Submillimeter wave spectroscopy of biological
macromolecules
Tatiana Globus
Department of Electrical and Computer Engineering, University of Virginia
[email protected]
This work is supported by the U.S. DOD under Contract DAAD19-00-1-0402,
and in part by U.S.Army NGIC Contract # DASC01-01-C0009
Other participants: Dwight Woolard (ARL, ARO), Boris Gelmont (UVA),
Tatyana Khromova (UVA), Maria Bykhovskaya (Lehigh University)
GRAs: Xiaowei Li, Ramakrishnan Parthasarathy (UVA)
N5-Applications of THz Radiation
American Physical Society Meeting 2005, Los Angeles
APS, March 2005
1
Absorption and Scattering Spectroscopy
Scattering and absorption spectroscopies utilize the interaction
of an applied electromagnetic field with the phonon (vibration)
field of the material to provide useful structural information .
Absorption
hνlight
I2
hνvibr.=hνlight
T.Globus, UVA
hν1
I1
hνvibr
hνlight
Raman scattering
Ground state
Exited state
hνvibr.
hν2
hνvibr. = hν1-hν2
hν1, hν2 >> hνvibr.
APS, March 2005
2
DNA Absorption Spectrum
Frequency, THz
3
2000
I
10
30
60
90
I
I
I
I
II -far IR
Absorption coefficient, cm-1
III -THz
I Visible & near IR
1500
‰ Regions I & II (Studied well
by IR & Raman)- resonances due
to short-range, high energy
interactions
1000
‰ in THz Region III – more
species specific spectral features
of bio molecules are found
500
0
0
1000
2000
Frequency, cm -1
3000
Absorption spectrum of DNA (our results).
FTIR spectroscopy produces high quality spectra in the region II,
and can separate overlapping subcomponents in the spectra
T.Globus, UVA
APS, March 2005
3
INTERNAL MOLECULAR VIBRATIONS
Vibrations of
Bonds
Frequencies
>21 THz (700 cm-1 )
Bond angles
6-27 THz (200-900 cm-1 )
Torsion angles
< 9 THz (<300 cm-1 )
In the region III (0.1-10 THz or 2 - 300 cm-1), absorption spectra reflect
low-frequency molecular internal motions or vibrations involving the
weakest hydrogen bonds and/or non-bonded interactions between different
functional groups within molecules or even between molecules.
The resonant frequencies of such motions – phonon modes- are strongly
dependent on molecular structure
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4
WEAK HYDROGEN BONDS
Weakest hydrogen bonds,
shown by dots:
-C-H ......O-C-H ......N-C ...... H-N-C ...... H-O-N-H......O=C- weak and have only ~ 5% of the strength of covalent bonds
- multiple hydrogen bonds stabilize the structure of bio-polymers
- hold the two strands of the DNA double helix together, or hold
polypeptides together in different secondary structure conformations.
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Why THz?
¾ THz spectroscopy reveals structural information quite different from all
other methods since it can directly detect weakest hydrogen bonds and
non-bonded interactions within biopolymers.
Liquid water absorption
¾ Less water absorption (at list 2 orders)
compare to IR and far-IR. Less
overlap with water or other analytes
absorption bands. Liquid samples can
be characterized.
¾ Absorption bands are more narrow
in the THz range than in the IR and
overlapping of neighboring bands is
less.
¾ The availability of multiple resonances for the sensitive measurement of
bio-molecule structure.
¾ Spectra are more species specific.
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“THE WORLD OF THE DEAD OR OF FUTURE PUNISHMENT“
M .N. Afsar &K.J.Button, “Infrared and Millimeter Waves,” V.12, Acad. Press, 1984
“Terahertz gap”
The spectral range between the upper end of the microwave and the
lower end of the extreme far IR
‰ Low energy of sources.
‰ Low absorption of biological material requires samples with
large area and thickness which is difficult to make because
samples are too fragile.
‰ Poor reproducibility of experimental results due to multiple
reflection in measurement systems, responsible for artificial
features; difficulties in sample preparation, instability of material.
‰ The absence of good commercially available laboratory
instruments
‰ Potentially promising laboratory techniques as time resolved
spectroscopy and photomixing technology are only recently
emerged
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MOTIVATION and GOALS
¾ There is a general need for faster and less expensive techniques
that can provide useful structural information on bio-materials.
¾ Our goal is to demonstrate that THz spectroscopy can be a
fruitful technique even with all mentioned difficulties and by using
commercially available instruments.
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Questions to answer:
‰ Is there something in the very far IR spectra? (initial prediction of
vibrational modes in polymer DNA in the 1-100 cm-1 frequency range [ E.W.Prohofsky, K.C. Lu,
L.L.Van Zandt and B. F. Putnam, Phys Lett., 70 a, 492 1979; K.V. Devi Prasad and
E.W.Prohofsky, Biopolymers, 23,1795, 1984].
‰
What are the reasons why researchers for 20 years failed to
achieve reproducible results? Experimental results are not reproducible and are
contradictive. It was not clear what to expect. Can we improve the results?
‰Can we use the observed features for DNA characterization,
identification and discrimination between species?
‰
The key to answer all these questions: we need to know of
what we are looking for.
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THEORETICAL PREDICTION OF THz ABSORPTION SPECTRA
Maria Bykhovskaia, B. Gelmont
IR active modes are calculated directly from the base pair
sequence and topology of a molecule.
Initial approximation was generated and optimized by the program packages
JUMNA and LIGAND (group of Prof. Lavery, Inst.Biologie Phys.Chim.Paris).
Energy minimum
Normal modes
Oscillator strengths
Spectra
QUESTIONS:
‰
‰
‰
What do we expect to find in the submillimeter wave range?
What is the predictive power of the method?
How sensitive are far IR absorption spectra to DNA structure?
Normal mode analysis is applicable to molecules with less than 30 base-pairs
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ENERGY MINIMIZATION AND NORMAL MODE ANALYSIS
(in internal coordinates of a molecule)
Maria Bykhovskaia, B. Gelmont
Molecular potential energy approximated as a function of dynamic
variables(q): torsion and bond angles.
Conformational energy
including long distance interactions :
E total = E Van der Waals + EElectrostatic + E HBonds + E Torsion + E Bond angles
Van der Waals and electrostatic interactions; the energy of hydrogen bonds deformations;
torsion rotation potentials; stretching deformations of bond angles and of bond length
Two B-helical conformation DNA fragments (TA)12 with different base pair
sequences:
AAAAAAAAAAAA
TTTTTTTTTTTT
ATATATATATAT
TATATATATATA
and the A-helix of double stranded RNA Poly[C].Poly[G]
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LOW FREQUENCY NORMAL MODES
Poly (dA) Poly (dT)
Number of modes
15
Maria Bykhovskaia, B. Gelmont
15
10
10
5
5
0
0
200
400
600
0
800
0
50
100
150
200
250
50
100
150
200
250
Poly (dAdT) Poly (dTdA)
20
20
15
15
10
10
5
5
0
0
200
400
600
800
0
0
-1
Vibrational frequency (cm )
360 normal modes were found for each sequence with the density higher than 1
mode per cm-1. There is almost no overlap of weak bond modes with vibrations
of covalent bonds which have frequencies above 750 cm-1.
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Absorption Spectra vs. Base Pair Sequence
α(ω) ∼ γω2 Σ (pk)2 / ( (ωk2 - ω2 )2 + γ2 ω2 ),
the dipole moment
p=
∑e a
i i
/ mi
the oscillator decay γk=2 cm-1 ,
,
i
Poly(dAdT)Poly(dTdA)
Poly(dA)Poly(dT)
Absorption
Observed (Powell et al., 1987)
0
100 200 300 400 500
0
100 200 300 400 500
-1
Frequency (cm )
The spectrum of optical activities is very sensitive to the DNA
base pair sequence
APS, March 2005
T.Globus, UVA
Maria Bykhovskaia, B. Gelmont
13
A double stranded 12 base pair RNA homopolymer fragment
Poly[C]-Poly[G] (Maria Bykhovskaia, B. Gelmont)
80
γ =0.5 cm
80
α(x+y)
αz
-1
Absorption coefficient, rel. units
Absorption coefficient, rel. units
Absorption spectra for two values of oscillator decay γ =0.5 cm -1 and γ =1
cm –1,
60
40
20
0
10
12
14
16
18 20
-1
22
α (x+y)
αz
-1
γ =1 cm
60
40
20
0
24
10
12
14
16
18
20
22
24
-1
Frequency, cm
Frequency, cm
for electric field E perpendicular to the long axes of a molecule ( αx+y) and
parallel to the long axes (α z).
For this fragment, the maximum absorption corresponds to E perpendicular to
the long molecular axis z.
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Short artificial DNA
Dry
In gel High salt
Absorption, relative units
0.010
BB
BA, g=0.5 cm-1, x+y
0.008
(Maria Bykhovskaia, B. Gelmont)
B-form: 5'-d(CCGGCGCCGG)-3‘, 10 base pairs per turn,
right-handed, has major and minor grooves.
A-form: 5'-d(CCCGGCCGGG)-3‘is adopted by dehydrated
DNA; it has 11 base pairs per turn, and the base pairs are
tilted with respect to the helix axis. A-form is sensitive to
humidity and can be changed to B-form.
Z-form: 5'-d(GCGCGCGCGC)-3‘- is a left handed DNA
helix in a zig zag pattern with12 base pairs per turn. It
adopted in solution at high salt concentration and when
reduce salt content it can be changed from left-handed to
right-handed.
It has no documented biological relevance. DNA exerts
a regulatory activity when in Z conformation.
0.006
0.004
0.002
0.000
10
12
14
16
18
20
22
24
The two DNA strands are held together by base pairing
(hydrogen bonding) between complementary bases.
Cytosine ( C ) is always hydrogen bonded to guanine ( G ).
Frequency, cm -1
Optical characteristics
depend on conformation
APS, March 2005
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Experimental set up
ƒ Bruker IFS-66 spectrometer (Hg- lamp source, He cooled Sibolometer @ 1.7 º K). Vacuum systems are not shown.
ƒ Attachment for reflection measurements.
ƒ Resolution 0.2 cm-1.
ƒ Range of interest throughout 10 cm-1 – 25 cm-1.
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Martin-Pupplett Polarizing Spectrometer
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THz Fourier-Transform (FT) spectroscopy
What is important?
Good Instrument performance:
‰ Spectral resolution at least 0.25 cm-1 to measure features with 0.5 cm-1
band width
‰ High sensitivity (signal to noise) and reproducibility to provide
standard deviation better than 0.3% to measure small signals
Sensitivity
‰ High transparency of substrates
Resolution
0.006
1.01
7 samples of BG (5 mg each)
0.99
0.97
0.004
Transmission
Standard deviation
0.005
0.003
CT DNA, 5 mg
100%-line
PC-membrane, d=5 mkm
0.95
0.93
0.91
0.002
0.89
0.001
10
12
14
16
18
20
22
24
0.87
10
15
Frequence,cm-1
7 different samples
20
25
-1
Frequency, cm
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Challenging Problem
Serious problem at THz - all kinds of multiple reflections
or standing waves in most of measurement systems because of large
wavelengths of radiation.
These effects cause artificial false resonances.
1.01
# 206.83, Gor-Tex, d=3 mm
Transmission
1.00
Check for false resonances using
thick plates from material with low
absorption is important. Ideally
spectrum is close to cos form.
0.99
0.98
0.97
10
12
14
16
18
20
22
24
Frequency, cm-1
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Material for study
‰
Herring, salmon and calf thymus DNA sodium salts with 6 %
Na content, from Sigma Chemical Co.
‰ Artificial short-chained oligonucleotides of known basepair sequences from Sigma Chemical Co:
• Single stranded RNA - potassium salts with the different nucleotide
composition: poly (G), poly (C), poly (A), poly (U),
(Guanine (G), Cytosine (C), Adenine (A), Uracyl (U)).
• Double helical RNA - sodium salts: Poly [C] * Poly [G] and
Poly [A] * Poly [U].
• DNA, as 5'-d(CpCpGpGpCpGpCpCpGpGp)-3‘ and others with different
sequencing, in A, B and Z conformation.
‰ Spores, plasmids, proteins, cells
‰ Bio-materials in solutions
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Sample preparation
Simple techniques has been developed to fabricate large area, thin,
stable samples
‰ Free-standing films and films on substrates are prepared from
the water gel. Film thickness 2 µm - 250 µm.
‰ The ratio of water to dry material in the gel from 5:1 to 30:1.
‰ Thin polycarbonate membranes, polyethylene or Teflon films
with ~98% transmission are used as supporting substrates in some
cases.
‰ To receive good resolution, samples of at least 1/2" diameter are
fabricated.
‰ Samples are aligned to receive preferable orientation of long
molecule axes in one direction. Good alignment enhances the
sensitivity.
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T.Globus, UVA
Reproducibility vs. orientation and interference effects
Same orientation
0.65
0.60
n=2.2
Poly[A], d~160 µm
n=1.9
0.45
0.35
02/04
02/05
0.58
Transmission
Transmission
0.55
d=0.125 mm
d=0.205 mm
m=2
0.56
0.54
0.52
0.25
8
10
12
14
16
18
Frequency, cm-1
20
22
24
m=1
0.50
8
10
12
14
16
18
20
22
24
Frequency, cm-1
Resonance features are resolved on the envelope of the wide interference
pattern.
λextr - the wavelength of transmission extrema
m λextr
nd =
d - the film thickness
4
m - the order of extremum.
n- refractive index ~ 1.7 - 2.3
These kind of interference features were initially considered as resonance
modes in bio materials.
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Absorption coefficient
0.95
PolyC
PolyG
PolyC-PolyG
Transmission
0.85
0.75
0.65
PolyA
PolyU
PolyA-PolyU
0.55
0.45
10
12
14
16
18
20
-1
22
24
Frequency, cm
The interference pattern is not obvious in transmission of thin films
(thickness between 15 and 70 µm).
Absorption coefficient spectra are derived by interference spectroscopy technique
(IST) for proper modeling of the multiple reflection behavior.
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Material texture
Image of the Salmon DNA sample in
polarizing microscope (free standing).
Film thickness about 10 µm.
Gel concentration 1:10.
DNA, as a rod-like polymer, spontaneously forms ordered liquid crystalline
phases in aqueous solution with the long molecular axis preferentially
aligned in one direction.
In drying process, DNA solution undergoes a series of transitions and film
samples are characterized by their microscopic textures with periodic
variations in refractive index and fringe patterns observed in polarizing
microscope.
The film texture depends on the concentration of molecules in solution and
on drying conditions.
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Change with sample rotation.
‰ Documented strong anisotropy of optical characteristics of
biological molecules at THz.
Absorption coefficient, cm-1
150
E
x+y
Poly [A]-Poly[U], d=16 µm
z
E
E perp Z
E//Z
z
x+y
100
a
b
50
0
10
15
20
Frequency, cm-1
25
Sample position with electric field (a)
perpendicular and (b) parallel to the
long-axis of the molecule z.
Absorption coefficient at
two orientations
For Poly[A]-Poly[U] fragments, absorption is higher and resonance structure
is much more pronounced with electric field of radiation E perpendicular to
the long axes of molecules Z.
APS, March 2005
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Experiment and Modeling
Many of the initial successful measurements of the THz absorption properties of
biological materials have been performed at the University of Virginia.
Evidence of multiple resonances in THz transmission spectra with a high
degree confidence in recognition of bio- molecules has been demonstrated.
From the width of the vibrational modes:
oscillator decay γ =0.5 cm -1
relaxation time τ =7 10-11 s
T.Globus, UVA
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Absorption coefficient, cm-1
Direct comparison of experimental
spectrum (red) with theoretical
prediction (blue) for a short chain
DNA fragment with known structure.
Reasonably Good correlation
validates both, experimental and
theoretical results.
measured
Poly [C]- Poly [G]
d=6.5 µm
calculated, γ=0.5 cm-1, E perp Z
calculated, E // Z
30
20
10
0
10
12
14
16
18
20
22
Frequency, cm-1
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26
24
Long term reproducibility
‰ Improved technique for solid film sample preparation:
¾ good alignment
¾ reduced amount of material from 15-20 mg to 1- 3 mg for one sample
¾ reproducibility better than 0.5%
0.84
Salmon DNA
0.82
Transmission
0.80
#220.29, 10/31/02
#220.39, 10/31/02
#222.12, 11/07/02
#225.23, 11/14/02
#227.07, 11/21/02
#230.10, 12/11/02
0.78
0.76
0.74
Period of 45 days
0.72
0.70
0.68
10
12
14
16
18
20
22
24
Frequency, cm-1
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Single- and double stranded Salmon-DNA
3.1
double stranded
2.4
single stranded
80
60
3.0
2.3
2.9
2.2
2.8
40
2.7
2.1
10
10
12
14
16
18
20
22
24
12
14
16
18
20
22
24
Wavenumber, cm-1
Wavenumber, cm -1
¾ Absorption for ss-DNA ~ 20 % higher than for ds-DNA.
¾ Additional peaks in α at 11.2 cm-1,13.4 cm-1 and 14.8 cm-1 for ssDNA.
¾ Higher n for ss-DNA.
THz spectra are sensitive to conformation change that can be used
for monitoring folding-unfolding of DNA
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Refractive Index (ss)
double stranded, d = 24.3 µm
single stranded, d = 5.8 µm
Refractive Index (ds)
Absorption Coefficient, cm-1
100
THz spectra of Biopolymers in water (gel)
Importance:
all living matter is in a liquid form
Lysozyme
0.34
™
Biological materials in an aqueous
form (or gel) can be characterized
as well as in solids.
™Signature is strong. Relative change
0.32
Transmission
0.30
0.28
0.26
0.24
in the peak up to 10-30%.
0.22
™ Spectra
are not disturbed by
water absorption at THz (except at
30 deg
90 deg
120 deg
0.20
0.18
10
12
14
16
18
20
22
24
18.6 cm-1).
Frequency, cm-1
Disturbance at 18.6 cm-1 is due to
absorption of water vapor in air.
™
™ High sensitivity of spectra to
orientation.
Possible applications: structural
characterization of proteins
™
and DNA at THz; monitoring biological processes.
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Structural phase transition
Time, min
3
7
13
18
23
28
33
38
Herring DNA, gel 1:12
0.12
Transmission
0.10
0.08
0.06
0.04
0.02
10
12
14
16
18
Frequency,cm
20
22
24
700
Herring DNA , gel 1:10
Absorption coefficient, cm-1
0.14
600
500
400
300
200
100
24.1 cm-1
23
15.95
10.95
0
100
-1
1000
Time, sec
Changing transmission spectrum of liquid
herring DNA sample with time after defrosting.
Several vibrational modes
™Temperature of structural transitions is close to RT and is sensitive to
environmental conditions, including humidity.
™Very high reproducibility of resonant features up to transmission level 90 %.
Sensitivity limit in aqueous form as well as the possibility to measure
polarization effects require clarification.
APS, March 2005
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Possible applications of THz spectroscopy
‰ Wide-range of biomedical applications based on close
relationship between structure and spectra, including
monitoring changes in molecular conformation in real time
ƒ
ƒ
ƒ
ƒ
ƒ
real-time analysis of protein binding for transport
protein binding with antibodies,
specificity interactions between proteins and nucleic acids
binding stability studies of drug-protein and vitamin-protein systems
disease diagnostic
‰ Systems for bio-detection and identification
‰ Remote sensing of biochemical agents
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Bio-Medical Application: Disease diagnostic
Cancer cells suspended in buffer solution (Phosphate-Buffered
Saline) with the ratio of dry material to liquid ~ 1:10 were measured
Cancer Cells frozen
0.54
bladder
prostate
Transmission
0.52
0.50
0.48
0.46
0.44
10
12
14
16
18
Frequency, cm
20
22
24
-1
Spectra of prostate and bladder cancer cells
THz spectroscopy appears to be a promising approach toward discriminating
between different tumor phenotypes.
Can we use tissue for cancer characterization?
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REFERENCES
M. Bykhovskaia, B. Gelmont, T. Globus, D.L.Woolard, A. C. Samuels, T. Ha-Duong, and K. Zakrzewska, “Prediction of DNA Far IR
Absorption Spectra Basing on Normal Mode Analysis”, Theoretical Chemistry Accounts 106, 22-27 (2001).
T. Globus, L. Dolmatova-Werbos, D. Woolard, A.Samuels, B. Gelmont, and M. Bykhovskaia, "Application of Neural Network
Analysis to Submillimeter-Wave Vibrational Spectroscopy of DNA Macromolecules ,” Proceedings of the International
Symposium on Spectral Sensing Research, p.439 (2001), Quebec City, Canada, 11-15 (June 2001).
D. L. Woolard, T. R. Globus, B. L. Gelmont, M. Bykhovskaia, A. C. Samuels, D. Cookmeyer, J. L. Hesler, T. W. Crowe, J. O. Jensen,
J. L. Jensen and W.R. Loerop. “Submillimeter-Wave Phonon Modes in DNA Macromolecules", Phys. Rev E. 65, 051903 (2002).
D.L. Woolard, E. R. Brown, A. C. Samuels, T. Globus, B. Gelmont, and M. Wolski “Terahertz-frequency Spectroscopy as a
Technique for the Remote Detection of Biological Warfare Agents ,” Proceedings to the 23th Army Science
Conference, Orlando, Florida, 2-5 (2002).
T. Globus, D.Woolard, M. Bykhovskaia, B. Gelmont, L. Werbos, A. Samuels. “THz-Frequency Spectroscopic Sensing of DNA
and Related Biological Materials”in Selecting Topics in Electronics and System, Vol 32: Terahertz Sensing Technology, Vol 2,
pp.1-34, World Scientific (2003) .
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Journal of Physics D: Applied Physics 36, 1314-1322 (2003).
T. R. Globus, D. L. Woolard, T. Khromova, T. W. Crowe, M. Bykhovskaia, B. L. Gelmont,. Hesler, and A. C. Samuels, “ThzSpectroscopy of Biological Molecules,” Journal of Biological Physics, V29, 89-100 (2003).
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and liquid phases”, Chemical and Biological Standoff Detection in the Terahertz Region, Providence, RI, Oct 2003, Proceedings of
SPIE, Vol. 5268-2, pp.10-18 (2004).
T. Globus, D. L. Woolard, A.C. Samuels, T. Khromova, J. O. Jensen “Sub-Millimeter Wave Fourier Transform Characterisation of
Bacterial Spores”. Proceedings of the International Symposium on Spectral Sensing Research, Santa Barbara, California, 2-6 June
2003, file://F:\isssr2003\index.htm (2004).
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of biological-warfare-agent simulants”, International Symposium on Defense and Security: Orlando, FL, Apr.2004, Proceedings of
SPIE Vol. 5411-5 (2004).
T. Globus, R. Parthasarathy, T. Khromova, D. Woolard, N.Swami, A. J. Gatesman, J. Waldman, “Optical characteristics of biological
molecules in the terahertz gap”, Proceedings of SPIE Vol. 5584-1 “Chemical and Biological Standoff Detection II” OE04 Optics
East, Phyladelphia (Oct. 2004).
T. Globus, D. Theodorescu, H. Frierson , T. Kchromova , D. Woolard, “Terahertz spectroscopic characterization of cancer cells”,
Proceedings of SPIE, The Advanced Biomedical and Clinical Diagnostic Systems III, v. 5692-42, San Jose (2005).
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