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Raman Chemical Imaging Provides Rapid, Non-Invasive and

Reagentless Biothreat DetectionSession VIII: Technology Forum Focus Groups

Group I: Chemical/Biological/Explosive Detection & Security

Steven Vanni ChemImage Corporation

7301 Penn AvenuePittsburgh, PA 15208

m: (412) 478-3504f: (412) 241-7311

vanni@chemimage.comwww.chemimage.com

ChemImage Copyright

Raman Chemical Imaging Provides Rapid, Non-Invasive and

Reagentless Biothreat Detection

Steven VanniChemImage Corporation

vanni@chemimage.comwww.chemimage.com

Homeland Security19th Annual NDIA

Security Division Symposium and Exhibition18 June 2003Reston, VA

In cooperation with:

Dr. Ted HadfieldDr. Kathy KalasinskyDr. Vic KalasinskyArmed Forces Institute of Pathology

Dr. Steve ChristesenDr. Alan SamuelsJanet JensenUS Army, Edgewood Chemical & Biological Center

Dr. Jay EversoleNaval Research Laboratory

Dr. Patrick TreadoDr. Matthew P. NelsonDr. Charles W. GardnerJulianne WolfeDr. Robert SchweitzerJason NeissChemImage Corporation

ChemImage CopyrightChemical Imaging - Molecular spectroscopy and digital imaging for chemical analysis of materials

Why Chemical Imaging?• Spectrum provides intrinsic contrast• No need for dyes, stains or reagents• No sample preparation• Fast, noncontact & nondestructive• Spectroscopy provides fingerprint for material

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Chemical ImagingMassively Parallel Spectroscopy

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Polystyrene Raman Spectrum

Wavenumber (cm-1)

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Nylon Raman Spectrum

Conventional Imaging of Low Contrast Object

Components:• Polystyrene• Nylon

Raman image of Component A

Chemical Image Contrast based on Composition, Structure & Concentration

Raman image of Component B

ChemImage Copyright

Polymer BlendMicroscope Image

HOW DOES CHEMICAL IMAGING WORK?

Raman SpectraChemical Image

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Wavenumber (cm-1)

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• High performance plastics (ex. car bumpers) are blends of polymers

• Chemical imaging improves cost performance

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Chemical Imaging Techniques

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Feature Size (m)

• Chemical Imaging integrates multiple, orthogonal detection strategies

ChemImage Copyright

hνο

RayleighScattering (II)

hνο

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Raman Spectra are Specific

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E. coli from Agar plate

Yeast

BG (bacilus subtilis) spores

Cryptosporidium spores

Raman Spectroscopy• Inelastic scattering phenomenon• Laser based technique• Probes energy of molecular vibrations - provides a “fingerprint”

Advantages• H2O not an interference (compatible with aqueous systems)• Little to no sample preparation• Small amount of sample required• Operates in ‘reflectance’ mode• Diverse experimental conditions (fiber optics & microprobes)• Suitable for in situ monitoring (no vacuum required)• 50 – 4,000 cm-1 coverage in single instrument• Glass optics including fiber optics and fiberscopes• Usually nondestructive and noninvasive• Suitable for aqueous, gaseous and solid samples• Inorganic and organic material analysis• High spatial resolution (250 nm) imaging

Disadvantages:• Weak phenomena – 1 out of 106 photons a Raman photon• Moderate sensitivity (0.1-1 wt%)• Fluorescence interference (can be minimized)• Not quantitative unless internal standard is used

ChemImage Copyright

ConventionalMicroscopy

Conventional TechnologyAcquisition Time: 2.8 Hours

Information Content: 1024 Pixels

ChemImage TechnologyAcquisition Time: 10 Seconds

Information Content: 256,000 Pixels

Chemical Imaging vs. Competitive Chemical Imaging Technology

Why is ChemImage’s Technology Unique?• Requires No Sample Pre-Treatment• Rapid Analysis Time: Typically 5 Minutes or Less• Valid Results: No Need for Additional Tests• Not Limited to a Specific Biothreat Agent• Has the Ability to Detect Multiple Agents• Readily Adaptable to New BiothreatAgents

1µm

BG BS

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MacroMicro Remote

• Large surface area analysis• Macro/Micro zoom optics• NIR, Raman, PL, Fluorescence, Color

• Real-time video imaging• Laser Raman spectroscopy• NIR, Fluorescence & Raman

Chemical Imaging

• Dispersive Raman platform• High definition imaging• 250nm spatial resolution• Entry level systems• Volumetric imaging capable• Raman, PL, Fluorescence, NIR, Color

U.S

. Patent N

o. 6,002,476

Patent P

ending

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Software

ChemImage Instrumentation Platforms

ChemImage Copyright

Counterfeit $10 Bill

• Latent fingerprint not previously detectable with existing technology

• Technique is non-destructive

Conventional Microscopy Raman Chemical Image

10 µm

Drug Particles

• Drug particle size not detectable with any other existing technology

Particle Size (Max. Chord, µm)

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Particle Size Distribution (PSD)

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Mean Particle Size = 1.79±1.33

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Raman Image

Implant: 2.0 x 1015 As cm-2

Optical Image

• Raman imaging has unparalleled sensitivity for ion implantation

3D Raman Chemical Image (Normalized Data)

3D Raman Chemical Image (Deconvolved Data)

15 µm

• Volumetric Raman Chemical Imaging provides non-destructive whole object molecular imaging

Dark Background ExtractLight Background Extract

Forensics: fingerprint detection Pharmaceuticals: drug particle size

Polymers: 3D blend imagingSemiconductors: ion implant imaging

ChemImage Application Examples

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Biothreat Detection• Molecular analysis complicated and difficult for mixtures of really small things

• Bioagents are the hardest: small, complex organisms, in cluttered backgrounds

• Bioagents are usually invisible, odorless, taste-free; human senses can not recognize when exposure has occurred

How are We Addressing the Problem?• Molecular chemical imaging technology has demonstrated great promise in addressing this

problem

• Works even for single bacteria … and… orders of magnitude faster than conventional techniques

• Chemical molecular identification possible – now being validated with Government Labs (AFIP, ECBC, NRL)

ChemImage Copyright

BacteriaAnthraxPlague

Rabbit FeverDiptheria

RickettsiaeTyphus

Spotted FeverQ-Fever

VirusesYellow FeverDengue Fever

Influenza

Chemical-Biological Warfare Threat Detection and Identification Methods

Biological Chemical

ToxinsBotulinum Toxins

MycotoxinsStaphylococcus

Saxitoxin

ChemicalsSarin

SomanMustard

0.0001µm(1nm)

0. 1µm(100 nm)

0.25 x 1 µm1 x 4 µm

• Chemical Imaging & Spectroscopy• MALDI-TOF Mass Spectrometry• Flow cytometry• Polymerase chain reaction• Whole-cell immunosensors

• Colorimetric• DNA-based• Particle tag-based• Gravimetric• Electrochemical

• Chemical Imaging & Spectroscopy• Mass spectrometry• 2D gel electrophoresis• Immunosensors

• DNA-based• Colorimetric (ELISA)• Nanoparticle tag

• Quantum dot• Upconverting phosphor

• Gravimetric• Electrochemical

• Chemical Imaging & Spectroscopy

• Ion mobility spectrometer• Mass spectrometry• SAW sensor with sorption

coating• FPW sensor with sorption

coating• FTIR

0.0001µm(1nm)

Matthias-Maser, S; “Primary Biological Aerosol Particles,” in Atmospheric Particles, ed. By R. M. Harrison and R. Van Grieken (Wiley, New York, 1998), p. 349.

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Bacillus cereus (BC)Bacillus thurigiensis (BT)Bacillus anthracis (BA)Dipicolinic AcidClostridium sporogenes (botulism sim.)

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Raman Spectra of Anthrax SporesUsing a ChemImage FALCON Raman Chemical Imaging Microscope Bacillus anthracis (BA)

1 µm

• Raman analysis rapidly provides molecular fingerprint (

ChemImage Copyright

AFIP Samples – B. Anthracis in Sporulation BrothDispersive Raman Spectroscopy – 10 Different Regions of Interest

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ROI 1ROI 2ROI 3ROI 4ROI 5ROI 6ROI 7ROI 8ROI 9ROI 10

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• Statistical Analysis (F-Test) indicates reproducibility to 95% confidence level• Collected with FALCON Raman Chemical Imaging Microscope• Data Acquisition Time: 60 sec/spectrum

Raman Spectra are reproducible

ChemImage CopyrightRaman Spectra of RAAD Program BG SporesUsing a ChemImage FALCON Raman Chemical Imaging Microscope

Raman Shift (cm-1)

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5µm

NRLMIT LL

Performed in Collaboration with• Dr. Jay Eversole , NRL• Dr. Steve Christesen, ECBC• Dr. Antonio Sanchez, MIT LL

• Interlaboratory results are reproducible• BG traceable to Dugway

ChemImage CopyrightRapid Automated Detection of BG Spores

1 second Acquisition Time

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Raman Shift (cm-1)

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Dispersive Spectra

Target SpectrumBG library Spectrum

Spectral Library Search Dialog Box

• True positive identified within 1 sec, using automated approach

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Single Spore Detection in 25 sec

Bacillus Anthracis Limit of DetectionUsing Raman Chemical Imaging

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Spore Density (# of Spores/cm2 x 105)

Fused Raman/Optical Image

Single SporeS/N ~ 1.91

y = 2.771x - 2.0461

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Raman Shift (cm-1)

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Aspergillus terreus(Fungal spore)

MS2 bacteriophage(Virus sim)

Diesel Soot

BG (Anthrax sim)

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Egg White (Ricin sim )

Raman Image

2 µm

BGconidia of A. terreus

Raman Analysis of BG/Aspergillus terreus MixtureUsing a ChemImage FALCON Raman Chemical Imaging Microscope

ChemImage Copyright

False Positive Rate

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BA among Non-Bacillus_anthracis

True Negatives

Preliminary Receiver Operator Characteristic (ROC) CurveBacillus anthracis Discrimination Assessment

• Using un-optimized discrimination approach, at 90% probability of detection, 5X improvement in false alarm rate demonstrated

ChemImage CopyrightChemical Imaging Analysis of Mixtures

• Mixtures are not homogeneous on the microscopic scale• Spectra obtained from different sample locations are different• Chemical Imaging (i.e. spatially-resolved spectroscopy) rapidly provide a set of spectra

incorporating these variations• Data analysis tests set of mixture spectra against all of the compounds in its library and

determines the compounds in its library likely to be present• A ranking system selects and reports the compounds present

Spatially Resolved Spectra

Reflectance images with sampling grid1 2 3 4

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Raman Shift (cm -1)300 900 1500 2100 2700 3100

Specimen is white powder containing (Talc, Tylenol, BG spores)

Results of Automated Identification

ChemImage Copyright

Mucin, BG, and OnionRaman Spectra

MucinOnionBG

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Raman Chemical Imaging of Anthrax on Food & Bodily FluidsUsing a ChemImage FALCON Raman Chemical Imaging Microscope

Optical Image

Raman Chemical Image

Optical Image

Raman Chemical Image

3 µm

Threats to FoodBG and Onion

Threats to HumansBG and Mucin

ChemImage Copyright

Blind Study of BG/Mucin Samples

Sample 1*Sample 2Sample 3*Sample 4Sample 5*

Sample 6Sample 7*Sample 8Sample 9Sample 10*

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*Sensitivity (true positive rate): %Sensitivity = (TP/(TP+FN))*100, where TP = true positives and FN = false negatives.*Selectivity (true negative rate): %Selectivity = (TN/(FP+TN))*100, where TN = true negatives and FP = false positives.

Sensitivity* = 100%Selectivity* = 100%

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• ChemImage has successfully demonstrated its EAGLE Transportable Microscope System

• Anthrax (simulant) detected in seconds – comparable to FALCON

• EAGLE can automatically identify presence of biothreat by using Biothreat Database

• Features• Fluorescence, Colorimetric Chemical Imaging (targeting)• Dispersive Raman Chemical Imaging (identification)• Wireless and Remotely Controlled• Live Digital Video

EAGLE Transportable Microscope System

EAGLE – 4 SecsFALCON – 60 Secs

Fluorescence Chemical Imaging BG/Diesel Soot/Road Dust Mixture

Fluorescence Chemical Image Fluorescence Spectra

Wavelength (nm)

Inte

nsity

480 510 540 570 600 630 660 690 720

Bacillus globigi (BG)Diesel SootRoad Dust (non fluorescing)

ChemImage Copyright

Conclusions• Chemical Imaging detection is non-invasive, non-contact and does not require significant sample

preparation or reagents

• ChemImage technology can allow users (physicians, law enforcement, soldiers, researchers) see and identify materials (cancer, biothreats, evidence) that you can’t detect now

• Chemical Imaging is inherently orthogonal, integrating multiple detection strategies into the same system

• Normal Raman spectroscopy is highly selective and sensitive (single spore detection demonstrated) when targeted

• Optical imaging and fluorescence imaging sensitive means for targeting

• Chemical Imaging provides excellent sampling statistics, which compensates for spore to spore variability and enables morphometric assessment

• Widefield illumination important for Chemical Imaging

• Near term deployment of Chemical Imaging technology conceivable, based on mature, commercially available Raman technology

• Technology scalable

– Field transportable technology demonstrated

– Fully portable technology feasible

– Basis for a hand held point Chemical Imaging sensor