ToF-SIMS for Biological Research Sample Preparation Techniques

Transcription

1 ToF-SIMS for Biological Research Sample Preparation Techniques Peter Sjövall SP Technical Research Institute of Sweden Borås, Sweden Göteborg Borås

2 SP Technical Research Institute of Sweden 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 Fire technology

3 TOF-SIMS instrument at SP TOF-SIMS IV, purchased december 1999 Bi n LMIG source C 60 source Heating and cooling (LN 2 ) in loadlock and main chamber

4 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, )

5 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

6 Outline 1. Introduction to ToF-SIMS of biological samples 2. Sample preparation strategies and techniques Freezing /drying Surface preparation 3. Examples Tissues Cells

7 Current methods used for biological samples Fluorescence TEM FE-SEM Mass spectrometry Histology

8 Advantages of TOF-SIMS Mass Spectrum 5 x TOF detector Intensity Primary ions (Bi 3 +, 25 kev) Secondary ions / u + 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!

9 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

10 ToF-SIMS analysis of biological samples Challenges: - Analysis in vacuum! Freezing, drying? - Sample specificity! How to expose the relevant structures on the sample surface? Sample preparation! - Chemical complexity of biological samples? - Quantification, identification, image interpretation? Data interpretation!

11 Preparation strategies the vacuum problem Biological structures (e.g. cell membranes) depend on water Analysis requires dried or frozen sample Air drying will cause chemical rearrangements Freeze drying necessary Normal freezing gives rise to crystallization, which may damage the structures and cause chemical redistribution Amorphous ice produced by Plunge freezing (> K/s), or High-pressure freezing Slow freeze drying at low temperatures (water recrystallization occurs at around C)

12 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

13 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 Ion sputtering (C 60+ ) Plunge freezing High-pressure freezing Separation of the two substrates Analysis in frozen hydrated state or after freeze drying Freeze dried or frozen hydrated sample Material removal by Ion sputtering Analysis of sputtered sample

14 Tissue examples 1. Mouse brain tissue 2. Adipose tissue from patients with chronic kidney disease (CKD)

15 Tissue preparation Plunge freezing or high-pressure freezing often difficult/impossible Crystallization may be accepted Cryosectioning at C provides flat tissue surfaces without smearing typically 15 µm thick successive 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

16 Mouse brain tissue, negative TOF-SIMS spectrum Fatty acids Cholesterol 6 x10 x 15 Detected lipids: x 300 Phosphatidylcholine (PC) Cholesterol Sphingomyelin Sulfatides Phosphatidylinositol (PI) Vitamin E / u 3 x ST 20:0 ST h20:0 ST 24:1 ST 18:0 ST h18:0 ST 22:0 PI 38:4 ST 24:0 ST h24:0 PI 36:4 ST h22:0 ST h24:1 Intensity sulfatide / u

17 TOF-SIMS images from mouse brain tissue 7 x 7 mm 2 Palmitate, C 16 H 31 O 2 - Cholesterol, (M-H) - ST 24:0/1+h24:0/1, (M-H) - Sphingomyelin PI 38:4, (M-H) - Palm+chol+sulf

18 Sagittal section, caudate putamen 11 x 11 mm x 500 µm 2 Phosphocholine (+) Cholesterol (-) ST 24:0/1+h24:0/1 (-) CN+CNO (-) 100 x 100 µm 2 Phosphocholine (+) Cholesterol (+) CH 4 N+C 4 H 8 N (+) Colocalization of cholesterol and sulfatide Complementary localization with PC Spots in N-containing ion images shows cell nuclei

19 Temperature-controlled measurements T = 30 ºC T = -110 ºC CNO (-) phosphocholine (+) cholesterol (-) sulfatide (-) Lipid migration: Cholesterol migrates to surface at T> ~0C

20 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

21 Adipose tissue from CKD patients, positive TOF-SIMS spectrum Intensity 5 x x 10 DAG triglycerides x / u Triglycerides (TAG): Main ingredient in animal fat Energy storage in adipose tissue Detected lipids: TAG, Diacyl glycerol (DAG) and Fatty acids (FA) Phosphatidylcholine

22 Adipose tissue from CKD patients, positive TOF-SIMS spectrum Intensity 5 x x 10 DAG triglycerides x / u 4 x DAG(16+16):Z Z= Intensity DAG(14+16):Z Z= DAG(16+18):Z Z= DAG(18+18):Z Z= / u

23 Principal component analysis (PCA) Identifies correlations and systematic variations in large data sets Score plot: Differences between samples Loadings plot: Identifies the differences Scores on PC 2 (10.47%) C4 C4 C4 C4 C9 C9 C7 Samples/Scores Plot of TAGtabeller xls P12 C9 P12 P12 C2 C2 C11 C11 C9 C7 C2 C2 C11 C11 P8 P3 P8 P8 P12 C6 C6 C6 P3 P3 P10 C7 C6 P1 P1 P1 C7 P1 P5 P5 P5 P5 P10 P10 P10 Loadings on PC 1 (75.93%) :3 30:2 Variables/Loadings Plot for TAGtabeller xls 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 36:0-2.5 P8 P Scores on PC 1 (75.87%) Variable Each individual has a characteristic pattern/distribution of glycerol lipids Compared to the controls, patients tend to have higher relative signal from unsaturated DAGs

24 Positive TOF-SIMS images of adipose tissue Phosphocholine Unsaturated DAG Saturated DAG Field of view: µm 2 Sample A Sample B Phosphocholine and DAG complementary localized Different spatial distributions for saturated and unsaturated DAGs

25 TOF-SIMS analysis of cells Aim: Chemical composition of subcellular structures Mapping of (inhomogeneous) spatial distribution of lipids on cell membrane

26 Cell examples 1. Surface-adhering htert cells (fibroblasts) 2. Chemical imprinting of PMLN cells (leukocytes)

27 TOF-SIMS analysis of cells Sample preparation: 1. htert (fibroblasts) on SiO 2 substrate 2. Removal of salt by rinsing in NH 4 HCOO 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

28 TOF-SIMS analysis of htert cells CNO - Palmitate - Oleate - PO 3 - Video image of cell sample phosphocholine + Na 2 Cl + K 2 Cl +

29 ToF-SIMS of htert cells 199 x 199 µm 2 Na + K + K 2 Cl + m/z 58 m/z 86 m/z Phosphatidylcholine fragments

30 ToF-SIMS of htert cells 199 x 199 µm 2 Na + K + K 2 Cl + m/z 58 m/z 86 m/z K inside cell, Na outside Some K leakage out from cell Phosphatidylcholine not redistributed

31 htert, profile 125x125 µm, m/z Profile: signal intensity along line in image % 70 Intensity µm µm % Distance (µm) Sharpness of cell edge: ca 1.4 µm Lipids in filopodia detectable

32 3D analysis of cells How to expose cell interior for TOF-SIMS analysis? Ion etching with C 60+ ions (Breitenstein et al) Freeze fracturing (Winograd et al) Cryosectioning (Arlinghaus et al) Chemical imprinting Removal of cell membrane by Triton-X detergent

33 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

34 TOF-SIMS images of cell imprint 77x77 μm 2 phosphocholine u (Ag-cholesterol) u Ag u CH 4 N + 30 u (Ag-cholesterol 2 ) u Total ion image

35 TOF-SIMS images of cell imprint Nuclear membrane Plasma membrane Ag

36 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

37 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 ) NANOBIOMAPS Swedish Government - Ministry of Industry Swedish Agency for Innovation Systems

38 Thank you! and Good luck!