Elemental Microanalysis of Bacillus Anthracis Spores from the Amerithrax Case Joseph R. Michael and Paul G. Kotula Materials Characterization Department 1822 Sandia National Laboratories, Albuquerque, NM 87185 Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under contract DE-AC04-94AL85000.
Outline � Tools for elemental microanalysis � Spectral imaging � Microanalysis of Leahy and NYP with SEM � Microanalysis of Leahy, NYP and Daschle with STEM and TOF-SIMS � Are the letter powders unique with respect to elemental signatures? � Summary
•Variability between fields of view •Signals from Individual Spores •Variability within bulk material Signature Statistics 1 μ m
Comparison of SEM and STEM SEM – scanning electron microscope STEM – scanning transmission electron microscope
Comparison of SEM and STEM SEM • STEM • Imaging – 0.6 nm currently • High Resolution Imaging – 0.2 nm • Microanalysis – about 1 μ m • Microanalysis – 1-2 nm spatial • Elements – limited to >Be resolution • Diffraction for crystallography • Elements – limited to >Be • No sample preparation may be • Diffraction for crystallography required • Electron transparent (thin) samples STEM SEM In this study we make use of 1 nm the characteristic x-rays 1 nm generated by the electron/sample interactions. 100 nm Volume excited Volume excited ~ 1 μ m 3 ~ 10 -8 μ m 3
Automated Spectral-Image Analysis: Why? 1 μ m • How do you comprehensively survey the chemistry of large sample areas? 1 • Point analyses can be subjective– where to take them from and how many. 2 • 2D distributions of chemical phases are needed but simple mapping alone is not the 3 answer. Mapping has potential artifacts and requires fore-knowledge. STEM image of spores ‘ Chemical component images ’ are needed–a spectrum from each component and an image describing where in the microstructure it’s found
What are x-ray spectral images? X-ray spectrum: chemical information from sample Spectral Image Data Set Focused Ti 0.5 Electron 0.4 0.3 Probe 0.2 0.1 energy 0 0 2 4 6 8 10 X-ray Signal pixel x y What do we do with all that data? Thin foil or Typically 10’s of millions of pieces of bulk sample data
Spectrum imaging for elemental forensic signatures Spectrum imaging Statistical Analysis Tools Focused electron Distribution of elemental • Rapid decomposition of huge data sets probe x-ray signals • Unbiased—no input guesses needed • Elemental associations shown Co-L Ni Ni-L * • Ability to find “needle in haystack” Co + energy 0 5 10 15 = W-M * W-L + x y Sn-L * Keenan, M. R., and Kotula, P. G.,(2003) Apparatus and System for Multivariate Spectral Analysis., US Patent #6584413. (filing date June 1, 2001). Keenan, M. R., and Kotula, P. G.,(2004) Method of Multivariate Red = C- -support support Red = C Spectral Analysis., US Patent #6675106. (filing date June 1, Green = alumina Green = alumina 2001) Blue = FeCo FeCo Blue = Kotula, P. G., Keenan, M. R., Michael, J. R. (2003), “Automated Cyan = Ca- -S S- -Si Si- -O O Cyan = Ca Analysis of SEM X-ray Spectral Images: A Powerful New Black = shadowed support Black = shadowed support Microanalysis Tool, Microscopy and Microanalysis ; Feb. 2003; vol.9, no.1, pp.1-17.
Preparation of samples for STEM or SEM Access sample Sample fixation/ inactivation Image sample either Dry SEM and dust on stub in uncoated (variable powder Gamma irradiation (4Mrad) or disposable glove pressure SEM) or sample bag 1 %Osmium tetroxide (1 hour) or after conducive coating Glutaraldehyde (96 hours) Access sample and Dry dust on TEM grid in Rinse in Millonig’s buffer powder ( (S)TEM disposable glove S sample ) T (S)TEM bag E M Dry Access sample Move thin Mount sample in powder and mount on stub sample to FIB and ion mill in disposable sample carbon film thin sample from glove bag on TEM Grid spore(s) Dehydration (30% ethanol) Embedding Stain 50% ethanol 1:1 propylene oxide:resin 70% ethanol 100% resin Section and collect Uranyl Acetate 90% ethanol Place in mold with fresh on TEM grid Lead citrate 100% ethanol resin and cure (oven) 100% propylene overnight oxide Performed at Performed at Sandia USAMRIID or NBFAC National Laboratories
Bacillus Thuringiensis treated with Silica nano- particles for flow improvements 700 0 Si 600 0 500 0 Counts O 400 0 300 0 C 200 0 100 0 P Mg Ca Secondary electron image of 0 0 1 2 3 4 5 SiO nano-particles on Bt Energy (kV) spores. EDS acquired at 10 kV
SEM – Spectral images of weaponized surrogate material 100 μ m Spores Substrate 0.9 0.5 C Si-O 0.8 Si 0.45 0.7 0.4 0.6 0.35 0.3 0.5 0.25 0.4 0.2 0.3 0.15 0.2 O 0.1 0.1 0.05 0 0 0 1.00 2.00 3.00 4.00 5.00 0 5 10 15 20 0 10 20 30 40 0 1.00 2.00 3.00 4.00 5.00 0.16 Mg Mg-P Ca-P P 0.12 0.14 0.12 0.1 0.1 P 0.08 Ca 0.08 0.06 O 0.06 0.04 0.04 Na Cl K K Cl 0.02 0.02 0 0 0 10 20 30 0 10 20 30 0 1.00 2.00 3.00 4.00 5.00 0 1.00 2.00 3.00 4.00 5.00 keV keV
SEM of Leahy and New York Post Material letter material material New York Post Leahy letter
2500 New York Post material C 2000 Leahy letter material 20 kV 15 kV 5 kV Intensity (counts) Si Si = 1.2 - 2.3 wt% ±50% 1500 Ca =3.1 - 6.5 wt% ±50% Ca 1000 P S Mg 500 O Intensity (counts) Si P S Ca NaMg S 0 0 1 2 3 4 5 6 3500 1 2 3 4 C Energy (keV) 3000 New York Post material 5 kV= 300 nm 2500 15 kV = 2100 nm Si = 1.2 - 1.5 wt% ±50% 20 kV= 3300 nm 2000 Ca =2.7 – 3.1 wt% ±50% Lower voltages produce more 1500 surface elemental information. O 1000 Very small amount of Si detected at 5 kV therefore Si is locate away 500 Si Ca S P NaMg from the spore surface. 0 0 1 2 3 4 5 6 Energy (kV)
Bulk EDS Spectrum from Edgewood Report* Bacillus subtilis var. niger spores grown in Casein Digest (CD) Medium (no indication that an anti-foam agent was added). *L. F. Carey, D. C. St. Amant and M. A. Guelta, Production of Bacillus spores as a simulant for biological warfare agents, Edgewood Chemical Biological Center,ECBE-TR-372, April 2004.
SEM – Spectral images of Leahy spore material 10 μ m SEM Image of Leahy material Spore material Support material 1.2 0.1 O C 1 0.08 0.8 0.06 Si 0.6 Ca 0.04 P Na 0.4 Mg 0.02 S 0.2 0 0 0 2.00 4.00 6.00 0 5 10 15 0 2.00 4.00 6.00 0 5 10 15 10 μ m keV keV Spectral Image components of Leahy material
Summary of SEM Observations of spore materials Microanalysis in the SEM shows that Si is present in the Leahy and New York Post materials. But- microanalysis of bulk samples in the SEM lacks sufficient spatial resolution to show where the Si is located with respect to the spores. Low kV shows Si is mostly on the interior of the spores. Microanalysis in the SEM is can be made quantitative. But not from samples like the powder attack materials. Spectral imaging with component analysis provides some useful information.
Comparison of SEM and STEM SEM • STEM • Imaging – 0.6 nm currently • High Resolution Imaging – 0.2 nm • Microanalysis – about 1 μ m • Microanalysis – 1-2 nm spatial • Elements – limited to >Be resolution • Diffraction for crystallography • Elements – limited to >Be • Instrumentation is expensive • Diffraction for crystallography • No sample preparation may be • Instrumentation is really expensive required • Electron transparent (thin) samples STEM SEM In this study we make use of 1 nm the characteristic x-rays 1 nm generated by the electron/sample interactions. 100 nm Volume excited Volume excited ~ 1 μ m 3 ~ 10 -8 μ m 3
Preparation of samples for STEM or SEM Access sample Sample fixation/ inactivation Image sample either Dry SEM and dust on stub in uncoated (variable powder Gamma irradiation (4Mrad) or disposable glove pressure SEM) or sample bag 1 %Osmium tetroxide (1 hour) or after conducive coating Glutaraldehyde (96 hours) Access sample and Dry dust on TEM grid in Rinse in Millonig’s buffer powder ( (S)TEM disposable glove S sample ) T (S)TEM bag E M Dry Access sample Move thin Mount sample in powder and mount on stub sample to FIB and ion mill in disposable sample carbon film thin sample from glove bag on TEM Grid spore(s) Dehydration (30% ethanol) Embedding Stain 50% ethanol 1:1 propylene oxide:resin 70% ethanol 100% resin Section and collect Uranyl Acetate 90% ethanol Place in mold with fresh on TEM grid Lead citrate 100% ethanol resin and cure (oven) 100% propylene overnight oxide Performed at Performed at Sandia USAMRIID or NBFAC National Laboratories
Weaponized Bt Surrogate Bright Field TEM image Annular Dark Field STEM image Spore Si-O nanoparticles 1 μ m
Spectrum imaging for elemental forensic signatures • Fluidized agent has silica nano-particles • Ca-phosphate nano-particles present • Na, Ca and Cl associated with spore body See: L. N. Brewer, J. A. Ohlhausen, P. G. Kotula and J. R. Michael, Forensic imaging of bioagents by X-ray and TOF- SIMS hyperspectral imaging”, Forensic Science International, vol. 179, 2008, 98-106.
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