control the number of
applied matrix layers and the nozzle height over the sample. Our article
points the necessity of proper optimization of the process of sample
preparation, since it could help to obtain a substantial improvement of
the analysis parameters.

\textbf{2.1. The number of applied matrix layers}

In the positive ionization mode for DHB and norharmane matrices, the
peaks overall intensity gets higher with the higher number of matrix
layers, but only up to some optimum. After that point, the number of
layers does not increase the peaks intensity or does it insignificantly.
In the case of DAN, the higher number of layers may even be responsible
for losing the discrimination between the white and gray matter in the
discussed case. Thus, it is highly advisable to check this parameter
during sample preparation optimization.

In the negative ionization mode for 9AA and norharmane, the peaks
intensity increases with the number of the matrix layers, and we did not
observe the ``saturation effect.'' Nevertheless, the quality of the
obtained ion map could be reduced. Thus, this parameter must be
optimized before the final analysis. For NEDC matrix and lipid analysis,
the effect is very similar, except for small molecules such as glucose.
In this case, the lower number of matrix layers gives better results.

\textbf{2.2. The spraying nozzle height above the tissue sample}

The position of the spraying nozzle above the sprayed sample is
correlated with the intensity of obtained spectra, but the correlation
depends on the matrix type. In the case of DHB, the peaks intensities
are higher with the higher position of the nozzle (Z=5), but for
norharmane, most intensive peaks are obtained at the lowest position (Z
= 25). The same correlation is for 9AA (Z=5) and norharmane (Z=15) in
the negative ionization mode. Sometimes, the visual effect of matrix
application -- such as local tissue damage or irregular crystallization
-- could be observed. Such circumstances determine the proper matrix
application, as in the case of the NEDC matrix. Our study clearly shows
that such basic optimization is necessary. For example, a higher nozzle
height is frequently recommended for the lipid analysis, but
unexpectedly for the norharmane lower position works better.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image21/image21}

\end{center}
\end{figure}

\textbf{Fig. 21} The spectra of DHB, DAN and norharmane matrix

Considering quite similar performance of different matrices in positive
ionization mode, in our opinion DHB matrix could be chosen as the first
selection (see Fig. 21). Norharmane matrix could be rejected since it
changes the ion map -- probably as a result of the solvent properties.
As it was noted, for several m/z values, we obtained different ion maps
in comparison with DHB matrix. Generally, DHB matrix gives higher peaks
intensity and s/n ratio. Thus, for the measurement of lipids in the
positive ion mode, we recommended this matrix.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image22/image22}

\end{center}
\end{figure}

\textbf{Fig. 22} The mass spectra obtained with NEDC, norharmane, DAN
and 9AA matrix.

Considering the performance of different matrices in the negative
ionization mode (Fig. 22), norharmane matrix could be rejected since it
has low peaks intensity and, as a result, it losses the information from
the low MW range (e.g., peaks from fatty acids). NEDC is even worse for
low MW peaks, but at least it can provide the ion maps for specific
substances like glucose, n-acetyl-aspartic acid, glucose-6-phosphate,
glucose-3-phosphate, and glutamic acid. DAN matrix produces higher peaks
intensity in a lower mass range and seems to provide more information
than 9AA. Additionally, it provides quite high intensities and
signal-to-noise ratios. So, we recommended NEDC for particular tasks
such as glucose analysis, and DAN for the lipids analysis in negative
ionization mode.

\textbf{Table 1.} Summary of sample preparation optimization with the
aid of wet-interface matrix deposition device (SunCollect\selectlanguage{ngerman}®) for MALDI
MSI (L -- the number of matrix layers, Z - different spraying nozzle
height).\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Matrix} & \textbf{Matrix concentration} & \textbf{Solvent} &
\textbf{Z} & \textbf{L}\tabularnewline
\midrule
\endhead
DHB & 25 mg/ml & 50\% MeOH, 0.2\% TFA & 5 & 12\tabularnewline
Norharmane & 6 mg/ml & Chloroform:MeOH:H2O (1:1:1) & 1; 25 &
14\tabularnewline
DAN & 2.5 mg/ml & 50\% ACN & 5 & 14\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Matrix} & \textbf{Matrix concentration} & \textbf{Solvent} &
\textbf{Z} & \textbf{L}\tabularnewline
\midrule
\endhead
9AA & 7 mg/ml & 70\% EtOH & 5 & 14\tabularnewline
NEDC & 7 mg/ml & 70\% MeOH & 5 & 12\tabularnewline
Norharmane & 6 mg/ml & Chloroform:MeOH:H2O (1:1:1) & 15 &
12\tabularnewline
DAN & 2.5 mg/ml & 50\% ACN & 1 & 16\tabularnewline
\bottomrule
\end{longtable}

\textbf{Conclusions}

Wet-interface matrix deposition devices for MALDI mass spectrometry
imaging are convenient and highly effective in use. Proper optimization
of parameters such as the number of matrix layers and the nozzle height
over the sample allows substantial improvement of the analysis
parameters. Thus, the analysis of precious biological samples can be
performed with the best possible quality. However, the care must be
taken during the matrix selection to visualize the molecules of interest
without losing their spatial distribution. Moreover, our analysis shows
that the ion map obtained with the aid of a particular matrix might
differ significantly with the use of another. So, care must be taken
when comparing the results and drawing biological conclusions from such
analyses.

\textbf{Funding:} This work was supported by the Polish National Science
Center (NCN) grant number 2018/29/B/NZ4/02243 to ABK, statutory funds
from the Maj Institute of Pharmacology at the Polish Academy of Sciences
to PM, subsidy no 16.16.160.557 of the Polish Ministry of Science and
Education to ABK, PS, PK and IK.

\par\null

\textbf{Institutional Review Board Statement:} The study was conducted
according to the guidelines of the Declaration of Helsinki and approved
by the Local Ethics Committee (agreement number 137/2018).

\textbf{Data Availability Statement:} All the data present in the study
are available upon request from the corresponding author.

\textbf{Conflicts of Interest:} The authors declare no conflict of
interest. The funders had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the
manuscript, or in the decision to publish the results.

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