1. No category

ToF-SIMS for Biological Research –
Sample Preparation Techniques
Peter Sjövall
SP Technical Research Institute of Sweden
Borås, Sweden
Göteborg Borås
SI-Ontario Workshop
Toronto, March 20, 2008
SP Technical Research Institute of Sweden
www.sp.se
Chemistry and Materials
Technology
Polytechnical institute:
• Owned by the swedish government
• Ca 830 employees, ca 550 in Borås
• 8 technical divisions, including
• Measurement technology
• Energy technology
• Construction
and Mechanics
SI-Ontario Workshop
March 20, 2008
• FireToronto,
technology
TOF-SIMS instrument at SP
•
TOF-SIMS IV, purchased
december 1999
•
Bin LMIG source
•
C60 source
•
Heating and cooling (LN2) in
loadlock and main chamber
SI-Ontario Workshop
Toronto, March 20, 2008
TOF-SIMS group at SP
Ca 30 % contract work for industry
– Failure analysis, materials characterization, production
problems, quality control, …
– Medical device and pharmaceutical industry, manufacturing
industry, electronics, etc.
Ca 70 % research projects
– Collaboration with academic research groups
– Collaboration with industry
– Mainly bioscience (also combustion, polymers, coatings,
metrology, …)
SI-Ontario Workshop
Toronto, March 20, 2008
Examples of research projects (ToF-SIMS group)
•
ToF-SIMS imaging of biological samples
– lipid model systems, cells and tissue
– Improve lateral resolution, 3D analysis and identification of biomolecules
•
Geochemistry
– Detection and localisation of organic molecules (microorganisms) in
geobiological samples
– Detection of biomarkers in fluid inclusions
•
Marine antifouling
– Formulation, characterization and evaluation of new coatings
•
Biomaterals
– Surface modification for optimizing clinical function
– Characterization of implant surface and implant/tissue interface
SI-Ontario Workshop
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Outline
1. Introduction to ToF-SIMS of biological samples
2. Sample preparation strategies and techniques
•
Freezing /drying
•
Surface preparation
3. Examples
•
Tissues
•
Cells
SI-Ontario Workshop
Toronto, March 20, 2008
Current methods used for biological samples
Fluorescence
TEM
FE-SEM
Mass spectrometry
Histology
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Advantages of TOF-SIMS
Mass Spectrum
x10
5
1.6
1.4
Intensity
1.2
TOF detector
1.0
0.8
0.6
0.4
0.2
40
Primary ions
(Bi3+, 25 keV) +
60
80
/u
+
-
-
Secondary ions
+
Sample
Glass substrate
•
Identification of biomolecules by mass spectrometry (m/z < ~5000)
•
Mapping of biomolecules in biological samples (< 1 µm)
•
Chemical characterisation of small structures
Without labelling and staining!
SI-Ontario Workshop
Toronto, March 20, 2008
ToF-SIMS analysis of biological samples
Opportunities in biomedical research:
-
Basic knowledge about the chemical composition of
specific structures in cells and tissue
-
Disease-induced (or other stress-related) changes in
local chemical composition
-
Localization of pharmaceuticals and mapping of
drug-induced chemical changes
SI-Ontario Workshop
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ToF-SIMS analysis of biological samples
Challenges:
-
Analysis in vacuum! Æ Freezing, drying?
-
Sample specificity! Æ How to expose the relevant structures on the
sample surface?
Sample preparation!
-
Chemical complexity of biological samples?
-
Quantification, identification, image interpretation?
Data interpretation!
SI-Ontario Workshop
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Preparation strategies – the vacuum problem
Biological structures (e.g. cell membranes) depend on water
Æ Analysis requires dried or frozen sample
Air drying will cause chemical rearrangements
Æ Freeze drying necessary
Normal freezing gives rise to crystallization, which may damage
the structures and cause chemical redistribution
Amorphous ice produced by
Æ Plunge freezing (> 104-105 K/s), or
Æ High-pressure freezing
Slow freeze drying at low temperatures (water recrystallization
occurs at around -80 - -90C)
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Plunge freezing
Sample ”plunged” into liquid
nitrogen cooled ethane or
propane at -185 C
Prevents boiling at sample
surface, which otherwise
limits the heat transfer
Used by us for preparation of
lipid bilayer systems and
cell samples
SI-Ontario Workshop
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Preparation strategies – surface preparation
Cryomicrotoming
Frozen tissue
Cutting of sections
of frozen tissue
Section placed on
substrate
Analysis after
freeze drying
Freeze fracturing
Sample sandwiched
between two
substrates
Plunge freezing
High-pressure freezing
Separation of
the two substrates
Analysis in
frozen hydrated state
or after freeze drying
Ion sputtering (C60+)
Freeze dried or
frozen hydrated sample
Material removal by
Ion sputtering
Analysis of
sputtered sample
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Tissue examples
1. Mouse brain tissue
2. Adipose tissue from patients with chronic kidney
disease (CKD)
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Tissue preparation
Plunge freezing or high-pressure freezing often difficult/impossible
Æ Crystallization may be accepted
Cryosectioning at -15 - -20 C provides flat tissue surfaces without
smearing
Æ typically 15 µm thick
Æ successive sections can be used for ”3D analysis” or for
complementary analysis (histology, SEM, …)
Æ attachment of sections on substrate by ”finger-thawing” (or
pressed into indium substrate)
Æ risk for contamination (OCT,…)
Freeze drying
Analysis at room temperature or below
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Mouse brain tissue, negative TOF-SIMS spectrum
Fatty acids
x106
Cholesterol
Detected lipids:
x 15
• Phosphatidylcholine (PC)
• Cholesterol
• Sphingomyelin
• Sulfatides
• Phosphatidylinositol (PI)
• Vitamin E
1.5
x 300
1.0
0.5
200
x103
5.0
Intensity
4.0
400
ST 20:0
ST 18:0
ST h18:0
ST h20:0
600
800
/u
ST 24:1
PI 38:4
ST 22:0
ST h22:0
PI 36:4
ST 24:0
ST h24:0
ST h24:1
3.0
2.0
1.0
sulfatide
850
900
/u
SI-Ontario Workshop
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TOF-SIMS images from mouse brain tissue
7 x 7 mm2
Palmitate, C16H31O2-
Sphingomyelin
Cholesterol, (M-H)-
ST 24:0/1+h24:0/1, (M-H)-
PI 38:4, (M-H)-
Palm+chol+sulf
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Sagittal section, caudate putamen
500 x 500 µm2
11 x 11 mm2
Cholesterol (-)
Phosphocholine (+)
Cholesterol (+)
ST 24:0/1+h24:0/1 (-)
CN+CNO (-)
100 x 100 µm2
Phosphocholine (+)
•
•
•
CH4N+C4H8N (+)
Colocalization of cholesterol and sulfatide
Complementary localization with PC
Spots in N-containing ion images shows cell nuclei
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T = 30 ºC
T = -110 ºC
Temperature-controlled measurements
CNO (-)
phosphocholine (+)
cholesterol (-)
sulfatide (-)
Lipid migration: Cholesterol migrates to surface at T> ~0C
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ToF-SIMS analysis of adipose tissue from
kidney patients and controls
•
Subcutaneous fat tissue
•
7 kidney patients and 6 controls
•
Biopsies frozen, cryosectioned (15 µm thickness),
placed on glass and stored at -80 C
•
Freeze dried immediately before TOF-SIMS
analysis
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Intensity
Adipose tissue from CKD patients, positive TOF-SIMS spectrum
x10 5
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
DAG
x 10
triglycerides
x 100
200
400
600
800
/u
Triglycerides (TAG):
•
•
Main ingredient in animal fat
Energy storage in adipose
tissue
Detected lipids:
• TAG, Diacyl glycerol (DAG) and Fatty acids (FA)
• Phosphatidylcholine
SI-Ontario Workshop
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Intensity
Adipose tissue from CKD patients, positive TOF-SIMS spectrum
x10 5
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
DAG
x 10
triglycerides
x 100
200
400
600
800
/u
DAG(16+16):Z
Z= 3 2 1 0
x10 4
3.5
DAG(16+18):Z
3.0
Intensity
Z= 3 2 1 0
2.5
DAG(14+16):Z
2.0
DAG(18+18):Z
Z= 2 1 0
Z= 3 2 1 0
1.5
1.0
0.5
550
600
/u
SI-Ontario Workshop
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Principal component analysis (PCA)
Identifies correlations and systematic variations in large data sets
Score plot:
Differences between samples
Loadings plot:
Identifies the differences
Samples/Scores Plot of TAGtabeller 070815.xls
Variables/Loadings Plot for TAGtabeller 070815.xls
2.5
P12
C2
C2
1
P12
0.5
C7
0
C9
-0.5 C9
P1P1
P1
P1
P10
C2
C2
C11
C11
C7
C9
C9
C6
C6
-1
P3 P3C6
P8
P3
P8
P8
-1.5
-2
-2.5
P8
-6
-4
•
•
30:2
36:1
C7
P12
C11
C11
36:4 36:3 36:2
34:1
0.2
P5
C4C4 C4
C4
34:3 34:2
32:3 32:2
30:3
P5
-2
0
2
Scores on PC 1 (75.87%)
P10
P10
P10
Loadings on P C 1 (75.93% )
Scores on PC 2 (10.47%)
1.5
C7
P12
2
0.3
P5 P5
0.1
32:1
0
-0.1
30:1
-0.2
30:0
C6
32:0
36:0
34:0
-0.3
P3
4
6
-0.4
2
4
6
8
10
Variable
12
14
Each individual has a characteristic pattern/distribution of
glycerol lipids
Compared to the controls, patients tend to have higher relative
signal from unsaturated DAGs
SI-Ontario Workshop
Toronto, March 20, 2008
16
Positive TOF-SIMS images of adipose tissue
Field of view: 500 × 500 µm2
Phosphocholine
Unsaturated DAG
Saturated DAG
Sample ”A”
Sample ”B”
•
•
Phosphocholine and DAG complementary localized
Different spatial distributions for saturated and unsaturated DAGs
SI-Ontario Workshop
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TOF-SIMS analysis of cells
Aim:
•
•
Chemical composition of subcellular structures
Mapping of (inhomogeneous) spatial distribution of lipids on cell
membrane
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Cell examples
1. Surface-adhering hTERT cells (fibroblasts)
2. Chemical imprinting of PMLN cells (leukocytes)
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TOF-SIMS analysis of cells
Sample preparation:
1. hTERT (fibroblasts) on SiO2 substrate
2. Removal of salt by rinsing in NH4HCOO
3. Drying: plunge freeze + freeze drying
4. TOF-SIMS analysis
Or, alternatively
1. Fixation in glutaraldehyde
2. Rinsing in deionized water
3. Drying: plunge freeze + freeze drying
4. TOF-SIMS analysis
SI-Ontario Workshop
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TOF-SIMS analysis of hTERT cells
CNO-
Palmitate-
Oleate-
PO3-
Video image of cell sample
phosphocholine+
Na2Cl+
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K2Cl+
ToF-SIMS of hTERT cells
199 x 199 µm2
Na+
K+
K2Cl+
m/z 58
m/z 86
m/z 184
58+86+184
Phosphatidylcholine fragments
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ToF-SIMS of hTERT cells
199 x 199 µm2
Na+
K+
K2Cl+
m/z 58
m/z 86
m/z 184
•
•
•
58+86+184
K inside cell, Na outside
Some K leakage out from cell
Phosphatidylcholine not redistributed
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hTERT, profile
Profile:
signal intensity along line in image
125x125 µm, m/z 86
110
100
90
84%
80
Intensity
70
60
1.33
1.33 µm µm
50
40
30
16%
20
10
0
0
2
4
6
8
10
Distance
•
•
Sharpness of cell edge: ca 1.4 µm
Lipids in filopodia detectable
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12
(µm)
3D analysis of cells
How to expose cell interior for TOF-SIMS analysis?
•
Ion etching with C60+ ions
(Breitenstein et al)
•
Freeze fracturing
(Winograd et al)
•
Cryosectioning
(Arlinghaus et al)
•
Chemical imprinting
•
Removal of cell membrane by Triton-X detergent
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Cell imprinting:
•
Transfer sample molecules to substrate surface with retained
lateral distribution
•
Imaging TOF-SIMS of chemical imprint
Advantages:
– Optimised analysis conditions
– Access to intracellular regions
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77x77 μm2
TOF-SIMS images of cell imprint
phosphocholine+
184 u
CH4N+
30 u
(Ag-cholesterol)+
493-496 u
(Ag-cholesterol2)+
879-882 u
Ag3+
323 u
Total ion image
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TOF-SIMS images of cell imprint
Nuclear membrane
Plasma membrane
Ag
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Concluding remarks
•
Chemical analysis of biological samples with subcellular
resolution possible with ToF-SIMS
– Mass range 0 - ~2000 Dalton (lipids, peptides,
pharmaceuticals, …)
•
Sample preparation critical for obtaining relevant information
– Different methods should be applied based on the
requested information
•
Collaboration with biomedical research groups important
– Formulation of relevant biological questions
•
SI Ontario now very well equipped for ToF-SIMS analysis of
biological samples
SI-Ontario Workshop
Toronto, March 20, 2008
Acknowledgements
•
University of Glasgow (Cell work)
Mathis Riehle, Nicholai Gaadegard, Dimitrios Giannaras
•
Karolinska Institutet (Tissue work)
Björn Johansson, Martin Schalling, Dalila Belazi
•
SP
Jukka Lausmaa, Jakob Malm
Financial support:
European Community, FP6 (Contract no. 005045)
”NANOBIOMAPS”
Swedish Government - Ministry of Industry
Swedish Agency for Innovation Systems
SI-Ontario Workshop
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Thank you!
and
Good luck!
SI-Ontario Workshop
Toronto, March 20, 2008