tof-sims for biological research – sample preparation...

SI-Ontario WorkshopToronto, March 20, 2008

ToF-SIMS for Biological Research –Sample Preparation Techniques

Peter Sjövall

SP Technical Research Institute of SwedenBorås, Sweden

Göteborg Borås


SP Technical Research Institute of Sweden

www.sp.se

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• Fire technology

Chemistry and Materials Technology


TOF-SIMS instrument at SP

• TOF-SIMS IV, purchaseddecember 1999

• Bin LMIG source

• C60 source

• Heating and cooling (LN2) in loadlock and main chamber


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, …)


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


Outline

1. Introduction to ToF-SIMS of biological samples

2. Sample preparation strategies and techniques• Freezing /drying

• Surface preparation

3. Examples• Tissues

• Cells


Current methods used for biological samples

FE-SEM

TEM

Fluorescence

HistologyMass spectrometry


Advantages of TOF-SIMS

+Primary ions(Bi3+, 25 keV)

- -

+

TOF detector

/ u40 60 80

5x10

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Inte

nsity

Mass Spectrum

Secondary ions

+

Glass substrate

Sample

• 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!


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


ToF-SIMS analysis of biological samples

Sample preparation!

Data interpretation!

Challenges:

- Analysis in vacuum! Freezing, drying?

- Sample specificity! How to expose the relevant structures on the sample surface?

- Chemical complexity of biological samples?

- Quantification, identification, image interpretation?


Preparation strategies – the vacuum problem

Biological structures (e.g. cell membranes) depend on waterAnalysis requires dried or frozen sample

Air drying will cause chemical rearrangementsFreeze drying necessary

Normal freezing gives rise to crystallization, which may damagethe structures and cause chemical redistribution

Amorphous ice produced byPlunge freezing (> 104-105 K/s), orHigh-pressure freezing

Slow freeze drying at low temperatures (water recrystallizationoccurs at around -80 - -90C)


Plunge freezing

Sample ”plunged” into liquidnitrogen cooled ethane or propane at -185 C

Prevents boiling at samplesurface, which otherwiselimits the heat transfer

Used by us for preparation of lipid bilayer systems and cell samples


Preparation strategies – surface preparation

Freeze fracturing

Cryomicrotoming

Frozen tissue Section placed onsubstrate

Analysis after freeze drying

Cutting of sectionsof frozen tissue

Ion sputtering (C60+)

Freeze dried or frozen hydrated sample

Material removal byIon sputtering

Analysis of sputtered sample

Plunge freezingHigh-pressure freezing

Analysis in frozen hydrated stateor after freeze drying

Sample sandwichedbetween two

substrates

Separation ofthe two substrates


Tissue examples

1. Mouse brain tissue

2. Adipose tissue from patients with chronic kidneydisease (CKD)


Tissue preparation

Plunge freezing or high-pressure freezing often difficult/impossibleCrystallization may be accepted

Cryosectioning at -15 - -20 C provides flat tissue surfaces withoutsmearing

typically 15 µm thicksuccessive sections can be used for ”3D analysis” or for

complementary analysis (histology, SEM, …)attachment of sections on substrate by ”finger-thawing” (or

pressed into indium substrate)risk for contamination (OCT,…)

Freeze drying

Analysis at room temperature or below


Mouse brain tissue, negative TOF-SIMS spectrum

/ u850 900

3x10

1.0

2.0

3.0

4.0

5.0

Inte

nsity

ST 18:0 ST h18:0

ST 20:0 ST h20:0

ST 22:0ST h22:0

PI 38:4

ST 24:1

ST 24:0

ST h24:1

ST h24:0

PI 36:4

Detected lipids:

• Phosphatidylcholine (PC)• Cholesterol• Sphingomyelin• Sulfatides• Phosphatidylinositol (PI)• Vitamin E

x 15

x 300

/ u200 400 600 800

6x10

0.5

1.0

1.5

Fatty acids Cholesterol

sulfatide


TOF-SIMS images from mouse brain tissue7 x 7 mm2

Palmitate, C16H31O2- Cholesterol, (M-H)-

Sphingomyelin PI 38:4, (M-H)-

ST 24:0/1+h24:0/1, (M-H)-

Palm+chol+sulf


Sagittal section, caudate putamen

• Colocalization of cholesterol and sulfatide• Complementary localization with PC• Spots in N-containing ion images shows cell nuclei

500

x 50

0 µm

2

Phosphocholine (+) Cholesterol (-) ST 24:0/1+h24:0/1 (-) CN+CNO (-)

11 x 11 mm2

100

x 10

0 µm

2

Phosphocholine (+) Cholesterol (+) CH4N+C4H8N (+)


Temperature-controlled measurements

CNO (-) phosphocholine (+) cholesterol (-) sulfatide (-)

T =

-110

ºCT

= 30

ºC

Lipid migration: Cholesterol migrates to surface at T> ~0C


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


Adipose tissue from CKD patients, positive TOF-SIMS spectrum

x 10

x 100

/ u200 400 600 800

5x10

1.02.03.04.05.06.07.08.0

Inte

nsity

DAG triglycerides

Detected lipids:• TAG, Diacyl glycerol (DAG) and Fatty acids (FA)• Phosphatidylcholine

Triglycerides (TAG):• Main ingredient in animal fat• Energy storage in adipose

tissue


Adipose tissue from CKD patients, positive TOF-SIMS spectrum

/ u550 600

4x10

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Inte

nsity DAG(14+16):Z

DAG(16+16):Z

DAG(16+18):Z

DAG(18+18):ZZ= 2 1 0

Z= 3 2 1 0

Z= 3 2 1 0

Z= 3 2 1 0

x 10

x 100

/ u200 400 600 800

5x10

1.02.03.04.05.06.07.08.0

Inte

nsity

DAG triglycerides


Principal component analysis (PCA)

• Each individual has a characteristic pattern/distribution of glycerol lipids

• Compared to the controls, patients tend to have higher relative signal from unsaturated DAGs

Identifies correlations and systematic variations in large data sets

Score plot: Differences between samples

Loadings plot: Identifies the differences

-6 -4 -2 0 2 4 6-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

Scores on PC 1 (75.87%)

Sco

res

on P

C 2

(10.

47%

)

P1 P1 P1 P1

C2

C2 C2

C2

P3 P3

P3

P3

C4 C4 C4 C4

P5

P5

P5 P5

C6

C6 C6

C6

C7

C7

C7

C7

P8

P8

P8 P8

C9 C9

C9 C9

P10 P10 P10

P10 C11 C11

C11 C11

P12

P12

P12

P12

Samples/Scores Plot of TAGtabeller 070815.xls

2 4 6 8 10 12 14 16-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

VariableLo

adin

gs o

n P

C 1

(75.

93%

)

30:3

30:2

30:1

30:0

32:3 32:2

32:1

32:0

34:3 34:2 34:1

34:0

36:4 36:3 36:2

36:1

36:0

Variables/Loadings Plot for TAGtabeller 070815.xls


Positive TOF-SIMS images of adipose tissueField 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


TOF-SIMS analysis of cells

Aim:• Chemical composition of subcellular structures• Mapping of (inhomogeneous) spatial distribution of lipids on cell

membrane


Cell examples

1. Surface-adhering hTERT cells (fibroblasts)

2. Chemical imprinting of PMLN cells (leukocytes)


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


TOF-SIMS analysis of hTERT cells

PO3-

Palmitate-CNO- Oleate-

phosphocholine+ Na2Cl+ K2Cl+

Video image of cell sample


ToF-SIMS of hTERT cells

Phosphatidylcholine fragments

K2Cl+K+

m/z 58 m/z 86 m/z 184 58+86+184

Na+

199 x 199 µm2


ToF-SIMS of hTERT cells

K2Cl+K+

m/z 58 m/z 86 m/z 184 58+86+184

Na+

199 x 199 µm2

• K inside cell, Na outside• Some K leakage out from cell• Phosphatidylcholine not redistributed


hTERT, profile

125x125 µm, m/z 86

0 2 4 6 8 10 12

Distance (µm)

0

10

20

30

40

50

60

70

80

90

100

110

Inte

nsity

16%

84%

1.33 µm

Profile:signal intensity along line in image

1.33 µm

• Sharpness of cell edge: ca 1.4 µm• Lipids in filopodia detectable


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


• Transfer sample molecules to substrate surface with retainedlateral distribution

• Imaging TOF-SIMS of chemical imprint

Cell imprinting:

Advantages:– Optimised analysis conditions– Access to intracellular regions


TOF-SIMS images of cell imprint

phosphocholine+

184 u(Ag-cholesterol)+

493-496 uAg3

+

323 u

77x7

7 μm

2

CH4N+

30 u(Ag-cholesterol2)+

879-882 uTotal ion image


TOF-SIMS images of cell imprint

Plasma membrane

Nuclear membrane

Ag


Concluding remarks

• Chemical analysis of biological samples with subcellularresolution 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


Acknowledgements

Financial support:

European Community, FP6 (Contract no. 005045)”NANOBIOMAPS”

Swedish Government - Ministry of Industry

Swedish Agency for Innovation Systems

• University of Glasgow (Cell work)Mathis Riehle, Nicholai Gaadegard, Dimitrios Giannaras

• Karolinska Institutet (Tissue work)Björn Johansson, Martin Schalling, Dalila Belazi

• SPJukka Lausmaa, Jakob Malm


Thank you!

and

Good luck!

tof-sims for biological research – sample preparation...

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