www.elsevier.com/locate/apsusc

Applied Surface Science 252 (2006) 6966–6974

Localization of lipids in freeze-dried mouse brain sections

by imaging TOF-SIMS

Peter Sjovall a,*, Bjorn Johansson b, Jukka Lausmaa a

a SP Swedish National Testing and Research Institute, P.O. Box 857, SE-50115 Boras, Swedenb Department of Clinical Neuroscience, Karolinska Institutet, SE-17176 Stockholm, Sweden

Received 12 September 2005; accepted 15 February 2006

Available online 2 May 2006

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 mm 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 keVAu3+ or Bi3

+ primary ions, always keeping the accumulated ion dose below 4 � 1012 ions/cm2. Positive and

negative ion images over the entire mouse brain section and of analysis areas down to 100 mm � 100 mm 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 8C showed an abrupt increase in the cholesterol signal at T > 0 8C, indicating extensive migration of

cholesterol to the tissue surface under vacuum conditions.

# 2006 Elsevier B.V. All rights reserved.

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 biomole-

cules up to 100,000 u (including proteins) can be obtained,

however with a lateral resolution typically around 50 mm [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].

* Corresponding author. Fax: +46 33 103388.

E-mail address: peter.sjovall@sp.se (P. Sjovall).

0169-4332/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.apsusc.2006.02.126

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].

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 considera-

tion. 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

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–6974 6967

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 altera-

tions 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 phosphatidyli-

nositol [12]. The obtained images showed a pronounced

complementary localization of cholesterol and phosphatidyl-

choline (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 8C immediately after

dissection. Thin (14 mm) 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 8C. 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 8C (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, immedi-

ately evacuating the system and slowly allowing the sample to

warm up to room temperature during constant pumping

(<10�3 mbar). After approximately 30–40 min, the sample had

reached room temperature but pumping was normally

continued for another 20–30 min. 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 were recorded from analysis areas between

200 mm � 200 mm and 11 mm � 11 mm (bunched mode,

0.06 pA for Au3+ and 0.18 pA for Bi3

+). High image resolution

data were recorded at areas between 35 mm � 35 mm and

200 mm � 200 mm (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 � 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 freeze-drying

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 min of pumping (with the sample tempera-

ture 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 8C. The sample temperature was

increased at a rate of 0.5 K/s and kept constant at the stated

temperature for 4 min before start of the data acquisition. The

accumulated ion dose was <4 � 1010 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

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–69746968

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 8C, then quickly (<2 min)

lowered to approximately �100 8C and then gradually

decreasing to�120 8C. The analysis was started approximately

2 h 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� 5 min), fixed (99.5% ethanol for 2�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;

Fig. 1. TOF-SIMS images from a freeze-dried mouse brain section in the sagittal p

(184 u), (b) cholesterol� (385 u), (c) CN� (26 u) + CNO� (42 u), (d) phosphatidylin

been normalized to the total ion image. (f) An optical microscopy image of the same

some major brain structures indicated (co: cortex, cp: caudate putamen, th: thalam

xylene 2� 5 min). Finally, a coverslip was immediately

mounted on the sample slide with Pertex (Histolab, Goteborg,

Sweden) and allowed to 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

Fig. 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.

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

lane. The images show the signal intensity distributions of (a) phosphocholine+

ositol� PI 38:4 and (e) sulfatide� 24:1 + 24:0 + h24:1 + h24:0. All images have

tissue section after cresyl violet staining and (g) the phosphocholine image with

us, hi: hippocampus, ce: cerebellum). Field of view 11 mm � 11 mm.

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–6974 6969

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 mm � 3.0 mm.

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

Fig. 3. TOF-SIMS images at two different magnifications from the caudate putamen

recorded from the area indicated by the square in Fig. 1(a) and the lower images (100

phosphocholine image in this figure. The ion images have been normalized to the

cholesterol to the surface from the sample interior, which is

shown to occur in vacuum at temperatures >0 8C (see Section

3.3). Although the cholesterol signal thus 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.

Figs. 1 and 2 also show ion images of the added signal

intensities from the CN� and CNO� fragments. 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 micro-

scopy 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

region of the sagittal tissue section. The upper images (500 mm � 500 mm) were

mm � 100 mm) were recorded from the area indicated by the square in the upper

total ion image.

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–69746970

Table 1

Observed peaks and peak assignments in positive and negative TOF-SIMS

spectra from freeze-dried mouse brain sections (see spectra in Fig. 4)

Positive spectra Negative spectra

Mass Assignment Mass Assignment

478.39 Pg + 16:0 644.55 SM 18:0–N(CH3)3

(CH2)2

494.36 Pg + 16:0 + O 670.57 SM 18:0–N(CH3)3

496.37 Unassigned 673.53 PA 34:1a

504.41 Pg + 18:1 699.55 PC 34:1–N(CH3)3

506.45 Pg + 18:0 715.62 SM 18:0–CH3

520.41 Pg + 18:1 + O 771.81 Cholesterol2522.40 Pg + 18:0 + O 794.64 Unassigned

548.57 Ceramide 18:0a 806.60 Sulfatide 18:0

577.44 Unassigned 822.60 Sulfatide h18:0

630.53 Ceramide 24:1a 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 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

a Preliminary assignment.

Fig. 4. Positive (a) and negative (b) TOF-SIMS spectra from the cholesterol-

rich and phosphocholine-rich areas shown in Fig. 3.

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.

Fig. 3 shows TOF-SIMS images at two different magnifica-

tions 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 mm � 500 mm) were recorded with the instrument in

the bunched mode (high mass resolution, low lateral resolu-

tion), while the lower images (100 mm � 100 mm) 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.

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 mm � 100 mm 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 mm

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.

Fig. 4 shows (a) positive and (b) negative TOF-SIMS spectra

from the area shown in the 500 mm � 500 mm 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

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–6974 6971

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 mm � 200 mm.

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.

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 phosphatidylcho-

line and can therefore not be conclusively assigned to lysopho-

sphatidylcholine 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 sphingo-

myelin. 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 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

Fig. 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 quasimole-

cular peaks (e.g., 647 u) in the negative spectra.

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

Figs. 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 (approxi-

mately 10 8C), 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 8C.

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–69746972

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 8C and upon heating to T = 30 8C, respectively. Field of view 500 mm � 500 mm.

In Fig. 6, a comparison of TOF-SIMS images for CNO�,

positive phosphocholine, and negative cholesterol and sulfatide

signals between T = �110 8C and after annealing to T = 30 8Cshows 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.

Fig. 7. Normalized signal intensities of selected ions in TOF-SIMS spectra from free

of the sample from T = �130 to 50 8C. (a) The signal intensities in spectra from the

(see Fig. 6).

A more detailed account of the development of the ion signal

intensities during the annealing process is shown in Fig. 7.

Fig. 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.

In the cholesterol-rich area, the cholesterol signal is constant

at a relatively low level at temperatures below 0 8C, but

increases abruptly at T > 0 8C, while the phosphocholine signal

shows a small decrease up to T = 0 8C and a steeper decrease at

higher temperatures. In the phosphocholine-rich area, the

cholesterol signal shows a minor increase at T > �30 8C, while

the phosphocholine signal decreases gradually over the entire T

range. Sulfatide and PI show gradually decreasing signal

ze-dried mouse brain sections, as a function of sample temperature upon heating

cholesterol-rich area only and (b) the results from the phosphocholine-rich area

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–6974 6973

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 sections (anterior commissure, coronal orien-

tation, total area), as a function of sample temperature upon heating of the

sample from T = �130 to 60 8C. 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.

intensities with increasing temperatures both in the cholesterol-

rich and phosphocholine-rich areas.

After annealing and analysing the sample at 50 8C, the

sample temperature was decreased to T = �110 8C and again

analysed. The resulting images and spectra were not

significantly different from the data obtained during annealing

to 50 8C, 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 8C. 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.

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 8C or exposure to air between freeze

drying and analysis at low temperatures.

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 were recorded from equivalent areas (anterior commis-

sure) 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 were 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 8C) 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 8C are required.

4. Conclusions

� The lateral distributions of various lipids in different brain

structures were obtained using TOF-SIMS analysis of freeze-

dried mouse brain sections.

� T

he 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.

� T

wo different staining methods were used to obtain

information about structures observed in the TOF-SIMS

images.

� B

y using cresyl violet staining, large, elongated structures

observed in the CN�/CNO� TOF-SIMS images could be

identified as ribosome-rich regions in the sagittal mouse brain

section.

� E

osin 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.

� A

nalysis of freeze-dried mouse brain sections at varying

sample temperatures between �130 and 60 8C showed

changes in lipid signal intensities, indicating migration of

lipids on the tissue surface.

� T

he cholesterol signal intensity increases abruptly at

T > 0 8C, indicating extensive migration of cholesterol to

the tissue surface.

� T

he migration process was found not to be reversed by

decreasing the temperature, indicating that the lipid migra-

tion is a kinetically hindered process driven by the vacuum

environment inside the TOF-SIMS instrument.

P. Sjovall et al. / Applied Surface Science 252 (2006) 6966–69746974

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.

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