Download Localization of lipids in freeze-dried mouse brain sections by

Localization of lipids in freeze-dried mouse brain sections by
imaging TOF-SIMS
1
Peter Sjövall1, Björn Johansson2 and Jukka Lausmaa1
SP Swedish National Testing and Research Institute, P.O. Box 857, SE-50115 Borås, Sweden
2
Department of Clinical Neuroscience, Karolinska Institutet, SE-17176 Stockholm, Sweden
Abstract
Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to analyse the
lateral distributions of lipids on the surface of freeze-dried mouse brain sections. Tissue sections (14
µm thick) were prepared by cryosectioning, placed on glass or Si substrates, and desalinated by
submersion in NH3HCOO solution. Immediately prior to analysis, the samples were freeze dried by
thawing the sample in vacuum. TOF-SIMS analysis was carried out using 25 keV Au3+ or Bi3+
primary ions, always keeping the accumulated ion dose below 4 x 1012 ions/cm2. Positive and
negative ion images over the entire mouse brain section and of analysis areas down to 100 x 100
µm2 show characteristic distributions of various lipids. The signals from cholesterol and sulfatides
are primarily located to white matter regions, while the phosphocholine and phosphatidylinositol
signals are strongest in grey matter regions. By using two different staining methods, structures
observed in the TOF-SIMS images could be identified as ribosome-rich regions and cell nuclei,
respectively. Analysis of freeze-dried mouse brain sections at varying sample temperatures between
-130 and 60 ºC showed an abrupt increase in the cholesterol signal at T > 0 ºC, indicating extensive
migration of cholesterol to the tissue surface under vacuum conditions.
Keywords:
TOF-SIMS, lipids, tissue, brain, imaging
1. Introduction
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) provides a number of advantages
over any other single method used for chemical analysis of biological samples, such as parallel
identification and localization of unlabelled substances at high spatial resolution[1]. Local chemical
information about cells and tissues is typically obtained either by mass spectrometric analysis of
whole-tissue samples, isolated membranes or cultured cells[2, 3] or by fluorescence imaging of
labelled substances[4-6]. Using imaging MALDI, spatially resolved mass spectrometric detection of
biomolecules up to 100,000 u (including proteins) can be obtained, however with a lateral
resolution typically around 50 µm[7, 8]. Imaging of elements, isotopically labelled compounds and
certain organic fragments can be obtained with high sensitivity and lateral resolution below 100 nm
by dynamic SIMS[9-11]. The potential advantage of TOF-SIMS in the analysis of biological cells
and tissues, as compared to other methods, is to provide unambiguous identification, localization
and co-localization of organic molecules in the mass range ~200-2000 u, which is the mass range
that includes many of the most common lipids, peptides, metabolites, and drug substances. The
possibilities to develop TOF-SIMS into a powerful method in this respect have recently improved
dramatically due to the development of new cluster primary ion sources, which provide superior
useful secondary ion yields for organic compounds in this mass range, as compared to the previous
standard sources[12, 13].
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The fact that the TOF-SIMS analysis is made in vacuum and that classical fixation schemes are
often inappropriate to use since they alter the chemistry of the sample, means that sample
preparation for TOF-SIMS analysis requires careful consideration. The two main strategies that
have been used so far are to study the cell or tissue sample in the frozen hydrated state[14, 15], or to
freeze dry the sample prior to analysis[12, 13, 16]. The former method requires careful temperature
control and the analysis is made difficult by the presence of water in the sample. For freeze-dried
samples, the analysis is significantly easier to carry out, but one must consider that the drying
process changes the environment of the cellular and subcellular structures, most likely resulting in,
e.g., considerable alterations of the membrane structure[10]. In order to obtain relevant biological
information from TOF-SIMS images, it is therefore important to determine the effects of the drying
procedure on the spatial distributions of the investigated biomolecules.
An additional consideration in imaging TOF-SIMS analysis of cells and tissues is the
interpretation of ion images measured by TOF-SIMS in terms of known biological structures.
Fortunately, extensive knowledge about biological structures, from the subcellular to organized
tissue level, is available from histological and electron microscopy studies. If TOF-SIMS is to
contribute to the bioscience research field by providing new significant information of biological
relevance, it is required that the structures studied by TOF-SIMS are conclusively identified in
terms of structures studied by histology or other methods established in the field.
In previous work, we have shown that imaging TOF-SIMS may provide information about the
spatial distributions of a variety of specific lipids in mouse brain sections, including cholesterol,
phosphatidylcholine, sulfatide and phosphatidylinositol[12]. The obtained images showed a
pronounced complementary localization of cholesterol and phosphatidylcholine (PC), in which the
cholesterol signal was high in the white matter regions and PC was high in the grey matter regions.
Furthermore, the sulfatide images showed distributions with low signal in the regions with high
cholesterol signal (white matter), which is surprising since previous studies with other techniques
have shown that the sulfatide concentration is higher in white matter than in grey[17].
In the present work, results from mouse brain sections prepared using a slightly modified
freeze-drying procedure as compared to the one used in the previous work, show high signal
intensity from sulfatide in white matter regions, suggesting higher sulfatide concentration in white
matter as compared to in grey matter. Furthermore, it is shown that migration of cholesterol in
vacuum may occur at sample temperatures close to room temperature, which could explain the
previous contradictory results. Finally, the present study also shows that different structures
observed in the TOF-SIMS images could be identified by combining the analysis with two different
staining techniques.
2. Experimental
2.1. Sample preparation
The mouse brains were frozen to -80 ºC immediately after dissection. Thin (14 µm) tissue
sections were prepared using a cryosectioning device and placed on pre-cooled substrates (glass
slides except in the temperature studies, see below). In order to attach the tissue section to the
substrate, the back side of the substrate was gently warmed up using finger contact until the tissue
section just started to thaw, and then the sample was quickly refrozen to -80 ºC. In order to reduce
the salt content in the tissue sections, the samples were immersed in 0.15 M NH3HCOO solution
(room temperature) for approximately 30 s and then immediately refrozen, placed in plastic or glass
containers and stored at -78 ºC (dry ice) until freeze drying and subsequent analysis. The analysis
was normally carried out within less than 5 days after dissection of the mouse brain.
Freeze drying was normally done by placing the cold sample on precooled glass plates inside a
vacuum chamber, immediately evacuating the system and slowly allowing the sample to warm up
to room temperature during constant pumping (< 10-3 mbar). After approximately 30 – 40 minutes,
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the sample had reached room temperature but pumping was normally continued for another 20 – 30
minutes. After freeze drying, the samples were immediately introduced into the TOF-SIMS
instrument for analysis.
2.2. TOF-SIMS analysis
The analysis was done in a TOF-SIMS IV instrument (ION-TOF GmbH) equipped with a liquid
metal cluster primary ion source. Positive and negative ion spectra and images were recorded using
Au3+ or Bi3+ primary ions at 25 keV energy and electron flooding for charge neutralization. High
mass resolution data was recorded from analysis areas between 200 x 200 µm2 and 11 x 11 mm2
(bunched mode, 0.06 pA for Au3+ and 0.18 pA for Bi3+). High image resolution data was recorded
at areas between 35 x 35 µm2 and 200 x 200 µm2 (burst alignment mode, 0.03 pA, Au3+ only). The
primary ions were Bi3+ in the studies using different temperatures (see below) while all other results
were obtained using Au3+. The accumulated primary ion dose was always kept below 4 x 1012
ions/cm2.
2.3. Temperature-controlled analysis
For the analysis at varying sample temperatures, the tissue sections were deposited on Si wafers
in order to obtain good thermal contact with the sample holder, and the immersion in NH3HCOO
was omitted in order to keep the sample cold until analysis. Experiments using two different freezedrying procedures were carried out. In the first experiment, freeze drying was done in the separate
vacuum chamber as described above, with the exception that the chamber was vented after
approximately 20 minutes of pumping (with the sample temperature still slightly below room
temperature). The sample was then immediately mounted on a pre-cooled sample holder and
introduced into the TOF-SIMS instrument. Positive and negative ion images and spectra were
recorded at the same area of the mouse brain section (anterior commissure in the horizontal section)
after successively increasing temperatures between -130 and 60 ºC. The sample temperature was
increased at a rate of 0.5 K/s and kept constant at the stated temperature for 4 minutes before start of
the data acquisition. The accumulated ion dose was < 4x1010 ions/cm2 per spectrum which means
that the total accumulated primary ion dose for the whole experiment (10 positive and 10 negative
spectra) was less than 1012 ions/cm2. In the second experiment, freeze drying was carried out inside
the vacuum chamber of the TOF-SIMS instrument. For this, the cold sample was directly mounted
on the precooled TOF-SIMS sample holder, followed by immediate insertion into the load lock
vacuum chamber and pump down. The sample temperature during freeze drying was estimated to
be initially around -50 ºC, then quickly (< 2 minutes) lowered to approximately -100 ºC and then
gradually decreasing to -120 ºC. The analysis was started approximately 2 hours after start of the
freeze-drying procedure. By using this procedure, the sample was prevented from being warmed up
or exposed to air between freeze drying and analysis.
2.4. Staining procedures
For staining of the sample with eosin Y, the samples were immersed in phosphate-buffered
saline (PBS) containing 1 % eosin Y and then destained in PBS only, prior to submersion in
NH3HCOO solution. Eosin Y is a Br-containing organic molecule that provides staining to the
cytoplasm.
Cresyl violet staining was applied after TOF-SIMS analysis to the same sagittal tissue sample
that was used for recording of the data presented in figs. 1-4 (stored in exsiccator for 94 days
between TOF-SIMS analysis and staining). Cresyl Violet staining is considered to stain ribosomes
but also to some extent cell nuclei. For the cresyl violet staining, the sample was delipidated (xylene
2 x 5 min), fixed (99.5% ethanol for 2 x 5 min), hydrated (95% ethanol for 5 min; 70% 5 min;
dipped in H2O), and then stained in Cresyl Violet acetate (Sigma) with 0.4 M acetate buffer (pH
3.9) for 30 min. After staining, the sample was differentiated in H2O for 2 min and dehydrated (70%
ethanol 5 min; 95% 5 min; 100% 5 min; 100% 5 min; xylene 2 x 5 min). Finally, a coverslip was
immediately mounted on the sample slide with Pertex (Histolab, Göteborg, Sweden) and allowed to
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settle overnight. The optical image of the stained tissue was photographed with a Hamamatsu
C3077 CCD camera with a Micro-NIKKOR objective.
3. Results and discussion
3.1. TOF-SIMS analysis of sagittal mouse brain section
Figure 1 shows TOF-SIMS images from an entire mouse brain section in the sagittal plane.
Different structures, such as the cortex, caudate putamen, hippocampus, thalamus, and the
cerebellum, can be easily localized in the images (see fig. 1(g)). The thalamus region is specifically
highlighted in fig. 2, which shows ion images from a separate measurement of this region.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
co
co
cp
co
th hi
co
co
ce
Fig. 1. TOF-SIMS images from a freeze-dried mouse brain section in the sagittal plane. The images
show the signal intensity distributions of (a) phosphocholine+ (184 u), (b) cholesterol- (385
u), (c) CN- (26 u) + CNO- (42 u), (d) phosphatidylinositol- PI 38:4, and (e) sulfatide- 24:1 +
24:0 + h24:1 + h24:0. All images have been normalized to the total ion image. Figure (f)
shows an optical microscopy image of the same tissue section after cresyl violet staining and
(g) shows the phosphocholine image with some major brain structures indicated (co-cortex,
cp-caudate putamen, th-thalamus, hi-hippocampus, ce-cerebellum). Field of view 11 x 11
mm2.
The cholesterol images in figs. 1 and 2 show high intensity in the white matter regions while the
phosphocholine signal displays a higher signal in the grey matter regions. This is an expected result
since the white matter regions consist primarily of nerve cell fibers, axons, surrounded by myelin,
which are rich in cholesterol. Grey matter, in contrast, consists of nerve cell bodies and glial cells,
whose plasma membranes also contain cholesterol, but in lower concentrations than myelin.
The sulfatide images show generally higher signal intensity in the white matter regions, also in
agreement with what can be expected from previous studies[17], but the signal is “smeared out”
into the grey matter regions to a larger extent than the cholesterol signal is. The bright area in the
lower left part of the sulfatide image in fig. 1 is outside the tissue section and is most likely caused
by deposition of sulfatide from the tissue during placement of the sample onto the substrate surface.
The phosphatidylinositol image (fig. 1(d)) shows a homogeneous intensity distribution in the grey
matter regions and a slightly reduced signal in the white matter regions.
As discussed in section 3.3 below, the strong cholesterol signal in figs. 1 and 2 is most likely
partly due to migration of cholesterol to the surface from the sample interior, which is shown to
occur in vacuum at temperatures > 0 ºC (see section 3.3). Although the cholesterol signal thus
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cannot be considered to show the cholesterol concentration on the original tissue sample surface,
the lateral distribution in the cholesterol images can be expected to reflect the cholesterol
distribution in the surface region of the tissue sample.
(a)
(c)
(b)
(d)
Fig. 2. TOF-SIMS images from the thalamus region of the sagittal tissue section shown in fig. 1.
The images show the signal intensity distributions of (a) CN- + CNO-, (b) PO2- (63 u), (c)
cholesterol-, and (d) sulfatide- 24:1 + 24:0 + h24:1 + h24:0. All images have been
normalized to the total ion image. Field of view 3.0 x 3.0 mm2.
Figures 1 and 2 also show ion images of the added signal intensities from the CN- and CNOfragments. These fragments are non-specific signatures of nitrogen-containing organic compounds
in the tissue section. They can therefore be expected to show higher signal intensity in regions with
high concentrations of nitrogen-rich substances, such as proteins, peptides and nucleotides.
Inspection of the CN/CNO images shows increased intensity in several rounded linear structures
just outside the thalamus region (the two bright irregular spots on each side of the thalamus region
are due to cracks in the tissue section). The origin of the high CN/CNO intensity in these structures
can be rationalized from fig. 1(f), which shows an optical microscopy image of the same tissue
section from which the TOF-SIMS images were obtained, after subjecting the sample to cresyl
violet staining. The microscopy image shows strong staining in the same rounded structures around
the thalamus region as can be seen in the CN/CNO images. In addition, strong staining is also
evident around the cholesterol-rich tree-like structures of the cerebellum and close inspection of fig.
1(c) shows increased intensity also of the CN and CNO TOF-SIMS signal in these areas. Since
cresyl violet specifically stains ribosomes, the positive correlation between the microscopy image in
fig. 1(f) and the CN/CNO images in fig. 1(c) indicates that the structures with increased CN/CNO
signal intensity reflect regions with high concentration of ribosomes. Considering the fact that
ribosomes are the locations for protein synthesis in the cells, the high CN/CNO signal intensity in
the ribosome-rich structures can be rationalized by the expected high concentration of proteins in
these structures. In addition to the structures discussed above, fig. 1 (c) shows increased CN/CNO
signal along the right edge of the tissue section (cortex). This area does not show any considerable
staining by cresyl violet in fig. 1 (f) and the origin of the strong CN/CNO signal in this area is not
known.
Figure 3 shows TOF-SIMS images at two different magnifications from the area in the caudate
putamen region that is indicated by the square in fig. 1(a). The upper images (field of view 500 x
500 µm2) were recorded with the instrument in the bunched mode (high mass resolution, low lateral
resolution), while the lower images (100 x 100 µm2) were recorded in the burst alignment mode
(high lateral resolution). The ion images were normalized to the total ion image in order to reduce
the image contrast due to topographic variations on the sample surface. An indication of the sample
topography can be seen in the total ion image in fig.3.
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500 x 500 µm2
(a)
100 x 100 µm2
CN-+CNO(e)
CH4N++C4H8N+
(b)
Phosphocholine+
(f)
Phosphocholine+
(c)
Cholesterol(g)
Cholesterol+
(d)
Sulfatide- (24:0/1+h24:0/1)
(h)
Total ion+
Fig. 3. TOF-SIMS images at two different magnifications from the caudate putamen region of the
sagittal tissue section. The upper images (500 x 500 µm2) were recorded from the area
indicated by the square in fig. 1(a) and the lower images (100 x 100 µm2) were recorded
from the area indicated by the square in the upper phosphocholine image in this figure. The
ion images have been normalized to the total ion image.
As in figs. 1 and 2, the images in fig. 3 show complementary localization of the phosphocholine
and cholesterol signal intensities, indicating complementary localization of cholesterol and
phosphatidylcholine also in structures at sizes down to the micrometer range. The 100 x 100 µm2
cholesterol image, however, shows a more inhomogeneous distribution than phosphocholine,
indicating accumulation of cholesterol in point-like structures in the micrometer range (or less). The
sulfatide image shows high signal intensity in the regions with high cholesterol signal, indicating
higher concentration of sulfatide in the axons, and/or the myelin sheaths surrounding the axons,
than in the nerve cell bodies and glial cells.
The CN-+CNO- and CH4N++C4H8N+ images, both representing nitrogen-rich organic
compounds (e.g., proteins), show characteristic structures with spots in the ~7-10 µm range. A
possible origin of these structures are cell nuclei, since cell nuclei contain high amounts of
nitrogen-rich compounds (proteins, nucleotides) and their size is consistent with the size of cell
nuclei. This interpretation is also corroborated by results using eosin Y staining described below.
Figure 4 shows (a) positive and (b) negative TOF-SIMS spectra from the area shown in the
500x500 µm2 images in fig. 3. Spectra have been extracted separately from the phosphocholine-rich
and cholesterol-rich areas in the images. The most prominent peaks in these spectra and
assignments are listed in table 1.
The negative spectra contain peaks that can be identified as molecular ion peaks of a number of
different lipids. Peaks that have been identified previously[12] include cholesterol (monomer at 385
u and dimer at 771 u) and a number of different sulfatide and phosphatidylinositol compounds. The
variable fatty acid chains in the two latter types of compounds are specified with regards to the
number of carbon atoms, X, and double bonds, Y, in the X:Y notation in table 1.
Phosphatidylinositol and phosphatidylcholine contain two variable fatty acids, and here the X:Y
notation specifies the total numbers of carbon atoms and double bonds in the two fatty acid chains,
while sulfatide, sphingomyelin and ceramide has one variable and one fixed fatty acid. The variable
fatty acid of sulfatide is connected to the head group either with an amide bond or a hydoxylated
alpha-carbon (containing an extra O-atom next to the keto group), the latter of which is notated with
an h preceding the fatty acid notation in table 1.
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Positive spectra
Negative spectra
Mass
Assignment
Mass
Assignment
478.39
Pg + 16:0
644.55
SM 18:0 –
N(CH3)3(CH2)2
2 .0
494.36
Pg + 16:0 + O
670.57
SM 18:0 – N(CH3)3
1 .0
496.37
unassigned
673.53
PA 34:1*
504.41
Pg + 18:1
699.55
PC 34:1 – N(CH3)3
506.45
Pg + 18:0
715.62
SM 18:0 – CH3
1 .5
520.41
Pg + 18:1 + O
771.81
Cholesterol2
1 .0
522.40
Pg + 18:0 + O
794.64
unassigned
0 .5
548.57
Ceramide 18:0*
806.60
Sulfatide 18:0
577.44
unassigned
822.60
Sulfatide h18:0
630.53
Ceramide 24:1*
834.62
Sulfatide 20:0
734.57
PC 32:0
850.66
Sulfatide h20:0
753.61
Cholesterol2 – H3O
857.59
PI 36:4
760.59
PC 34:1
862.67
Sulfatide 22:0
788.62
PC 36:1
878.69
Sulfatide h22:0
796.51
unassigned
2
10
(a)
4 .0
Phosphocholine area
PC
PC
Intensity (counts)
3 .0
1 02
Cer
Ch
2 .0
500
1 03
0 .8
0 .6
Intensity (counts)
Cholesterol area
0 .4
0 .2
600
700
Phosphocholine area
(b)
800
/u
PI
SM
PC
1 02
Cholesterol area
4 .0
3 .0
S
Ch
2 .0
S
1 .0
885.67
PI 38:4
Pg – PC minus fatty acids:
C8H18NPO4
888.68
Sulfatide 24:0
16:0 – palmitate: C16H31O2
890.69
Sulfatide 24:1
18:1 – oleate: C18H33O2
904.69
Sulfatide h24:0
18:0 – stearate: C18H35O2
906.71
Sulfatide h24:1
* Preliminary assignment
700
800
900
/u
Fig. 4. Positive (a) and negative (b) TOF-SIMS
spectra from the cholesterol-rich and
phosphocholine-rich areas shown in fig. 3.
Table 1. Observed peaks and peak
assignments in positive and negative
TOF-SIMS spectra from freezedried mouse brain sections (see
spectra in fig. 4).
The positive spectra show peaks that previously have been identified as molecular ions of
phosphatidylcholine between 730 and 790 u. In addition, a number of peaks around 500 u were
observed, which can be identified as phosphatidylcholine with one of the fatty acids removed. The
same peaks were observed also in spectra from reference samples of pure phosphatidylcholine and
can therefore not be conclusively assigned to lysophosphatidylcholine compounds in the tissue
sample.
Based on reference spectra from pure sphingomyelin, several of the peaks between 640 and 720
u in the negative spectra can be identified as quasimolecular ions of sphingomyelin. The different
peaks originate from the same compound (SM 18:0) but correspond to the loss of variably large
portions of the phosphocholine head group.
The peaks at 548 and 630 u in the positive spectra (particularly strong in the spectrum from the
cholesterol-rich region) can, based on ESI mass spectra[18], tentatively be assigned to ceramide
18:0 and ceramide 24:1. The observation of these particular ceramide compounds (i.e. ceramide
18:0 and 24:1) is consistent with the known ceramide composition in neural tissue[19].
The assignment of the peak in the negative spectra at 673 u to phosphatidic acid is also based on
ESI mass spectrometry data[20]. It is, however, not possible to exclude that the peak can originate
from phosphatidylcholine (loss of N(CH3)3(CH2)2, as in the case of sphingomyelin).
When comparing the spectra from the phosphocholine-rich and cholesterol-rich areas in fig. 4,
significant differences can be observed. In the positive spectra, the signal intensity from the peaks at
548 u (ceramide 18:0), 771 u (cholesterol2) and the unassigned peak at 794 u are significantly larger
7
in the cholesterol-rich area, as compared to the phosphatidylcholine peaks. In the negative
spectrum, the signal intensity from phosphatidylinositol (857 and 885 u) and sphingomyelin (644,
670 and 715 u) are significantly stronger in the phosphocholine-rich area, as compared to the
sulfatide signal.
3.2. TOF-SIMS analysis of eosin Y stained mouse brain section
Figure 5 shows ion images from a tissue section that has been stained with eosin Y. The fact
that eosin Y contains four Br atoms per molecule makes it easy to detect by TOF-SIMS, providing
strong Br signals and easily identified quasimolecular peaks (e.g., 647 u) in the negative spectra.
(a)
(b)
(c)
(d)
Fig. 5. Negative TOF-SIMS images of (a) CN-+CNO-, (b) PO3-, (c) 79Br- + 81Br- and (d) a
quasimolecular ion of eosin Y at 647 u. Field of view 200 x 200 µm2.
The image of Br and the quasimolecular ion of eosin Y show essentially the same lateral
distributions, as expected since both ions represent the localization of eosin Y on the tissue surface.
The phosphate image, in contrast, shows a complementary distribution, i.e. weak intensity where
the Br signal is strong and vice versa. A tentative explanation for this observation is that the regions
with high signal intensity from eosin Y represent areas in which the cytoplasm is exposed on the
surface, which can be the case when the cells have been cleaved during the cryosectioning
procedure. The phosphate-rich regions on the other hand, would then represent regions where the
cryosectioning has occurred at the cell surfaces, thereby exposing the plasma membrane on the
sample surface, consistent with a higher concentration of phospholipids.
As in fig. 3, the image of the CN+CNO fragment ions in fig. 5(a) shows a spot-like distribution.
If the distribution of the spot-like structures in the CN/CNO image is compared to the other images
in fig. 5, it is evident that the CN/CNO structures almost exclusively are located in areas with strong
signal from eosin Y, i.e. according to the discussion in the previous paragraph, from regions with
cleaved cells. This observation corroborates the suggestion made in the discussion of fig. 3, namely
that the spot-like structures in the CN/CNO images originate from cell nuclei. If cell nuclei should
be observed in TOF-SIMS, they must be exposed on the surface and that can only occur if the cell
has been cleaved prior to analysis.
3.3. Sample temperature controlled TOF-SIMS analysis of mouse brain sections
Figure 6-8 show results from experiments in which the effect of sample temperature on the
recorded TOF-SIMS images and spectra was studied. In the first experiment, the tissue sample was
freeze dried in a separate vacuum chamber, allowed to reach a temperature just below room
temperature (approximately 10 ºC), and then mounted on a cold stage sample holder, inserted in the
TOF-SIMS instrument, and finally analysed at increasing temperatures from -130 to 50 ºC.
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T = -110 ºC
T = 30 ºC
CNO-
Phosphocholine+
Cholesterol-
Sulfatide-
Fig. 6. TOF-SIMS images of selected ions from mouse brain section (including the anterior
commissure, coronal orientation) after freeze drying in separate vacuum chamber and
analysis at T = -110 ºC and upon heating to T = 30 ºC, respectively. Field of view 500 x 500
µm2.
In fig. 6, a comparison of TOF-SIMS images for CNO-, positive phosphocholine, and negative
cholesterol and sulfatide signals between T = -110 ºC and after annealing to T = 30 ºC shows that
the sample temperature has a significant effect on the lipid distributions on the tissue surface. The
sulfatide image shows significantly stronger signal intensity and more distinct localization to the
anterior commissure region at the lower temperature, while the opposite is true for cholesterol, i.e.
stronger signal and more distinct localization to the same region at the higher temperature.
Furthermore, the phosphocholine signal distribution shows a clearer separation between the anterior
commissure region and its exterior at the higher temperature than at the lower. These observations
indicate that cholesterol migrates from the interior of the sample out to the surface of the tissue
sample during annealing of the sample.
cholesterol (x1)
sulfatide (x6)
PI (x150
PC (x1.5)
(a)
4000
2000
0
-120
-80
-40
Temperature (C)
0
40
Normalized TOF-SIMS Signal (arb. units)
Normalized TOF-SIMS Signal (arb. units)
A more detailed account of the development of the ion signal intensities during the annealing
process is shown in fig. 7. Figure 7(a) shows the signal intensities (normalized to the total ion
intensity) as a function of sample temperature from the cholesterol-rich (anterior commissure)
region and fig. 7(b) shows the corresponding results from the phosphocholine-rich region.
6000
cholesterol (x50)
sulfatide (x50)
PI (x100)
PC (x1)
(b)
4000
2000
0
-120
-80
-40
Temperature (C)
0
40
Fig. 7. Normalized signal intensities of selected ions in TOF-SIMS spectra from freeze-dried
mouse brain sections, as a function of sample temperature upon heating of the sample from
T = -130 ºC to 50 ºC. Figure (a) shows the signal intensities in spectra from the cholesterolrich area only and fig. (b) shows the results from the phosphocholine-rich area (see fig. 6).
9
In the cholesterol-rich area, the cholesterol signal is constant at a relatively low level at
temperatures below 0 ºC, but increases abruptly at T > 0 ºC, while the phosphocholine signal shows
a small decrease up to T = 0 ºC and a steeper decrease at higher temperatures. In the
phosphocholine-rich area, the cholesterol signal shows a minor increase at T > -30 ºC, while the
phosphocholine signal decreases gradually over the entire T range. Sulfatide and PI show gradually
decreasing signal intensities with increasing temperatures both in the cholesterol-rich and
phosphocholine-rich areas.
After annealing and analysing the sample at 50 ºC, the sample temperature was decreased to T =
-110 ºC and again analysed. The resulting images and spectra were not significantly different from
the data obtained during annealing to 50 ºC, which indicates that the processes responsible for the
changes in the lipid distributions upon annealing are not reversible, but, instead, are the results of
kinetically hindered processes that transfers the system to an energetically more stable
configuration.
The results in fig. 7 indicate that there are two separate processes occurring; one in which the
concentration of sulfatide, PC and PI on the surface gradually decreases and one in which extensive
migration of cholesterol to the sample surface occurs at temperatures above 0 ºC. The decrease in
the cholesterol signal in the phospholcholine rich regions may indicate a third process, involving
lateral diffusion of cholesterol into the anterior commissure. These two first processes can be
rationalized by the changed environment that the membrane structures are subjected to prior to
analysis, from a hydrated (hydrophilic) environment surrounded by water to a vacuum
(hydrophobic) environment, and that the energetically most stable configurations in the two
different environments involve different compounds at the surface. It is quite reasonable to assume
that sulfatide and the phospholipids due to their more polar nature are more stable than cholesterol
at the surface in a hydrated environment but that this may be reversed in vacuum. At low
temperatures, the system may be kept in the original (hydrated) configuration due to kinetic energy
barriers for migration, but upon increasing the sample temperature, these barriers may be surpassed
and thereby allowing the cholesterol migration to the surface to occur.
Normalized TOF-SIMS Signal (arb. units)
In order to investigate possible lipid migration during transfer of the sample from the separate
vacuum chamber (after freeze drying) to the TOF-SIMS instrument, an experiment was carried out
in which the sample was freeze dried in situ inside the TOF-SIMS vacuum chamber and, thus, not
subjected to temperatures above ~ -50 ºC or exposure to air between freeze drying and analysis at
low temperatures.
Cholesterol (i) (x8)
Cholesterol (ii) (x8)
PC (i) (x1)
PC (ii) (x2)
CNO (i) (x1)
CNO (ii) (x2)
6000
4000
2000
0
-140
-120
-100
-80
-60
-40
-20
Temperature (C)
0
20
40
60
Fig. 8. Normalized signal intensities of CNO-, positive phosphocholine, and negative cholesterol
ions in TOF-SIMS spectra from the same region in two differently prepared mouse brain
10
sections (anterior commissure, coronal orientation, total area), as a function of sample
temperature upon heating of the sample from T = -130 ºC to 60 ºC. Sample (i), filled
symbols, was freeze-dried in a separate vacuum chamber and thawed before analysis at low
temperature, while sample (ii), open symbols, was freeze-dried in the TOF-SIMS chamber
and not thawed or exposed to air before analysis.
In fig. 8, the temperature dependences of the CN-, positive phosphocholine and negative
cholesterol signal intensities are compared for the two types of sample preparations, i.e. freeze
drying in a separate vacuum chamber or in situ. The TOF-SIMS data was recorded from equivalent
areas (anterior commissure) of the two different tissue samples. The diagram in fig. 8 shows that the
main features of the temperature dependences are similar for the two different methods of freeze
drying (the significance of the slightly different CN- curves is difficult to assess, in particular
considering that the data was obtained for two different tissue sections). This similarity indicates
that the process of freeze drying in a separate vacuum chamber, involving increase in sample
temperature (to ~ 10 ºC) and exposure to air during transfer of the sample to the TOF-SIMS
instrument after freeze drying, does not induce significant lipid migration in the tissue sample. For
cholesterol migration to occur, it thus seems that both high vacuum and sample temperatures above
0 ºC are required.
4. Conclusions
•
The lateral distributions of various lipids in different brain structures were obtained using TOFSIMS analysis of freeze-dried mouse brain sections.
•
The TOF-SIMS images of molecular ions of cholesterol and sulfatide, indicate higher
concentrations in the myelin-rich white matter regions, while the images of phosphocholine and
phosphatidylinositol indicate higher concentrations in the grey matter regions.
•
Two different staining methods were used to obtain information about structures observed in the
TOF-SIMS images.
•
By using cresyl violet staining, large, elongated structures observed in the CN-/CNO- TOFSIMS images could be identified as ribosome-rich regions in the sagittal mouse brain section.
•
Eosin Y staining of the cytoplasm was used to locate regions on the surface of the tissue
sections containing cells that had been cleaved during the cryosectioning procedure. In these
regions, cell nuclei were indicated by spot-like structures in the CN-/CNO- TOF-SIMS images.
•
Analysis of freeze-dried mouse brain sections at varying sample temperatures between -130 and
60 ºC showed changes in lipid signal intensities, indicating migration of lipids on the tissue
surface.
•
The cholesterol signal intensity increases abruptly at T > 0 ºC, indicating extensive migration of
cholesterol to the tissue surface.
•
The migration process was found not to be reversed by decreasing the temperature, indicating
that the lipid migration is a kinetically hindered process driven by the vacuum environment
inside the TOF-SIMS instrument.
Acknowledgements
This research was supported by EC FP6 funding (Contract no. 005045). This publication does not
necessarily reflect the views of the EC. The Community is not liable for any use that may be made
of the information contained herein.
11
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