Elemental Microanalysis of Bacillus Anthracis Spores from the - - PowerPoint PPT Presentation

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.

Joseph R. Michael and Paul G. Kotula Materials Characterization Department 1822 Sandia National Laboratories, Albuquerque, NM 87185

Elemental Microanalysis of Bacillus Anthracis Spores from the Amerithrax Case

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

Signature Statistics

• Signals from Individual Spores
• Variability between fields of view
• Variability within bulk material

1 μm

Comparison of SEM and STEM

SEM – scanning electron microscope STEM – scanning transmission electron microscope

Comparison of SEM and STEM

SEM

• Imaging – 0.6 nm currently
• Microanalysis – about 1 μm
• Elements – limited to >Be
• Diffraction for crystallography
• No sample preparation may be

required

• STEM
• High Resolution Imaging – 0.2 nm
• Microanalysis – 1-2 nm spatial

resolution

• Elements – limited to >Be
• Diffraction for crystallography
• Electron transparent (thin) samples

Volume excited ~ 1 μm3 Volume excited ~ 10-8 μm3 100 nm

1 nm 1 nm

SEM STEM

In this study we make use of the characteristic x-rays generated by the electron/sample interactions.

1 μm

Automated Spectral-Image Analysis: Why?

STEM image of spores

• How do you comprehensively survey the

chemistry of large sample areas?

• Point analyses can be subjective– where to

take them from and how many.

• 2D distributions of chemical phases are

needed but simple mapping alone is not the

• answer. Mapping has potential artifacts and

requires fore-knowledge.

1 2 3

‘Chemical component images’ are needed–a spectrum from each component and an image describing where in the microstructure it’s found

2 4 6 8 10 0.1 0.2 0.3 0.4 0.5

Ti

What are x-ray spectral images?

x y energy pixel

X-ray spectrum: chemical information from sample Spectral Image Data Set

X-ray Signal Focused Electron Probe

What do we do with all that data? Typically 10’s of millions of pieces of data

Thin foil or bulk sample

energy

y x

=

+ +

*

W-M

W-L

*

Co Ni

Co-L Ni-L

*

Sn-L

5 10 15

Spectrum imaging

• Rapid decomposition of huge data sets
• Unbiased—no input guesses needed
• Elemental associations shown
• Ability to find “needle in haystack”

Spectrum imaging for elemental forensic signatures

Statistical Analysis Tools

Focused electron probe Distribution of elemental x-ray signals Red = C Red = C-

• support

support Green = alumina Green = alumina Blue = Blue = FeCo FeCo Cyan = Ca Cyan = Ca-

• S

S-

• Si

Si-

• O

O Black = shadowed support Black = shadowed support

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 Spectral Analysis., US Patent #6675106. (filing date June 1, 2001) Kotula, P. G., Keenan, M. R., Michael, J. R. (2003), “Automated Analysis of SEM X-ray Spectral Images: A Powerful New Microanalysis Tool, Microscopy and Microanalysis; Feb. 2003; vol.9, no.1, pp.1-17.

Preparation of samples for STEM or SEM

Sample fixation/ inactivation Gamma irradiation (4Mrad) or 1 %Osmium tetroxide (1 hour) or Glutaraldehyde (96 hours) Rinse in Millonig’s buffer Dry powder sample Dry powder sample SEM Access sample and dust on stub in disposable glove bag Image sample either uncoated (variable pressure SEM) or after conducive coating Access sample and mount on stub in disposable glove bag Mount sample in FIB and ion mill thin sample from spore(s) Move thin sample to carbon film

• n TEM Grid

Dehydration (30% ethanol) 50% ethanol 70% ethanol 90% ethanol 100% ethanol 100% propylene

• xide

Embedding 1:1 propylene oxide:resin 100% resin Place in mold with fresh resin and cure (oven)

• vernight

Section and collect

• n TEM grid

Stain Uranyl Acetate Lead citrate ( S ) T E M (S)TEM Performed at USAMRIID or NBFAC Performed at Sandia National Laboratories Dry powder sample (S)TEM Access sample and dust on TEM grid in disposable glove bag

Bacillus Thuringiensis treated with Silica nano- particles for flow improvements

100 0 200 0 300 0 400 0 500 0 600 0 700 0 1 2 3 4 5

Counts Energy (kV)

Si P Ca O C Mg Secondary electron image of SiO nano-particles on Bt spores. EDS acquired at 10 kV

Si-O Mg-P

1.00 2.00 3.00 4.00 5.00 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 10 20 30 40 1.00 2.00 3.00 4.00 5.00 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 5 10 15 20

100 μm

keV

Si O C

1.00 2.00 3.00 4.00 5.00 0.02 0.04 0.06 0.08 0.1 0.12

P Cl K Ca

10 20 30

keV

1.00 2.00 3.00 4.00 5.00 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 10 20 30

P Mg Cl K Na O

Substrate

Ca-P

Spores

SEM – Spectral images of weaponized surrogate material

SEM of Leahy and New York Post Material

New York Post letter material Leahy letter material

500 1000 1500 2000 2500 1 2 3 4 5 6

Intensity (counts) Energy (kV)

500 1000 1500 2000 2500 3000 3500 1 2 3 4 5 6

C O Ca Si P S NaMg

Leahy letter material New York Post material

C O Ca Si S NaMg P

20 kV 15 kV 5 kV

Intensity (counts) 1 2 4 3 Energy (keV)

Ca P Si Mg S S

New York Post material

Lower voltages produce more surface elemental information. Very small amount of Si detected at 5 kV therefore Si is locate away from the spore surface.

Si = 1.2 - 2.3 wt% ±50% Ca =3.1 - 6.5 wt% ±50% Si = 1.2 - 1.5 wt% ±50% Ca =2.7 – 3.1 wt% ±50%

5 kV= 300 nm 15 kV = 2100 nm 20 kV= 3300 nm

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 Image of Leahy material

2.00 4.00 6.00 0.2 0.4 0.6 0.8 1 1.2 2.00 4.00 6.00 0.02 0.04 0.06 0.08 0.1 5 10 15 5 10 15

10 μm

C

keV keV

Ca P Si S O Na Mg

10 μm

Spore material Support material

SEM – Spectral images of Leahy spore material

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

• Imaging – 0.6 nm currently
• Microanalysis – about 1 μm
• Elements – limited to >Be
• Diffraction for crystallography
• Instrumentation is expensive
• No sample preparation may be

required

• STEM
• High Resolution Imaging – 0.2 nm
• Microanalysis – 1-2 nm spatial

resolution

• Elements – limited to >Be
• Diffraction for crystallography
• Instrumentation is really expensive
• Electron transparent (thin) samples

Volume excited ~ 1 μm3 Volume excited ~ 10-8 μm3 100 nm

1 nm 1 nm

SEM STEM

In this study we make use of the characteristic x-rays generated by the electron/sample interactions.

Preparation of samples for STEM or SEM

Sample fixation/ inactivation Gamma irradiation (4Mrad) or 1 %Osmium tetroxide (1 hour) or Glutaraldehyde (96 hours) Rinse in Millonig’s buffer Dry powder sample Dry powder sample SEM Access sample and dust on stub in disposable glove bag Image sample either uncoated (variable pressure SEM) or after conducive coating Access sample and mount on stub in disposable glove bag Mount sample in FIB and ion mill thin sample from spore(s) Move thin sample to carbon film

• n TEM Grid

Dehydration (30% ethanol) 50% ethanol 70% ethanol 90% ethanol 100% ethanol 100% propylene

• xide

Embedding 1:1 propylene oxide:resin 100% resin Place in mold with fresh resin and cure (oven)

• vernight

Section and collect

• n TEM grid

Stain Uranyl Acetate Lead citrate ( S ) T E M (S)TEM Performed at USAMRIID or NBFAC Performed at Sandia National Laboratories Dry powder sample (S)TEM Access sample and dust on TEM grid in disposable glove bag

Weaponized Bt Surrogate

Bright Field TEM image Spore Si-O nanoparticles

1 μm

Annular Dark Field STEM image

• Fluidized agent has silica nano-particles
• Ca-phosphate nano-particles present
• Na, Ca and Cl associated with spore body

Spectrum imaging for elemental forensic signatures

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.

Weaponized Surrogate Automated x-ray spectral image analysis

keV

2.00 4.00 6.00 2 4 6 8 200 600 1000

Si O Cl

Si-O particles are found on the exosporium 1 μm

5.00 10.00 0.05 0.1 0.15 0.2 5.00 10.00 0.05 0.1 0.15 0.2 0.25 0.3 20 40 60 80 100 20 40 60 80

Fe Si O Pb U Ni Os Os

500 nm

Si-O is on the spore coat and not the exosporium Elements from stain overlap

• ther possible elemental signals

STEM microanalysis of Daschle letter material Fixed, stained and ultramicrotomed section

keV keV

STEM ADF

SI5

5.00 10.00 0.05 0.1 0.15 0.2 0.25 0.3 5.00 10.00 0.05 0.1 0.15 0.2 20 40 10 20 30 40

keV

Si O Pb U Ni Pb Os U Ni Os

250 nm

Red = Stain Pb, U, C … Green = Coating Si, O… Blue = Carbon in spore and support

Spore

Carbon in support media

100 nm

Exosporium

Fe

STEM microanalysis of Leahy letter material

Fixed,stained and ultramicrotomed section

Focused Ion-Beam (FIB) Tool/ Scanning Electron Microscope (SEM) FIB/SEM

Electron source

Accelerator Scan coils Lens Sample G a+ S

• u

r c e

Ion column Electron column FIB allows unfixed/unstained site-specific TEM specimens to be prepared even of single spores

FIB Specimen Preparation

SEM of clump of spores.

Ion image of TEM specimen SEM of TEM specimen ready to be extracted <100nm thick STEM sample Region of TEM sample

Can also prepare specimens from isolated spores

STEM image

STEM Annular Dark- Field Image of spores in cross-section

5 μm

spore spore spore spore

Pt from FIB Cross-section sample made with FIB through irradiated, unfixed, unstained spores.

Leahy Letter FIB Cross-section

Leahy Letter FIB Cross-section – FIB prepared section

2.00 4.00 6.00 0.1 0.2 0.3 0.4 2.00 4.00 6.00 0.05 0.1 0.15 0.2 20 40 60 10 20 30

Si O Fe Ca K P Mg Na O

500 nm

3.00 4.00 5.00 6.00 7.00 0.005 0.01 0.015 0.02

Sn

keV keV keV

Sn Fe

Additional chemistry, Sn, revealed in the absence of fixative and heavy metal stains

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

10µm

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 5 10 15

Mass / Charge 110 120 130

0.0 0.1 0.2 0.3 0.4 0.5

Si K Ga Fe SiOH Ca Sn Sn

Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

Ga+ ion sputtering was used to remove surface of spore. Layer that contains Si and O also has trace amounts of Sn and Fe

2.00 4.00 6.00 0.1 0.2 0.3 0.4 0.5 2.00 4.00 6.00 0.1 0.2 0.3 0.4 0.5 2.00 4.00 6.00 0.2 0.4 0.6 0.8 1 1.2 1.4 10 20 10 20 30 20 40 60 80

Sn Si O C C N S O Ca

keV keV

2.00 4.00 0.02 0.04 0.06 0.08 0.1

Si P S O C Al Fe Sn

500 nm keV

P C O K Na Mg

New York Post Material – FIB prepared section

ADF STEM image

2.00 4.00 6.00 0.1 0.2 0.3 0.4 5 10 15 2.00 4.00 6.00 0.1 0.2 0.3 0.4 0.5 0.6 0.7 2.00 4.00 6.00 0.05 0.1 0.15 0.2 0.25 0.3 0.35 10 20 30 5 10 15 20 25

Ca P K O C S C O Si O C S Sn

keV keV

1 μm

Fe Na Mg

New York Post Material – FIB prepared section

2.00 4.00 0.02 0.04 0.06 0.08 0.1 1.00 2.00 3.00 0.05 0.1 0.15 0.2 10 20 30 20 40

O Si keV P + Os Cl Ca

Vegetative cell with endospore. Spore coat incorporates Si and O (Sn, Fe) within sporulating mother cell.

1 μm New York Post Microtomed, Unstained Section

2.00 4.00 6.00 0.1 0.2 0.3 0.4 0.5 20 40 60 80

Sn Si O C keV Fe

2.00 4.00 6.00 0.1 0.2 0.3 0.4 20 40 60

Si O Fe Sn

5.00 10.00 0.05 0.1 0.15 0.2 0.25 0.3 20 40 60 80

Fe Si O keV

Daschle Material New York Post Material Leahy Material

Leahy, New York Post and Daschle are indistinguishable

Spore coats on Leahy and New York Post samples are indistinguishable (both contain Si, Fe and Sn. Daschle appears the same (Si and Fe present, Sn is obscured by other elements in stain). Material from the Daschle letter was not made available for FIB sectioning.

Importance of Spore Count

• 10 spores per sample is not

sufficient for a reasonable comparison

• 100 spores is both

experimentally achievable and allow for reasonable comparison, but comparisons of spore count near 50% will lack real comparison power

• 1000 spores allow precise

comparisons but was experimentally unreasonable (~10 days and 10 TEM samples of analysis per bulk sample) until late development of new EDS detector for STEM in SEM.

X Number of spores with a particular chemical feature n Total number of spores analyzed

(Fraction of spores showing a certain chemical make-up)

Sample # Analyzed # with SiO % SPS02.266 124 97 76 SPS02.057 111 73 66 SPS02.088 141 91 65 040255-1 level 2 163 42 26 040255-1 level 5 161 17 11 040255-1 level 8 172 50 29 040030-2 level 2 94 6 6 040030-2 level 5 118 040030-2 level 8 113 7 6 040089-1 level 2 98 040089-1 level 5 115 040089-1 level 8 91 Leahy Daschle NYP

Analysis of fraction of spores with Si and O signature

RMR-1029 RMR-1029 RMR-1029 RMR-1030 RMR-1030 RMR-1030

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

500 nm

Si-O image

Bacillus anthracis Ames which was grown in shaker flasks at USAMRIID using Leighton-Doi media. Sample 1030

STEM images and Si and O component images of two samples of 040255

Note variability in number

• f spores with Si and O

elemental signature. BA Ames grown via fermentation (Dugway) using Leighton-Doi media

1 2 3 4 5 6 7 8 9 10

Energy (keV)

Si O Ni grid Si and O spectral component from spores

Sample # Analyzed # with SiO % NBFAC.071102.0001. 0221.0002 1051 197 18.7 NBFAC.071102.0001. 0228.0002 982 86 8.8 NBFAC.071102.0001. 0232.0002 986 40 4.4 NBFAC.071102.0001. 0230.0002 476 7 1.5 NBFAC.071102.0001. 0235.0002 989 12 1.2

Analysis of fraction of spores with Si and O signature

Sample was described as “evidence”, no further description given by FBI. Analyzed using STEM in SEM technique Si in the spore coat – new detector not sensitive to oxygen, STEM used to verify presence of oxygen

Red= Si, Green = Ca-P, Blue, Cl-S

SEM defines field of view for spectral image acquisition MSA identifies three chemical signatures Si-containing spore coat From this it is possible to count x and n

STEM in SEM of unstained, microtomed section

Background Coat Cortex Core

Previous studies have shown Si on the coat*

*Johnstone, K. et al., Location of metal ions on Bacillus megaterium spores by

high-resolution electron probe x-ray microanalysis, FEMS microbiology Letters,

• vol. 7, 1980, p 97-101.

Modifed CCY medium containing: MgCl26H2O, MnCl2 4H2O, FeCl36H2O, ZnCl2, CaCl26H2O, KH2PO4, K2HPO4, glutamine, acid casein hydrolysate, enzymatic casein hydrolysate, enzymatic yeast extract and glycerol.

• M. Stewart et al., Journ.

Bact., July 1980, p. 481- 491

Bacillus cereus

Si P Ca darkfield

Analysis of samples from a previous study

0.1 0.2 0.3 0.4 0.5 0.6

1 2 3 4 5 6 7 8 9 10

O Si Ni grid Intensity Energy (keV) Grown in modified liquid G media, no anti-foam used

Mg Si P S Ca Mn Cu from grid

Author’s noted: “considerable variation in Si content both within and between different spore preparations,… unlikely to be due entirely to contamination.”

Conclusions

The NYP, Leahy and Daschle materials are indistinguishable elementally at the spore level. NYP, Leahy and Daschle materials all have similar fraction of spores with Si-O in spore coat Si-O signature found on endospores in New York Post sample The letter powders are not unique with respect to Si and O elemental signatures. Examples from the literature and from samples grown for this study. SEM and STEM have proven useful for spore characterization