The J105 SIMS. A New Instrument for 3-Dimensional Imaging and Analysis. Paul Blenkinsopp, Ionoptika Ltd

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1 The J105 SIMS A New Instrument for 3-Dimensional Imaging and Analysis Paul Blenkinsopp, Ionoptika Ltd

2 The J105 SIMS

3 Why a new ToF Mass Spectrometer? The J105 ToF has been designed to allow us to separate out the Mass Spectrometry from the influence of both the Primary Ion Beam and the Sample itself, giving combined Mass and Spatial Resolution. High Mass Accuracy, with Mass Resolution and MSMS capabilities, aid identification on unknown peaks, regardless of sample height or topography changes. Limitations on primary ion beam are removed no fast pulsing is required, allowing any available ion source to be used for full analysis. There are no Etch Cycles with lost data when depth profiling. The J105 is always collecting data if the ion beam is on. The J105 is able to take full advantage of the imaging capabilities of our range of high performance Cluster Ion Sources, such as the IOG C60-40, and the GCIB 40.

4 Key Features of Mass Spectrometer Molecular imaging mass spectrometer, with high spatial resolution and high mass resolution at the same time Mass Accuracy and Mass Resolution are not affected by changes in the sample height Mass Spectrometry is independent of the Primary Ion Beam, allowing full use of any Cluster Source Optimised Sample Handling for organic samples Full computer control and maximum automation

5 J105 SIMS

6 J105 SIMS Operation

7 The J105 Sample Handling Large Glove Box with Argon purge for sample insertion LN2 sample cooling to 100K Mousetrap system for freeze fracture of samples. Motorised 3-axis stage with 1 micron precision. Insertion lock with low pump down volume

8 The J105 Mousetrap - Automated Freeze Fracture Cells can be grown on a hinged plate Plate is closed Sample is frozen, and mounted onto holder Sample is loaded using Glove Box Sample is pumped down, and during transfer hinge is sprung open

9 3D Cell Imaging - 2 Freeze fractured Frozen Hydrated Frozen hydrated analysis preserves 3D structure of the cells. Nuclues and lower mebrane only resolved when frozen. Dry

10 Large extraction gap aids analysis of rough samples Cr image of an M1.6 screw Cr

11 Large Depth of Field The J105 is able to analyse samples over a large area, even if they are highly curved. The images below show a frog s egg at various stages of development (2 cells, 8, 32, then many). The egg is a sphere about 1.1mm in diameter, and is analysed whole. The field of view is 1200 x 1200 microns. You can see from the images that the J105 has a good level of signal from almost the whole hemisphere, and the mass calibration is unchanged. Data courtesy of Hua Tian, University of Manchester µm 300 µm Mass 577 Total ion image of embryo after depth profiling

12 J105 Cluster Ion Beams 25 kv liquid metal cluster ion gun, to 150nm 40 kv C nm 40 kv GCIB, 3mm FLIG 5 Oxygen

13 Why we need Cluster Beams!

14 C60 impacting on a surface feature

15 The GCIB 40 Large Image is mm, 10 µm/pixel Detail is a 512mm image of tissue section at 2µm/pixel. Dose of 6e12, negative ion mode

16 The GCIB 40 Negative ion mode Spectra from a single 3.9mm pixel of cancerous tissue Positive ion image of a hair cross-section 90mm FOV 2.1mm resolution

17 Why it s important to have spatial resolution and mass resolution 2D Cell Imaging of Cheek Cells ToF-SIMS images of freeze fractured, freeze dried cheek cells deposited on steel. Total Ion m/z 184 The image was acquired using a primary ion dose of ions/cm 2. The field of view for the analysis in this case was approx µm 2. m/z m/z 103.0

18 3D Imaging on the J105 Etch Etch Σ Σ To enable 3D image generation on a reasonable time scale the conventional experiment uses interlaced imaging and etching cycles that wastes precious sample. The use of a continuous analysis beam on the J105 means that all the material is sampled.

19 3D Cell Imaging 1 - HeLa Dry Freeze fractured Frozen Hydrated Reconstructed 3D SIMS data shows the difference between dry (at room temperature) and freeze fractured frozen hydrated analysis. Fletcher et al. Rapid Commun. Mass spectrom., 2011

20 Organic Depth Profiling Irganox 1010 / Irganox 3114 thin layers with GCIB in single beam mode Room Temp Depth /nm Depth / nm 150 K

21 counts Organic Depth Profiling with the 40 kv GCIB A recent study looking at the analysis of a mixed Irganox sample made by NPL. J105 shows 12.8nm Depth Resolution on this sample MMK time

22 Inorganic Depth Profiling C60 has a high sputter rate for many materials, making it suitable for a wide range of materials. Metals, oxides, semi-conductors, and many organic materials can analysed, with good spatial and depth resolution. Nist NiCr sample, 66 and 53nm alternating layers.

23 Depth profiling a mixed oxide layer on a fuel cell µm 2 field of view, z depth approximately 200nm.

24 MS-MS of Haloperidol High gas X 10 MSMS spectra from the J105. Ratio of the m/z 123 and 165 is comparable to 40 ev collsion in quadrupole MS-MS experiments.

25 MSMS of Diacylglycerides in Fly brain DAG region of fly brain spectrum High energy CID (0.5 6 kev) Nitrogen collision gas ToF-ToF MSMS of m/z Povides detail on chain lengths and saturation and there increased specificity that tells us about the biology. x50

26 MS-MS of fatty acids aids identification of diacylglycerides from a fly brain Nhu Phan et al SIMSXIX

27 Tissue Imaging with Ar4000 on frozen hydrated tissue section All data was acquired using 40 kev Ar4000 +, ion dose: ions/cm2, negative ion mode. FOV is 5mm.

28 Mass Tissue Imaging with Argon GCIB (11 mm field of view) Mass Mass This Lipid is C40H80NO8P+ H + Na + K Each of these masses is within 3 ppm of theoretical mass for this lipid with these adducts.

29 Large Area Imaging of rat kidney Full mass spectrum available at each pixel 9 x 9 mm

30 GCIB 40 imaging of tissue samples. The J105 is ideally suited to the use of cluster ion beams for analysis, as there is no requirement for fast pulsing of the primary beam. The cluster beams have real advantages over other ion beams when analysing organic tissue, as damage to the sample molecules is minimised, allowing a volume of material to be analysed, and not just a static dose limit. Signals are higher, and backgrounds are lower, and the sample can be depth profiled without wasting material. In addition to this, it is clear that larger cluster beams tend to produce higher signal levels from most large molecules for the same dose. On the J105, the recommended ion beams are the IOG C60-40, a 40 kev C60 system capable of achieving a 300nm spot, and the GCIB 40, a 40 kv argon cluster beam, capable of achieving 3 microns with Ar4000 clusters. Brain section, analysed with 40 kv Ar4000, 13 pa, negative SIMS. 3 hours for full acquisition. 9.7x7.2mm Green is , Blue is , Red is 885.5

31 Brain section, analysed with 40kV Ar4000, 52pA, positive SIMS. 25 min for full acquisition Green is , Blue is , Red is Below is a 1mm area, 50 layer 3D view of Mass

32 Below is an example of some human hair (previously dyed different colours) that has been cross-sectioned and analysed with the GCIB 40. Total Ion & & & Overlay of the four masses

33 The J105 SIMS A Molecular Imaging mass spectrometer where Mass Resolution, Mass Calibration, and Mass Accuracy are not affected by the sample or the Ion Source A Large Extraction Gap, enabling the analysis of samples with significant height variations across the analysed area High performance cluster beams for 2D and 3D imaging High Mass Accuracy and Resolution, and MSMS capability, to enable better identification of an unknown peak Low Temperature Sample Handling capability, with sealed Glove Box for sample introduction

34 Acknowledgements Thank you for your attention I would like to thank the following (with their groups) for their help and collaboration: John C. Vickerman, SARC, The University of Manchester N. Winograd, PennState University Barbara J. Garrison, PennState University J. S. Fletcher, NCIMS, The University of Gothenburg Peter J. Cumpson, Newcastle University Dave Castner, NESAC/BIO, University of Washington

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