Download Combination technique of matrix assisted laser/desorption

Imaging of lipids in cultured mammalian neurons by matrix assisted
laser/desorption ionization and secondary ion mass spectrometry
Short title; Imaging of lipids in cultured mammalian neurons
by MALDI and SIMS
Hyun-Jeong Yang 1,2, Yuki Sugiura 1,2, Itsuko Ishizaki3, Noriaki Sanada3, Koji Ikegami2,
Nobuhiro Zaima 2, Kamlesh Shrivas2, and Mitsutoshi Setou 2
1
Department of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259
Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
2
Department of Molecular Anatomy, Hamamatsu University School of Medicine,
Handayama 1-20-1, Hamamatsu, Shizuoka 431-3192, Japan
3
ULVAC-PHI, 370 Enzo, Chigasaki, Kanagawa, 253-8522, Japan
* Correspondence to: Mitsutoshi Setou, Department of Molecular Anatomy, Hamamatsu
University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka 431-3192,
Japan. Email: [email protected] Telephone/Fax: +81-53-435-2292
* This work was supported by a Grant-in-Aid for SENTAN from Japanese Science and
Technology Agency (JST) and Young Scientists S (2067004) by Japan Society for the
Promotion of Science (JSPS) to Mitsutoshi Setou.
Keywords: MALDI, SIMS, imaging mass spectrometry, SCG, neuron
Abstract
Imaging mass spectrometry (IMS) provides a novel opportunity for visualization of
molecular ion distribution. Currently, there are two major ionization techniques,
matrix-assisted laser desorption/ionization (MALDI) and secondary ion mass
spectrometry (SIMS) are widely used for imaging of biomolecules in tissue samples.
MALDI and SIMS based-IMS have following features; measurable mass ranges
are wide and small, the spatial resolutions are low and high, respectively.
Best of our knowledge, this is a first report to identify the lipids in cultured
mammalian neurons by MALDI-IMS. Further, those neurons were analyzed with
SIMS-IMS in order to compare the distribution pattern of lipids and other derived
fragments. The parameters which influence the identification of lipids in cultured
neurons were optimized in order to get an optimum detection of lipid molecules.
The combined spatial data of MALDI and SIMS supported the idea that the
signals of small molecules such as phosphatidylcholine head groups and fatty acids
(detected in SIMS) are derived from the intact lipids (detected in MALDI-IMS).
Introduction
Imaging mass spectrometry (IMS) provides a novel opportunity for visualization of
cellular morphology via observation of molecular ion distribution in biological samples
[1]
. IMS is a practical molecular imaging tool which based on mass spectrometric
analysis of small organic molecules in the cells and tissues [2], and does not require any
chemical labeling or generation of probes such as antibodies, for each target molecules.
Today, there are two types of ionization methods, such as secondary ion mass
spectrometry (SIMS) and matrix-assisted laser desorption ionization (MALDI), which
are widely used for performing IMS in the biological [3] and medical samples [4, 5].
MALDI-IMS can measure wide mass ranges of ions at m/z in the range of 100-10000
Da, and it can also perform molecular identification via detailed structural analysis by
tandem mass spectrometry (MSn) [6]. Due to the general versatility, a variety of
application of MALDI-IMS has been reported for the visualization of biomolecules
from small metabolites [2, 7, 8] to large proteins [9, 10] in biological and medical samples.
However, the limitation of MALDI-IMS is regarding the lower spatial resolution
(10-100μm) , which is not compatible for imaging of single cell. This drawback can be
resolved by using a SIMS-based IMS, which has a much higher spatial resolution (a few
100 nm) due to the tightly focused primary ion beam. Another advantage of SIMS is an
elimination of an interference of matrix cluster ions by using the matrix-free ionization
method, which is particularly useful for the analysis of small molecules. Recently,
SIMS-IMS has been applied to surface imaging of small molecules in single cells [11, 12].
However, the disadvantage of SIMS is the identification of molecules of small mass
range (m/z <10000 Da.).
Since, the distribution pattern of metabolites within cellular organelles will provide
valuable information about the cellular metabolism and thus we expect to obtain such
information by applying IMS to the cultured cells. However, with MALDI-IMS, we can
only obtain a few pixels from the single cell which is insufficient to visualize
subcellular structures (Figure 1, upper part). In other words, IMS allows for
visualization of relatively large-sized cells to know the molecular distribution within the
distinct cellular compartment. The superior cervical ganglion (SCG) explant culture is
much larger than a single cell and the cluster of cultured neurons are highly polarized,
which allows the comparison of biomolecular distributions between their cell-body and
neurites by both SIMS-IMS and even MALDI-IMS (Figure 1).
In this study, we report IMS of mouse SCG explant culture by using both SIMS-IMS
and MALDI-IMS for the mutual result interpretation. This is the first report to visualize
lipids in cultured mammalian neurons by MALDI-IMS. Further, those cells were
analyzed with SIMS in order to compare the distribution pattern of lipids and their
derived fragments in neurons. These combination supported the idea that the head
groups and fatty acids by SIMS-imaging could be derived from phosphatidylcholine
(PC) which are identified by MALDI-IMS and MS/MS.
Methods
Chemicals.
Methanol, trifluoroacetic acid (TFA), potassium acetate, and lithium acetate were
purchased from Wako Chemical (Tokyo, Japan). Calibration standard peptide and
2,5-dihydroxybenzoic acid (DHB) were purchased from Bruker Daltonics (Leipzig,
Germany).
PLL-Coating of ITO glass slides
Poly-L-lysine (PLL) coat is an essential step for neural cell culture and that can help
in elongating neurites. The steps for coating of PLL on the surface of ITO glass slide
(Bruker Daltonics ) are given. 1) ITO glass slides were irradiated with UV light
overnight in order to sterilize the slide surface. Then, slides were washed with 70% and
100% ethanol for several hours followed by sterilized water for several time. 2) After
air-dry inside the clean bench, autoclaved flexiPERM (Greiner Bio-One) were attached
to the slides. 3) ITO glass slides were coated with 1 mg/ml PLL in borate buffer at 37oC
overnight and washed with sterilized water for one time. 4) Then, slides were incubated
with 10 µg/ml laminin in Minimum Essential Medium (MEM) at 37 oC for at least 3
hours and washed with sterilized water for three times, and then used for the culture.
SCG explant cultures
For SCG explant cultures, ganglions were extracted from ICR pups of DIV 0 and
cultured as previously described [13]. Briefly, dissected SCG was divided in halves and
cultivated on ITO glass slide. The cultures were grown in a humidified atmosphere of
5% CO2/95% air at 37 °C in fresh feeding medium, MEM supplemented with 10% fetal
bovine serum, 50 ng/ml of nerve growth factor (NGF), and 15 μM fluorodeoxyuridine.
The cultures were treated with 6 μM aphidicolin at DIV 1 to eliminate non-neuronal
cells. At DIV 3-5, the cultures were used for experiments.
Sample preparation of SCG explant cultures for IMS
For sample preparations of both MALDI- and SIMS-IMS, the culture medium of
cells was carefully removed by an aspirator along the wall of the flexiperm and washed
three times with 1 mL of 0.1 M phosphate buffer (PB). At the final washing step, PB
was thoroughly removed by an aspirator.
To conserve cells in their native state under high vacuum condition in the mass
spectrometer, we employed the lyophilization method for the fixation of cells [14]. After
the washing step, cells were rapidly frozen by immersion into liquid nitrogen, and then
introduced in the lyophilizer.
Spray-coating of the matrix solution in MALDI-IMS
DHB solution (40 mg/mL DHB, 20 mM potassium acetate, 70% MetOH, 0.1% TFA)
was used as the matrix for imaging of PCs [15]. The matrix solution was sprayed over the
cell surface using a 0.2-mm nozzle caliber airbrush (Procon Boy FWA Platinum; Mr.
Hobby, Tokyo, Japan). The distance between the nozzle tip and the cell surface was
kept at 10 cm and the spraying period was for 5 minutes. Due to the tiny structure of the
SCG cells compared to the typical tissue sections used in MALDI-IMS studies (e.g.,
whole region of mouse brain section), the manipulation of spray-costing should also be
carried out with extreme care because a slight diffusion of analyte could result in the
detection of false localizations in cells. Matrices were applied simultaneously to the
cultured cells that were to be compared to equalize analyte extraction and
co-crystallization conditions [2].
MALDI-IMS measurement
MALDI-IMS was performed by using a MALDI TOF/TOF-type instrument
(Ultraflex 3 TOF/TOF; Bruker Daltonics). This instrument was equipped with a 355 nm
Nd:YAG laser. The data were acquired in the positive reflectron mode under an
accelerating potential of 20 kV using an external calibration method. Signals between
m/z 400 and 1000 were collected. Raster scans on tissue surfaces were performed
automatically using FlexControl and FlexImaging 2.0 software (Bruker Daltonics). The
number of laser irradiations was 100 shots in each spot. Image reconstruction was
performed using FlexImaging 2.0 software.
TOF-SIMS-IMS measurement
The lyophilized cells were directly measured by a Time-of-Flight
(TOF)-SIMS-instrument (Figure 2). The SIMS-imaging was performed by a TRIFT IV
apparatus (ULVAC-PHI). The bunched 60 keV Bi32+ primary ion beam was employed,
and the beam diameters of the Bi32+ were routinely set below 600 nm. The analyzed area
was a square region of 500 μm - 500 μm. The ion dose was 2.4×1011 ions/cm2.
Tandem mass spectrometry
Tandem mass spectrometry was carried out by Q-TOF instrument(Q-star Elite,
Applied Biosystems) in the SCG culture, prepared with same protocol for MALDI-IMS.
Results
Optimization of sample preparation procedure of SCG neuron for both MALDI- and
SIMS-IMS
Until today, how to prepare the samples from cultured cells for the IMS measurement
has still remained to be discussed. While established preparation protocols for
traditional immunostainings and other dye-stainings are available, the procedures to
prepare cultured cells for IMS are still under development; indeed, there are several
problems to be overcome. For example, cells are cultured in the medium which contains
rich nutrition components, however, such molecular components may disturb the
MALDI process [9]. We found that removal of the medium and wash with phosphate
buffer contributes to getting clear signals of PCs in cultured neurons by IMS
measurement (Figure 2 A and B). Without washing procedures, the remnant medium
components formed undesirable crystals on the cells (as shown in Figure 2A), and we
failed to detect any mass peaks (m/z) (Figure 2B). On the other hand, the washing of
cells drastically improved the mass spectrum to detect several mass peaks derived from
phospholipids of the SCG cellular membrane (Figure 2B). The detected molecules in
figure 2B were verified by tandem mass spectrometry and assigned as species of PC
(data not shown).
Another problem is associated with the fixing of brittle cells on the glass slides.
Reagents generally used for cell fixation such as paraformaldehyde (PFA) might cause
signal reduction due to the generation of cross-linkage among the molecules. For small
molecular analysis, loss or migration of small molecules from original location might be
occurred during the fixation treatment. Alternative method should achieve
morphological preservation of cells, avoidance of MS signal contamination, and
efficient ionization of analyte molecule. Although lyophilization has been used in SIMS
[14]
, there has been no report to successfully measure PCs of cultured mammalian
neurons with MALDI-IMS by using this technique. Here, we lyophilized the cultured
neurons after the careful removal of the washing buffer. The lyophilization procedure
did not alter the neuronal morphology in both cell body and neurite (Supplementary
material 1). The lyophilization in combination with above washing step, we succeeded
in obtaining clear signal of PCs in cultured mammalian neurons by MALDI-IMS.
MALDI- and SIMS- imaging of subcellular components of SCG explant cultures
With the sample preparation procedures above, we successfully visualized the
distribution of intact phospholipids in the fan-shaped region of the SCG culture, which
includes the cell-body and full length of neurites, by MALDI-IMS. As a result, we
found that two ions were complementarily distributed between the cell-body and neurite
of SCG neurons (Upper panel of Figure 3). These mass peaks, identified to be derived
from intact PC by tandem mass spectrometry (data not shown), showed considerably
distinct distribution patterns such asion at m/z 734 was localized in the neurite region,
while ion at m/z 826 was only found from cell body region.
SCG neurons were also visualized by SIMS imaging. By this technique, relatively
small fragment molecules such as PC head group or fatty acid, conceivably derived
from the intact phospholipids were mainly detected, and on the other hand, signals for
intact lipids such as phosphatidylcholines were detected as low intensities. The fragment
molecules were visualized with much higher spatial resolution than MALDI-IMS. The
lower panels of figure 3 show high resolution images for four kinds of phospholipid
fragments. In the positive ion detection mode, ions of phospholipid head group and
intact PC molecule ion were detected, while fatty acids such as stearic-acid were
detected in the negative ion detection mode. We identified ion of each mass was
correspondent to each indicated molecule by tandem mass spectrometry using standard
PC samples (data not shown). Intact PC (m/z 782) could be imaged, though only poor
image could be obtained because of the low sensitivity. The MS/MS fragment pattern at
m/z 782 showed that the ion was derived from PC (diacyl-16:0/18:1) (Figure 4). The
ions at m/z 59 and m/z 124 presented the head group of PC, while the ions at m/z 282
and m/z 256 displayed the fatty acid components; oleic acid and palmitic acid,
respectively.
Discussions
Establishment of sample preparation procedures for both MALDI- and SIMS-IMS
In this study, we demonstrated the practical sample preparation procedures for both
MALDI- and SIMS-IMS of murine SCG explant cultures. Our results indicate that the
removal of the culture medium and the wash with PB, prior to IMS, lead to obtain clear
signals of PCs (Figure 2). We also showed the effectiveness of lyophilization as a
cell-fixation procedure for MALDI- and SIMS-IMS (Figure 3, Supplementary material
1).
Regarding the sample preparation procedure for peptide imaging in neuronal model
Aplysia californica by MALDI-IMS, Sweedler’s group has explored several cell
fixation procedures, which are air substitution, FC-43 and mineral oil substitution,
glycerol stabilization, and PFA-fixation [16]. Among them, they found that glycerol
stabilization showed the best signal quality. However, for IMS of small organic
compounds, we have to explore an alternative method, because the glycerol stabilization
might cause generation of a thin layer of the remnant glycerol which covers the cultured
cells, and it requires an additional step to remove the remnant glycerol. It can also bring
an artifact to imaging results because the treatment with glycerol solution can cause
migration of the small molecules from their original location. The washing procedure
using PB buffer after the removal of the culture medium made us obtain high intensity
of intact PCs and their fragment molecules in MALDI and SIMS, respectively.
Further, Sweedler’s group also tried the deionized water and found that no salt
crystals are visible, but the MS signal was of low intensity, possibly from losing
molecules during the hypoosmotic environment [16]. In this study, we were able to keep
the neurons in isotonic environment during the washing procedure, by using 0.1 M PB
buffer.
In addition, it should be mentioned that our trial in MALDI-IMS is a novel one that
challenges to visualize cultured mammalian neurons, which different from the above
mentioned approach where the non-mammalian neuronal model is used.
As another aspect of small metabolite analysis by IMS, we have to pay a special
attention to inhibit activities of enzymes thorough the preparation procedures, because
several metabolites are degraded within short time (even within a minute) [17]. By
immersion of the cells on the glass slides into liquid nitrogen, cells are frozen in a
moment without the metabolite degradation. During the lyophilization process, by
reducing the air pressure, the frozen water in the samples sublimates and is completely
lost from the cells without shrinkage or toughening of the samples [18]. Therefore, even
after the lyophilization, at the room temperature, any enzymatic decomposition of
analyte metabolites could be inhibited, thus the lyophilization is a useful method as a
fixation procedure for IMS. Our results showed that the lyophilization can be a common
fixation for MALDI-IMS as well as SIMS-IMS to visualize neuronal cells (Figure 3).
Although cell preparation methods using lyophilization have been known in SIMS [14],
there have been few reports testing it for cell cultures in MALDI [16] and no reports
which succeed in obtaining clear PC signals in cultured mammalian neurons using
MALDI by this lyophilization. Additionally, as we confirmed this sample preparation is
also available for SIMS (Figure 3), our sample preparation for SCG neuronal cultures
can be used for both analyses to provide complementary information about subcellular
distribution of target molecules.
Application of MALDI- and SIMS-IMS for cultured SCG explant neurons
Here, we introduced the SCG explant neurons to IMS. The SCG neuronal cultures
have been widely used for the study of neurodegeneraiton and axonal growth [13],
because their long and highly polarized axons provide excellent opportunities to study
them. This is also case for IMS, particularly for MALDI-IMS. As described above,
MALDI-IMS successfully visualized the distribution of intact biomolecules, especially
for the membrane phospholipids of the SCG neurons. Our results showed that the two
PC molecules at m/z 734 and m/z 826 form characteristic distribution patterns
depending on the location of neurons (Figure 3 upper panel). Such distribution
information of lipids will uncover novel aspects of lipid-metabolism within the
subcellular components of the neurons, and further helpful in improving the spatial
resolution of MALDI-IMS.
The ion images with much higher resolution were obtained by SIMS-IMS (Figure 3
lower panel). We could even visualize bundle structures of axons and distribution of
ions of fatty acids. Some of these fatty acids were presumably derived from
phospholipids because PC signals in SIMS were quite lower than MALDI in the same
sample (Figure 3, Supplementary material 3). On the other hand, fatty acid signals in
MALDI were quite lower than SIMS (data not shown). Intact PC was fragmented in
SIMS condition but not in MALDI condition (Figure2, 3, Supplementary material 3).
Further, the signals of other PC fragments like polar heads were detected in SIMS but
not so much in MALDI (Figure 3 and Supplementary material 3). Thus, it is highly
possible that some parts of fatty acid are not original intact state but derived from PC.
This suggests that SIMS gives the in-source fragmentation information in this SCG
specimen. This effect is already discussed elsewhere [17, 19], but yet not tested in the
same biological sample. Several fatty acids, particularly poly unsaturated fatty acids
(e.g., arachidonate and docosahexaenoate) are metabolized as bioactive-molecules
which regulate the various biological phenomenon [20]. Imaging of such fatty acids at
high resolution will largely contribute to the field of lipid biochemistry.
Although abnormal lipid homeostasis in neurodegenerative pathology has been
visualized by using several staining reagents, such as oil red O, which is dissolved in
intracellular triglyceride [21], and there is a limitation in understanding the pathogenesis
of lipid-related diseases, within the conventional staining method. Applying the
combinations of two IMS techniques for visualization of lipid molecules provides
access to the critical knowledge about which lipid species are changed in the wide area
by MALDI-IMS and about which signaling molecules are altered in specific area of
interest by SIMS-IMS. We consider that our sample preparation methods for the
visualization of intact phospholipids by MALDI-IMS as well as fragment ions of
phospholipids by SIMS-IMS using cultured neurons offer a novel approaching strategy
in the two techniques for the biomedical researches of lipids. As the SCG neuronal cultures
have been extensively used for research of neurite outgrowth as well as neurite degeneration, the
success of both IMS techniques will offer in understandings about the characteristics of neural lipid
metabolism which has been insufficient in previous researches due to the technical limitations.
Acknowledgments
This work was supported by a Grant-in-Aid for SENTAN from
Japanese Science and Technology Agency (JST) and Young Scientists S (2067004) by
Japan Society for the Promotion of Science (JSPS) to Mitsutoshi Setou.
References
[1]
M. Stoeckli, P. Chaurand, D. E. Hallahan, R. M. Caprioli. Nat Med. 2001,7,493.
[2]
Y. Sugiura, M. Setou. J Neuroimmune Pharmacol. 2009.
[3]
T. Harada, A. Yuba-Kubo, Y. Sugiura, N. Zaima, T. Hayasaka, N. Goto-Inoue, M. Wakui, M.
Suematsu, K. Takeshita, K. Ogawa, Y. Yoshida, M. Setou. Anal Chem. 2009,81,9153.
[4]
S. Shimma, Y. Sugiura, T. Hayasaka, Y. Hoshikawa, T. Noda, M. Setou. J Chromatogr B Analyt
Technol Biomed Life Sci. 2007,855,98.
[5]
Y. Morita, K. Ikegami, N. Goto-Inoue, T. Hayasaka, N. Zaima, H. Tanaka, Uehara, T. Setoguchi,
T. Sakaguchi, H. Igarashi, H. Sugimura, M. Setou, H. Konno. Cancer Science. 2010,in press.
[6]
S. Shimma, Y. Sugiura, T. Hayasaka, N. Zaima, M. Matsumoto, M. Setou. Anal Chem.
2008,80,878.
[7]
T. Hayasaka, N. Goto-Inoue, Y. Sugiura, N. Zaima, H. Nakanishi, K. Ohishi, S. Nakanishi, T.
Naito, R. Taguchi, M. Setou. Rapid Commun Mass Spectrom. 2008,22,3415.
[8]
Y. Sugiura, S. Shimma, Y. Konishi, M. K. Yamada, M. Setou. PLoS One. 2008,3,e3232.
[9]
Y. Sugiura, S. Shimma, M. Setou. Anal Chem. 2006,78,8227.
[10]
I. Yao, Y. Sugiura, M. Matsumoto, M. Setou. Proteomics. 2008,8,3692.
[11]
S. G. Ostrowski, C. T. Van Bell, N. Winograd, A. G. Ewing. Science. 2004,305,71.
[12]
A. F. Altelaar, I. Klinkert, K. Jalink, R. P. de Lange, R. A. Adan, R. M. Heeren, S. R. Piersma.
Anal Chem. 2006,78,734.
[13]
K. Ikegami, T. Koike. Neuroscience. 2003,122,617.
[14]
A. F. M Altelaar, I. Klinkert, K. Jalink, R. P. de Lange, R. A. H. Adan, R. M. A. Heeren, S. R.
Piersma. Anal Chem. 2006, 78, 734
[15]
Y. Sugiura, M. Setou. Rapid Commun Mass Spectrom. 2009,23,3269.
[16]
S. S. Rubakhin, W. T. Greenough, J. V. Sweedler. Anal Chem. 2003,75,5374.
[17]
Y. Sugiura, Y. Konishi, N. Zaima, S. Kajihara, H. Nakanishi, R. Taguchi, M. Setou. J Lipid Res.
2009,50,1776.
[18]
R. W. Dudek, G. V. Childs, A. F. Boyne. J Histochem Cytochem. 1982,30,129.
[19]
P. Sjovall, J. Lausmaa, H. Nygren, L. Carlsson, P. Malmberg. Anal Chem. 2003,75,3429.
[20]
M. Murakami, Y. Nakatani, G. Atsumi, K. Inoue, I. Kudo. Crit Rev Immunol. 1997,17,225.
[21]
L. Wang, G. U. Schuster, K. Hultenby, Q. Zhang, S. Andersson, J. A. Gustafsson. Proc Natl Acad
Sci U S A. 2002,99,13878.
[22]
M. Setou Edited, Imaging Mass Spectrometry, Japan, Springer, 2010.
[23]
M. Setou Edited, Sitsuryoukenbikyouhou, Japan, Springer, 2008.
Figure legend
Figure 1. Size comparison of typical laser spot diameter of matrix-assisted laser
desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS)
instrument and cultured cells.
(Top panel) Microscopic observation of “burned-out” region of tissue section coated
with 2,5-dihydroxybenzoic acid (DHB) matrix. In this case, the diameter of the spot is
approximately 60 µm. (Bottom panel) Size comparison of the burned-out spot size and cultured
cells, which are a Ptk2 cell, hippocampal neuron, and superior cervical ganglia (SCG) neuron.
(Partly modified from the figure introduced in the author’s recent text book [22])
Figure 2. Washing neurons with PB contributes to getting clear signals for
MALDI-IMS.
Pictures (A) and spectra (B) of lyophilized SCG neurons, with/without washing
procedure by phosphate buffer (PB). (Figure 2A is partly modified from the figure introduced
in the author’s recent text book [22])
Figure 3. MALDI and SIMS images of cultured SCG neurons.
Normarski image in culture and an image of MALDI-IMS (top panel). SIMS images
measured in positive (middle panel) and negative mode (lower panel). (Partly modified
from the figure introduced in the author’s recent text book [22,23])
Figure 4. Product ion mass spectra of ion at m/z 782
We performed the MS/MS of ion at m/z 782 and obtained the shown product ion mass
spectra. Neutral losses (NL) of 59, 124 corresponding to tri-methyl amine and
cycrophosphate, respectively, demonstrate that the ion was PC molecular species.
Furthermore, NL of 256 and 282 from the tri-methyl amine-lost PC indicates that this
molecule is containing palmitic (C16:0) and oleic acid (C18:1).
Supplementary material 1. Significant morphological changes were not observed
before and after freeze-drying
The morphology of cultured SCG neurons was shown by staining with DiI before and
after freeze-drying. Images were acquired by confocal microscope. The cell bodies and
the major thick neurites were preserved before and after the procedure.
Supplementary material 2. The spectra of PC standard by SIMS
On positive mode, standard PC compound was measured by SIMS. Intact PC shows low
intensity, while its fragmented ions show high intensity.
Supplementary material 3. The spectra of cultured SCG neurons by MALDI and
SIMS
On positive mode, cultured SCG neurons were measured by MALDI and SIMS. The
spectra of SIMS show low intensity in high mass range (shown as black and dark grey
bars on X axis), while those of MALDI present high intensity. In addition, some spectra
of SIMS demonstrate high intensity in low mass range (shown as white and light grey
bars on X axis).