nanoSIMS: a new analytical tool for ultra-fine feature analysis using secondary ion emission R. Stern* and Peta Clode The University of Western Australia Centre for Microscopy and Microanalysis rstern@cmm.uwa.edu.au

nanoSIMS labs #10

SIMS Secondary Ion Mass Spectrometry probing analytical technique for measuring the surface or near surface chemical or isotopic composition of solids uses probe ions to sputter target ions in laterally microscale or smaller regions (ion microprobe) analytical characteristics: nm depth resolution lateral resolution 10’s nm to mm most sensitive of the probing analytical techniques, ppb-ppm entire periodic table, H to U simple sample preparation Although the instrument is new, the analytical technique has been widely utilized for decades This of course is SIMS or secondary ion mass spectrometry. The NanoSIMS is a specialized form of ion microprobe.

SIMS Data Basic Dimensions: 1 dimension 2 dimensions 3 dimensions ratios 18 16 O/ O 24Mg/28Si Mass spectrum cps,ppm Se Basic from tiny mass consumed Dimensions: 1 dimension ‘depth profile’: composition along a line normal to the surface ‘line scan’: composition along a line parallel to the surface 2 dimensions ‘image’ (map) of the surface distribution of secondary ion species 3 dimensions a stacked series of secondary ion images collected at discrete depths into the target Image Depth profile Line scan

SIMS analytical Energy Analyzer Mass Analyzer Ion source Secondary ion Collection Optics Detector Primary ion column The typical layout of a SIMS instrument is shown here. There are two basic components: the ion probe and the analyzer. The ion probe comprises a source of ions ( O,Cs,Ar,Ga), a column to accelerate and focus the primary ions to a fine spot, and the sample under vacuum. As a consequence of bombarding the sample with primary ions, secondary ions of the target (which must be a solid) are produced, and these are subsequently analyzed within the second major component, the mass analyzer, or more technically, the secondary ion mass spectrometer. The analytical technique is thus commonly referred to as SIMS. . Sample under vacuum

High mass resolution analyzer Detector array: 5 isotopes acquired simultaneously Ion sources: O-, Cs+ Normal, co-axial primary & secondary ions samples Sample introduction

Features of NanoSIMS dynamic SIMS probe lateral resolution for bulk sample ion chemical/isotopic characterization not for molecular identification or oxidation state probe lateral resolution 50 nm for 133Cs+ 200 nm for 16O- ability to scan the primary beam and record secondary ion images at ultra-fine-scale lateral resolution very low eroson rate, ~nm/hr (sample preservation) simultaneous multicollection of up to 5 isotope species routine high mass resolution resolve isobaric interferences

Ion imaging of TiCN alloy demonstrating sub-50 nm edge resolution with Cs+ 3 x 3 um, 26CN 10 x 10 um, 24C2

BIOMATERIALS Engineered Materials Minerals Semiconductors Ceramics Alloys Minerals Extraterrestrial dust Geochronology Ore minerals BIOMATERIALS sub-cellular imaging biochemical function cancer research pharmaceuticals

NanoSIMS labs bio-SIMS program 0. Harvard Medical School/ MIT, Boston USA National Resource for Mass Spectrometry Imaging, NIH NCRR 1. Washington University, Saint Louis, MO 2. Max Planck Institute, Mainz, Germany 3. Institut Curie, Paris, France 4. Oxford University, U.K 5. Lawrence Livermore National Lab, Livermore, CA, USA University of California, Davis 6. National Institute for Materials Science, Tsukuba, Japan 7. LAM, Centre de Recherche Public-GL, Luxembourg 8. ExxonMobil, New Jersey, USA 9. University of Tokyo 10. The UWA 11. Rouen Univ, France bio-SIMS program bio-SIMS program bio-SIMS program

Non-metallic elements used in SIMS biological analysis Naturally-occurring isotopes 12C – all cell organelles 14N – DNA, RNA, proteins 31P – DNA, RNA (nucleic acids) 32S – proteins Artificial isotopes (stable or radioactive) 14C – turnover/pathways of C 15N – turnover/pathways of labelled amino acids (proteins), nucleic acids 17F – Fluoracil, cancer drug targetting chromosomes 81Br – in Bromodeoxyuridine (BrdU), specifically incorporated into DNA during DNA synthesis 123, 125, 127I – in Iododeoxyuridine

Examples of metallic elements used in SIMS biological analysis Li, Na, K, Rb, Cs Be, Mg, Ca, Sr, Ba Ti, Cr, Mn, Fe, Ni, Pb, Al, In Au, Ag Sc, lanthanides, actinides to date, few studies, poor sensitivity and spatial resolution

O- primary ion bombardment, positive secondary ions, and a silicon matrix

Cs+ primary ion bombardment, negative secondary ions, and a silicon matrix Compared to other probe techniques, SIMS has the highest sensitivity.

Sample preparation must be dehydrated, +/- fast freezing, chemical fixation (glutaraldehyde, Os tetroxide), epoxy embedding, cryo-sectioning (0.3 – 1 µm) will feature of interest be preserved? other documentation techniques to map features (confocal, VPSEM, TEM, etc.) Example of human hair cross-section deposited on Si (Hallegot and Corcuff, 1993) Scanning electron microscopy images cortex cuticle Important to emphasize that the N50 should not be used for mapping internal structures of cells, which can be done by other methods (confocal microscopy, TEM, SEM, etc). The idea is to use the instrument for process tracing, particularly using tagged molecules in experimental conditions.

nanoSIMS samples Sample holder for 1 cm samples Thin membranes on Si wafer

Imaging of resin embedded biological tissues 12C14N 32S A mucous-producing cell in coral tissue Scale = 3um Primary ions: Cs+ 150 mm 12C14N 31P A symbiotic algae in coral tissue Scale = 2um Primary ions: Cs+

Imaging isotopic tracers in resin embedded biological tissues 150 mm 44Ca / 40Ca 44Ca used as a tracer of calcium (40Ca) to determine pathways of movement & uptake 1. 2. 3. Region 44Ca/40Ca Natural abundance 0.02 1. Mucous cell 0.05 2. Spiked Seawater 0.7 3. Epithelial cells 0.37 4. Stinging cell 4. Coral epithelium 44Ca 1 min Scale = 5um FOV = 35um Primary ions: O- Significant 44Ca uptake

Mouse cochlea cells

Imaging of As within human hair Audinot et al. (2003) Heavy metal exposure An example of where the NanoSIMS is capable, but can be done with other instrumentation, and therefore not particularly cost effective

Analytical Issues: will the metal ions be detectable? are there non-metallic ion labels that can be used as proxies for the metals? quantitation: how important? sample preparation to avoid element migration or loss

nanoSIMS & ARC Metals in Medicine is capable of playing a vital role the analytical capabilities and experience with metals in biological media needs to be developed R. Stern and P. Clode are here to enter into research partnerships with you