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 1822Sandia 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 STEMIn 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

0 2 4 6 8 100

0.1

0.2

0.3

0.4

0.5 Ti

What are x-ray spectral images?

xy

energypixel

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

yx

= ++*

W-M

W-L

* CoNiCo-L

Ni-L

*Sn-L

0 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 ToolsFocused electron probe

Distribution of elemental x-ray signals

Red = CRed = C--supportsupportGreen = aluminaGreen = aluminaBlue = Blue = FeCoFeCoCyan = CaCyan = Ca--SS--SiSi--OOBlack = shadowed supportBlack = 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 SEMSample 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

SEMAccess 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 on TEM Grid

Dehydration

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

Embedding1:1 propylene oxide:resin100% resinPlace in mold with fresh resin and cure (oven) overnight

Section and collect on TEM grid

Stain

Uranyl Acetate Lead citrate

(S)TEM

(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

0

100 0

200 0

300 0

400 0

500 0

600 0

700 0

0 1 2 3 4 5

Cou

nts

Energy (kV)

Si

P Ca

O

C

MgSecondary electron image of SiO nano-particles on Bt spores.

EDS acquired at 10 kV

Si-O

Mg-P0 1.00 2.00 3.00 4.00 5.00

0

0.050.1

0.150.2

0.250.3

0.350.4

0.450.5

0 10 20 30 40 0 1.00 2.00 3.00 4.00 5.000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15 20

100 μm

keV

Si

O

C

0 1.00 2.00 3.00 4.00 5.000

0.02

0.04

0.06

0.08

0.1

0.12 P

Cl K

Ca

0 10 20 30

keV0 1.00 2.00 3.00 4.00 5.00

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 10 20 30

P

Mg

Cl KNa

O

Substrate

Ca-P

Spores

SEM – Spectral images of weaponized surrogate material

SEM of Leahy and New York Post MaterialN

ew Y

ork

Post

le

tter m

ater

ial

Leah

y le

tter

mat

eria

l

0

500

1000

1500

2000

2500

0 1 2 3 4 5 6

Inte

nsity

(cou

nts)

Energy (kV)

0

500

1000

1500

2000

2500

3000

3500

0 1 2 3 4 5 6

C

OCaSi P SNaMg

Leahy letter material

New York Post materialC

O

CaSiSNaMg P

20 kV15 kV5 kV

Inte

nsity

(cou

nts)

1 2 43Energy (keV)

CaP

Si

MgS

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

0 2.00 4.00 6.000

0.2

0.4

0.6

0.8

1

1.2

0 2.00 4.00 6.000

0.02

0.04

0.06

0.08

0.1

0 5 10 150 5 10 15

10 μm

C

keV keV

CaP

Si

S

O

NaMg

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

Preparation of samples for STEM or SEMSample 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

SEMAccess 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 on TEM Grid

Dehydration

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

Embedding1:1 propylene oxide:resin100% resinPlace in mold with fresh resin and cure (oven) overnight

Section and collect on TEM grid

Stain

Uranyl Acetate Lead citrate

(S)TEM

(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-Onanoparticles

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 SurrogateAutomated x-ray spectral image analysis

keV0 2.00 4.00 6.00

0

2

4

6

8

200 600 1000

SiO

Cl

Si-O particles are found on the exosporium

1 μm

0 5.00 10.000

0.05

0.1

0.15

0.2

0 5.00 10.000

0.05

0.1

0.15

0.2

0.25

0.3

20 40 60 80 100 0 20 40 60 80

Fe

SiO

Pb

U

Ni

OsOs

500 nm

Si-O is on the spore coat and not the exosporium

Elements from stain overlap other possible elemental signals

STEM microanalysis of Daschle letter material

Fixed, stained and ultramicrotomed section

keV keV

STEM ADF

SI5

0 5.00 10.000

0.05

0.1

0.15

0.2

0.25

0.3

0 5.00 10.000

0.05

0.1

0.15

0.2

0 20 4010 20 30 40

keV

Si

O

PbU

Ni

Pb

OsU

Ni

Os

250 nm

Red = StainPb, U, C …Green = Coating Si, O…Blue = Carbon in spore and supportSpore

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 sourceAcceleratorScan coilsLens

Sample

Ga+Sou

rce

Ion columnElectron 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

<100nmthick 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

sporespore

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

0 2.00 4.00 6.000

0.1

0.2

0.3

0.4

0 2.00 4.00 6.000

0.05

0.1

0.15

0.2

0 20 40 600 10 20 30

Si

O

Fe

Ca

K

P

MgNa

O

500 nm

3.00 4.00 5.00 6.00 7.000

0.005

0.01

0.015

0.02

Sn

keV

keV

keV

Sn FeAdditional chemistry, Sn, revealed

in the absence of fixative and heavy metal stains

00.10.20.30.40.50.60.70.80.91

10µm

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

5

10

15

Mass / Charge

110 120 1300.0

0.1

0.2

0.3

0.4

0.5

Si

KGa

Fe

SiOHCa

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

0 2.00 4.00 6.000

0.1

0.2

0.3

0.4

0.5

0 2.00 4.00 6.000

0.1

0.2

0.3

0.4

0.5

0 2.00 4.00 6.000

0.2

0.4

0.6

0.8

1

1.2

1.40 10 20

0 10 20 30

20 40 60 80

Sn

Si

OC

C

NSO

Ca

keV

keV

0 2.00 4.000

0.02

0.04

0.06

0.08

0.1Si

P S

OC

Al

Fe

Sn

500 nm

keV

P

C

O

KNaMg

New York Post Material – FIB prepared section

ADF STEM image

0 2.00 4.00 6.000

0.1

0.2

0.3

0.4

0 5 10 15

0 2.00 4.00 6.000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2.00 4.00 6.000

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 10 20 30

5 10 15 20 25

Ca

PK

O

C

S

C

O

Si

O

C

S Sn

keV

keV

1 μm

Fe

NaMg

New York Post Material – FIB prepared section

0 2.00 4.000

0.02

0.04

0.06

0.08

0.1

1.00 2.00 3.00

0

0.05

0.1

0.15

0.2

0 10 20 30

0 20 40

OSi

keV

P + Os

ClCa

Vegetative cell with endospore.

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

1 μm

New York Post Microtomed, Unstained Section

0 2.00 4.00 6.000

0.1

0.2

0.3

0.4

0.5

20 40 60 80

Sn

Si

OC

keV

Fe

0 2.00 4.00 6.000

0.1

0.2

0.3

0.4

0 20 40 60

Si

O

FeSn0 5.00 10.00

0

0.05

0.1

0.15

0.2

0.25

0.3

0 20 40 60 80

Fe

SiO

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 featuren Total number of spores analyzed

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

Sample # Analyzed # with SiO %SPS02.266 124 97 76SPS02.057 111 73 66SPS02.088 141 91 65

040255-1 level 2 163 42 26040255-1 level 5 161 17 11040255-1 level 8 172 50 29

040030-2 level 2 94 6 6040030-2 level 5 118 0 0040030-2 level 8 113 7 6

040089-1 level 2 98 0 0040089-1 level 5 115 0 0040089-1 level 8 91 0 0

Leahy

Daschle

NYP

Analysis of fraction of spores with Si and O signature

RMR-1029RMR-1029RMR-1029

RMR-1030RMR-1030RMR-1030

0

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 of spores with Si and O elemental signature.

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

0 1 2 3 4 5 6 7 8 9 10

Energy (keV)

SiO

Ni grid

Si and O spectral component from spores

Sample # Analyzed # with SiO %NBFAC.071102.0001.

0221.00021051 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-SSEM 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: MgCl2 6H2O, MnCl2 4H2O, FeCl3 6H2O, ZnCl2, CaCl2 6H2O, 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

PCa

darkfield

Analysis of samples from a previous study

0 0.1 0.2 0.3 0.4 0.5 0.6

0 1 2 3 4 5 6 7 8 9 10

OSi

Ni grid

Inte

nsity

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