Post on 26-Dec-2015
Periannan Kuppusamy, PhDCenter for Biomedical EPR Spectroscopy &
ImagingDavis Heart & Lung Research InstituteOhio State University, Columbus, OH
E-mail: Kuppusamy.1@osu.edu
EPR Spectroscopy Spying on unpaired electrons - What information can we
get?
Sunrise Free Radical SchoolOxygen-2002 ::Nov 21, 2002
±½
En
erg
yElectron Paramagnetic Resonance (EPR)Electron Spin Resonance (ESR)Electron Magnetic Resonance (EMR)
EPR ~ ESR ~ EMR
Ms = +½
Ms = -½
E=h=gB
B = 0
Magnetic Field (B)
Bpp
B > 0
h = gB= (g/h)B = 2.8024 x B MHz
for B = 3480 G = 9.75 GHz(X-band)for B = 420 G = 1.2 GHz (L-band)for B = 110 G = 300 MHz(Radiofrequency)
What is EPR?
h Planck’s constant 6.626196 x 10-27 erg.sec frequency (GHz or MHz)g g-factor (approximately 2.0) Bohr magneton (9.2741 x 10-21 erg.Gauss-
1)B magnetic field (Gauss or mT)
EPR is the resonant absorption of microwave radiation by paramagnetic systems in the presence of an applied magnetic field
MS=±½
MS=-½
MS=+½
ElectronS(½)
MI=+½
MI=-½
MI=-½
MI=+½
NucleusI (½)
Selection RuleMS = ±1; MI = 0
Magnetic Field
S=½; I=½
Doublet
hfc
Hyperfine Coupling
MS=±½
MS=-½
MS=+½
ElectronS (½)
MS = ±½
MI=+1
MI=-1
MI=-1
MI=+1
NucleusI (1)
MI=0,±1
Selection Rule MS = ±1; MI = 0
Magnetic Field
S=½; I=1
Triplet
hfc
Hyperfine Coupling
MI= 0
MI= 0
hfc
We can detect & measure free radicals and paramagnetic species
Direct detection e.g.: semiquinones, nitroxides, trityls
Indirect detectionSpin-trapping
Species: superoxide, hydroxyl, alkyl, NOSpin-traps: DMPO, PBN, DEPMPO, Fe-DTCs
Chemical modificationsSpin-formation : hydroxylamines (Dikhalov et al)Spin-change : nitronylnitroxides ( Kalyanaraman et al)Spin-loss : trityl radicals
• High sensitivity (nanomolar concentrations)
• No background
• Definitive & quantitative
What do we do with EPR?
What do we do with it?Yes! Intact tissues, organs or whole-body can be measured.But there is a catch!
What is the optimum frequency? - depends on sample size
Biological samples contain large proportion of water. They are aqueous and highly dielectric. Conventional EPR spectrometers operate at X-band ((9-10 GHz) frequencies, which result in (i) ‘non-resonant’ absorption (heating) of energy and (ii) poor penetration of samples. Hence the frequency of the instrumentation needs to be reduced.
Can we use EPR to measure free radicalsfrom biological systems (in vivo or ex vivo)?
Frequency ~300 MHz ~750 MHz 1-2 GHz ~3 GHz 9-10 GHz
Penetration Depth
> 10 cm 6-8 cm 1-1.5 cm 1-3 mm 1 mm
Objects Mouse, rat Mouse Mouse, rat heart
Mouse tail
Topical (skin)
In vitro samples (~100 uL vol.)
Pioneers Halpern et al
Krishna et al
Zweier et al Hyde et al
Swartz et al
Zweier et al
Hyde et al
Zweier et al
What else can we do with EPR?
• A known free radical probes is infused or
injected into the animal
• The change in the EPR line-shape profile,
which is correlated to some physiological
function, is then monitored as a function of time
or any other parameter.
• The measurements can be performed in real-
time and in vivo to obtain ‘functional
parameters’.
We can use free radicals as “spying probes” to obtain information from biological systems
ampl
itudeRedox status
Cell viabilityTissue
perfusion
width
Oxygen, pO2
Acidosis, pHViscosityMolecular
motion
Splitting
Oxygen, pO2
Redox status
Acidosis, pH
Thiols (GSH)
Cell viability
Viscosity
Tissue perfusion
Molecular
motion
Functional parameters from an EPR spectrumIn vivo EPR spectroscopy is capable of providing useful physiologic and metabolic (functional) information from tissues
Can we image free radicals in biological systems?
Spatially-resolved information (mapping) can
be obtained using EPR imaging (EPRI)
techniques
Can we image free radicals in biological systems?
NMR EPR
Spin Probes Tissue protons Free
radicals
(endogenous) (>50 M) (<< nM)
Probe Stability Ideal <
nanoseconds
Relaxation time (LW) m sec < sec
(LW: 1 G)
No suitable endogenous spin probes for EPRI Nothing to
image
Fast electronics needed for EPRI. No way to
image
Eaton & Eaton
(1989)
EPRI is capable of measuring the distribution of paramagnetic and free radical species in tissues
EPR SpectroscopySpatially-unresolved
0 + 1 dimensional
Spatial ImagingSpatially-resolved
Spin density3 + 0 dimensional
Spectral-spatial ImagingSpatially-resolved
Spectral shape
3 + 1 dimensional
1D1D SPATIAL SPATIAL 2D2D SPATIAL SPATIAL 3D3D SPATIAL SPATIAL
Gradient Magnetic Field
Homogeneous(Spectroscopy)
Inhomogeneous(Not useful)
Gradient(Imaging)
<----- Magnetic Field
Distance ---> Distance ---> Distance --->
Gradient Magnetic Field
ISO
FIE
LD L
INE
B1 B2
B1 > B2
Gradient Vector
In the conventional CW EPR sweep mode, spins at B1 will come into resonance first.
s2
s3
s1
s = cos + y sin
p()
s
Projectio
n
x
y
Projection
Field Sweep0 2 0 0 2 0 0 3 0
Fie
ld S
wee
p
02
00
30
02
0
Field Sweep
002030101
Field
Sweep
10
10
20
20
1
Field Gradient
Ray
Spin density
Projection
= 0o) = 45 o)
=
90o
)
=
135o )
ProjectionAcquisition
0 2 0 0 2 0 0 3 0
02
00
30
02
0
00
20
30
10
1 10
10
20
20
1
2 2 2 2 2 2 1 3 32 6 2 2 8 2 2 9 22 2 2 2 2 2 3 4 11 2 2 2 2 3 2 3 13 8 3 3 10 3 3 9 32 2 1 3 2 2 1 3 1
3 2 1 1 2 1 1 3 22 8 2 2 6 2 2 8 21 2 3 1 3 1 2 3 1
P ( =45 o)P ( =0o)
P (
=90
o)
P ( =
135
o )
1 2 3 4 5 6 7 8 9
1
2
3
4
5
6
7
8
9
xy
Image Reconstruction by Backprojection
12 mm
0 256 0 256
12 mm12
mm
3D composite view A 2D projection of the image
Proj., 1024; gradient, 10 G/cm; acq. time, 51.6 min; resolution, 100 mm.
A pack of three identical tubes (i.d.: 3 mm) and a polyethylene tubing (id: 1.1 mm) wound around the pack. The tubes were filled with 0.5 mM solution of
TAM.
3D IMAGING OF A SPIRAL PHANTOM
A BPAC
LAD
LV apex
Ao
LV
LM
Ao
LV apex
3D EPR image of an ischemic rat heart infused with glucose char suspension oximetry label. A: Full view B: A longitudinal cutout showing the internal structure of the heart. Ao, aortic root; C, cannula; PA, pulmonary artery; LM, left main coronary artery; LAD, left anterior descending artery; LV, left ventricular cavity.
3D Image of a rat heart perfused with glucose char
Gated Imaging of Rat Heart
Transverse slices
systolic diastolic
Longitudinal slices
A1 A2 A3
A6A5A4
a
c
d
b
B1 B2 B3
d
a c
Representative slices (24x24 mm2,
thickness, 0.19 mm) obtained from a 3D
spatial image.A1-A6: Vertical slices
B1-B3: Transverse slices
a - cannulab - renal artery
c - cortexd - calyses
IMAGING OF RAT KIDNEY
PERFUSED WITH TAM
Proj, 1024;grad, 25.0 G/cm;
acq. time, 76.8 min; resolution, 200 um Intensity
NO
RM
AL
RIF
-1S
CC
VII
SLICES (0.3 MM) FROM 3D IMAGES OF MURINE TUMORS (3-CP; 100 mg/kg)
C3H mice; gradient 20 G/cm;144 projections;10x10 mm2
Intensity -->
3D IMAGING OF NITROXIDE DISTRIBUTION IN TUMORS
Kuppusamy, P., et al. Cancer Research, 58, 1562-1568 (1998).
2p (O)2p (O) 2p(O)2p(O)
y*=z*
y=z
x*
x
Molecular oxygen has two unpaired electrons
Oxygen gives strong EPR
signals in the gas phase
However, no EPR spectrum has
been reported for oxygen
dissolved in fluids. (too broad!)
Thus, there seems to be no
possibility for direct detection of
oxygen in biological systems
using EPR
However, molecular oxygen can
be measured and quantified
indirectly using spin-label EPR
oximetry
Molecular oxygen is paramagnetic
EPR oximetry probes
Particulate (Solid) probes
Lithium phthalocyanine (LiPc)Sugar charsFusiniteCoalIndia ink
What is reported?
• pO2 (mmHg/Torr)• Localized Measurement• Resolution < 0.2 mmHg• Repeated Measurements• Stable for days to weeks• Independent of medium
• Concentration (M) of dissolved oxygen in the bulk volume
• Resolution 2-10 mmHg
Soluble probes
NitroxidesTrityl radicals
.N
O
SL
Ö2Ö2
Ö2
Ö2
Ö2
Bimolecular collision between SL and oxygen
leads to Heisenberg spin
exchange
R
Principle of EPR oximetry
which translates to EPR line-broadening as
= 4Rp(DSL + DO2) [O2]
The collision frequency , according to the hard sphere theory of Smoluchowski is
w = k DO2 [O2]
Mapping of Oxygen
Object
Spectral-Spatial EPR
image
xy
Data
Spin density
Information
e.g. Oxygen
Mapping of oxygen in biological tissues is possible by spectral-spatial or ‘spectroscopic’
EPR imaging.
Spin Density Map
Phantom
of tubes with15N-PDT
Amplitude Map Oxygen Map
38% 38%
21% 21%
[Oxygen]
0.50 0.25
0.25 0.50
[PDT, mM]
EPR Oxegen Mapping (EPROM)
40%
0%
10%
20%
30%
Oxy
gen
S. Sendhil Velan, R. G. S. Spencer,J. L. Zweier & P. Kuppusamy, Magn. Reson. Med. 43, 804-809 (2000)
Cutaneous layer
Ventral veinVentral artery
Bone
Tendon Lateral veins
Lateral arteries
Spin Density Map
300
0
75
150
225
Oxy
gen
Co
nce
ntr
atio
n (M
)Oxygen Map
Mapping ofarterio-venous oxygenationin a rat tail, in vivo
Amplitude Map
3-D
spe
ctra
l-sp
atia
lL
-ban
d, 3
-CP
prob
e
Sendhil Velan, S., Spencer, R.G.S., Zweier, J. L. & Kuppusamy P. Magn. Reson. Med. 43, 804-809 (2000)
N N
NN
N
N N
N
Li
LiPc (Lithium
Phthalocyanine)Oxygen sensitive (T2) EPR probe
Time (sec)0 10 20 30 40 50 60
21%
0 %
Oxy
gen
Oxygen Time Response Air Air AirN2 N2
Oxygen (pO2)
0 25 50 75 100 125 150
EP
R L
ine-
wid
th (
G)
0.0
0.2
0.4
0.6
Response to Oxygen
slope = 8.9 mG / mmHg
Ilangovan, G., Li, H., Zweier, J.L., Kuppusamy, P. J. Phys. Chem. B 104, 4047 (2000); 104, 9404 (2000); 105, 5323 (2001)
Oxygenation of RIF-1 Tumor(Carbogen-breathing)
pO2 (
mm
Hg)
Time (min)0 20 40 60 80 100 120
0
20
40
60
80
100
120
Room Airbreathing
Carbogenbreathing
Room Airbreathing
Carbogenbreathing
Carbogenbreathing
Magnetic Field (Gauss)
444 446 448
Carbogen-breathing
Air-breathing
RESONATOR
20 m
m x
20
mm
RIF-1 Tumor(14x 16 mm)
Normal LegTissue
pO2 = 4.6 mmHgpO2 = 17.6 mmHg
Oxygen Measurements usingLiPc, a Particulate EPR Probe
Co-imaging of the oximetry probe in the
tumor tissue
Image of LiPc powder deposit
Perfusion image of a nitroxide
Days Post-Implantation of LiPc
0 2 4 6 8
Tis
sue
pO
2 (m
mH
g)
0
5
10
15
20
25
30
Normal Muscle Tissue of RIF-1 Mice
Tumor Tissue of RIF-1 Mice
Control : Muscle Tissue of Non-tumor bearing mice
In vivo measurements of pO2 from tumor and normal
gastrocnemius muscle tissues of RIF-1 tumor-bearing mice.
LiPc particles were implanted in the tumor on the right leg and normal muscle on the left leg and tissue pO2 values were
repeatedly measured on the same animals for up to 8 days using EPR oximetry. (N = 5)
Redox Status
Redox State describe the ratio of the interconvertible oxidized and reduced form of a specific redox couple
GSSG/2GSH GSSG + 2H+ + 2e- 2GSHEhc = E0– (RT/nF) log([GSH]2/[GSSG])Redox State = Reduction potential x concentration
Schaefer, F. Q. & Buettner, G. R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191-1212 (2001)
Redox Status applies to a set of redox couples.It is the summation of the products of the reduction potential and reducing capacity of all the redox couples present
GSSG/2GSHNADP+/NADPHTrxSS/Trx(SH)2
Redox Status = [Reduction potential x concentration]i
Nitroxides as probes of tissue redox status
Time (min)
EP
R I
nten
sity
t½ ~ min
Nitroxide Hydroxylamine
Redox conversionin tissues
EPR 'active' EPR 'inactive'
+ e-
- e-
Swartz et al, Free Radic. Res. Commun., 9, 399-405 (1990)Kuppusamy et al, Cancer Research, 58, 1562-1568 (1998)Krishna et al, Breast Disease, 10, 209-220 (1998)
N
O
NH2
O
N
OH
NH2
O
NORMAL TISSUE (LEFT LEG)
RIF-1 TUMOR (RIGHT LEG)
Reduction of 3-CP in theNormal & Tumor Tissue
C3H mice with RIF-1 tumor; ~30 g bw; dose: 100 mg/kg, iv; Measured in vivo using surface resonator at L-band (1.25 GHz); Images: 10x10 mm2
3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0 16.5
Time (min)
Intensity -->
Kuppusamy, P., et al. Cancer Research, 58, 1562-1568 (1998).
Inte
nsi
tyTime
‘kx,y’
Intensity Map Reduction constant, kx,y Redox Map
Reconstruction of Tissue ‘Redox Status’ Image
[64x64] [64x64]8-12 points, first order
x,yx,y
Kuppusamy, P., & Krishna, MC., Curr. Topics in Biophys. (2002)
3.6 min 5.8 min 8.2 min 10.5 min
13.5 min 15.8 min 18.2 min 20.5 min
Fre
que
ncy
0.0 0.1 0.2 0.30
0.4
5
Reduction Rate (min-1)
25
120
20
15
10
B
CD
100
A
+ 4 XO
TPL
+ XO (x2)
+ XO
0.00 min-1
0.43 min-1
REDOX MAPPING BY EPR IMAGING(VALIDATION EXPERIMENT)
A B
CD
+ XO
+ 2 XO+ 4 XO
TPL
TPL TPHX + XO
GSH
Fre
quen
cy
0
10
20
30
40
RIF-1
0
0.0 0.05 0.10 0.15Rate constant (min-1)
RIF-1+BSO
0.05 0.10Rate constant (min-1)
0.150.0
13253850637588
Fre
quen
cy
Redox Mapping of Tumor: Effect of BSO
(GSH Depletion)
Median: 0.054 min-1
Median: 0.030 min-1
Kuppusamy et al Cancer Research (2002)
GSH levels in leg muscle (Normal) and RIF-1 tumors of untreated and BSO-treated (6-hrs post-treatment of 2.25 mmol/kg of BSO, ip) tumor-bearing mice. (N=7)
NormalMuscle
RIF-1 RIF-1+BSO
GS
H (m
ol/g
Tis
sue
)
0
1
2
3
4
5
GSH Level
NormalMuscle
RIF-1 RIF-1+BSO
Rat
e C
onst
ant
(min
-1)0.00
0.02
0.04
0.06
0.08
Rate constants of nitroxide clearance in leg muscle (Normal) and RIF-1 tumors of untreated and BSO-treated (6-hrs post-treatment of 2.25 mmol/kg of BSO, ip) tumor-bearing mice. (N=5)
Redox Status
REDOX STATUS & GSH LEVELS IN RIF-1 TUMOR
EFFECT OF DIETHYLMALEATE (DEM) ON TUMOR REDOX STATUS
0
1
2
3
4
0.08 0.12 0.160 0.04
Fre
qu
ency
Reduction rate (min-1)
Tumor
Median 0.053 min-1
1
2
3
00.08 0.12 0.160 0.04
Fre
qu
ency
Reduction rate (min-1)
Tumor+DEM
Median 0.034 min-1
0.0
0.5
1.0
1.5
2.0
2.5
GS
H (
µg/
mg
tissu
e)
GSH in Tumor Tissue
Tumor+DEM(N=8)
Tumor(N=5)
Yamada et al Acta Radiol (2002)
Nitroxide intensity ->
Car
boge
n
0.0 0.05 0.10 0.15
Rate constant (min-1)
Rate constant (min-1)
Fre
quen
cy
0.05 0.100
20
40
60
0.150.0
Roo
m a
ir
Fre
quen
cy
0
10
20
30
40
Redox mapping of tumor: Effect of Carbogen-Breathing
(Oxygenation)
Ilangovan G., Li, H., Zweier, J. L., Krishna M. C., Mitchell J. B. Kuppusamy, P. Magn. Reson. Med. (2002))
EPR detection of SH-groups (ESR analogs of Ellman’s reagent)
Reaction with GSH
Berliner, L.J., et al, Unique In VivoApplications
of Spin Traps, Free Rad.Biol.Med.30(5): 489-
499.
Khramtsov,V.V. et al. 1997, J.Biochem.
Biophys. Methods 35: 115
Summary
1. EPR spectroscopy is a direct & definitive technique
for detection and quantitation of free radicals and
paramagnetic species.
2. Low-frequency EPR spectroscopy enables
measurement of free radicals
(endogenous/exogenous) in biological systems
including intact tissues, isolated organs and small
animals.
3. In vivo EPR spectroscopy and imaging methods
enable noninvasive measurement and mapping of
tissue pO2, redox status and pH.
BOOKSRosen, G. M., Britigan, B. E., Halpern, H. J., and Pou, S. Free Radicals: Biology and Detection by Spin
Trapping. New York: Oxford University Press, 1999Eaton, G. R., Eaton, S. S., and Ohno, K. EPR imaging and in vivo EPR: CRC Press, Inc, 1991.REFERENCESBuettner, G. R. Spin trapping: ESR parameters of spin adducts. Free Radic Biol Med, 3: 259-303, 1987.Berliner, L. J., Khramtsov, V., Fujii, H., and Clanton, T. L. Unique in vivo applications of spin traps. Free
Radic Biol Med, 30: 489-499, 2001.McCay, P. B. Application of ESR spectroscopy in toxicology. Arch Toxicol, 60: 133-137, 1987.Kuppusamy, P., Chzhan, M., Vij, K., Shteynbuk, M., Lefer, D. J., Giannella, E., and Zweier, J. L. Three-
dimensional spectral-spatial EPR imaging of free radicals in the heart: a technique for imaging tissue metabolism and oxygenation. Proc Natl Acad Sci U S A, 91: 3388-3392, 1994.
Kuppusamy, P., Ohnishi, S. T., Numagami, Y., Ohnishi, T., and Zweier, J. L. Three-dimensional imaging of nitric oxide production in the rat brain subjected to ischemia-hypoxia. J Cereb Blood Flow Metab, 15: 899-903, 1995.
Kuppusamy, P., Wang, P., and Zweier, J. L. Three-dimensional spatial EPR imaging of the rat heart. Magn Reson Med, 34: 99-105, 1995.
Kuppusamy, P., Chzhan, M., Wang, P., and Zweier, J. L. Three-dimensional gated EPR imaging of the beating heart: time-resolved measurements of free radical distribution during the cardiac contractile cycle. Magn Reson Med, 35: 323-328, 1996.
Kuppusamy, P., Wang, P., Samouilov, A., and Zweier, J. L. Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imaging. Magn Reson Med, 36: 212-218, 1996.
Kuppusamy, P., Wang, P., Zweier, J. L., Krishna, M. C., Mitchell, J. B., Ma, L., Trimble, C. E., and Hsia, C. J. Electron paramagnetic resonance imaging of rat heart with nitroxide and polynitroxyl-albumin. Biochemistry, 35: 7051-7057, 1996.
Khramtsov, V. V., Yelinova, V. I., Glazachev Yu, I., Reznikov, V. A., and Zimmer, G. Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label. J Biochem Biophys Methods, 35: 115-128, 1997.
Kuppusamy, P., Afeworki, M., Shankar, R. A., Coffin, D., Krishna, M. C., Hahn, S. M., Mitchell, J. B., and Zweier, J. L. In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model. Cancer Res, 58: 1562-1568, 1998.
Kuppusamy, P., Shankar, R. A., and Zweier, J. L. In vivo measurement of arterial and venous oxygenation in the rat using 3D spectral-spatial electron paramagnetic resonance imaging. Phys Med Biol, 43: 1837-1844, 1998.
Velan, S. S., Spencer, R. G., Zweier, J. L., and Kuppusamy, P. Electron paramagnetic resonance oxygen mapping (EPROM): direct visualization of oxygen concentration in tissue. Magn Reson Med, 43: 804-809, 2000.
Kuppusamy, P., Shankar, R. A., Roubaud, V. M., and Zweier, J. L. Whole body detection and imaging of nitric oxide generation in mice following cardiopulmonary arrest: detection of intrinsic nitrosoheme complexes. Magn Reson Med, 45: 700-707, 2001.
Ilangovan, G., Li, H., Zweier, J. L., Krishna, M. C., Mitchell, J. B., and Kuppusamy, P. In vivo measurement of regional oxygenation and imaging of redox status in RIF-1 murine tumor: Effect of carbogen-breathing. Magn Reson Med, 48: 723-730, 2002.
Kuppusamy, P., Li, H., Ilangovan, G., Cardounel, A. J., Zweier, J. L., Yamada, K., Krishna, M. C., and Mitchell, J. B. Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels. Cancer Res, 62: 307-312, 2002.
Yamada, K. I., Kuppusamy, P., English, S., Yoo, J., Irie, A., Subramanian, S., Mitchell, J. B., and Krishna, M. C. Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging. Acta Radiol, 43: 433-440, 2002.
WEB SITESThe Illinois EPR Research Center: http://ierc.scs.uiuc.edu/The National Biomedical EPR Center at the Medical College of Wisconsin :
http://www.biophysics.mcw.edu/bri-epr/bri-epr.htmlNIEHS - Spin Trap Database: http://epr.niehs.nih.gov/stdb.html
BOOKSRosen, G. M., Britigan, B. E., Halpern, H. J., and Pou, S. Free Radicals: Biology and Detection by Spin
Trapping. New York: Oxford University Press, 1999Eaton, G. R., Eaton, S. S., and Ohno, K. EPR imaging and in vivo EPR: CRC Press, Inc, 1991.REFERENCESBuettner, G. R. Spin trapping: ESR parameters of spin adducts. Free Radic Biol Med, 3: 259-303, 1987.Berliner, L. J., Khramtsov, V., Fujii, H., and Clanton, T. L. Unique in vivo applications of spin traps. Free
Radic Biol Med, 30: 489-499, 2001.McCay, P. B. Application of ESR spectroscopy in toxicology. Arch Toxicol, 60: 133-137, 1987.Kuppusamy, P., Chzhan, M., Vij, K., Shteynbuk, M., Lefer, D. J., Giannella, E., and Zweier, J. L. Three-
dimensional spectral-spatial EPR imaging of free radicals in the heart: a technique for imaging tissue metabolism and oxygenation. Proc Natl Acad Sci U S A, 91: 3388-3392, 1994.
Kuppusamy, P., Ohnishi, S. T., Numagami, Y., Ohnishi, T., and Zweier, J. L. Three-dimensional imaging of nitric oxide production in the rat brain subjected to ischemia-hypoxia. J Cereb Blood Flow Metab, 15: 899-903, 1995.
Kuppusamy, P., Wang, P., and Zweier, J. L. Three-dimensional spatial EPR imaging of the rat heart. Magn Reson Med, 34: 99-105, 1995.
Kuppusamy, P., Chzhan, M., Wang, P., and Zweier, J. L. Three-dimensional gated EPR imaging of the beating heart: time-resolved measurements of free radical distribution during the cardiac contractile cycle. Magn Reson Med, 35: 323-328, 1996.
Kuppusamy, P., Wang, P., Samouilov, A., and Zweier, J. L. Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imaging. Magn Reson Med, 36: 212-218, 1996.
Kuppusamy, P., Wang, P., Zweier, J. L., Krishna, M. C., Mitchell, J. B., Ma, L., Trimble, C. E., and Hsia, C. J. Electron paramagnetic resonance imaging of rat heart with nitroxide and polynitroxyl-albumin. Biochemistry, 35: 7051-7057, 1996.
Khramtsov, V. V., Yelinova, V. I., Glazachev Yu, I., Reznikov, V. A., and Zimmer, G. Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label. J Biochem Biophys Methods, 35: 115-128, 1997.
Kuppusamy, P., Afeworki, M., Shankar, R. A., Coffin, D., Krishna, M. C., Hahn, S. M., Mitchell, J. B., and Zweier, J. L. In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model. Cancer Res, 58: 1562-1568, 1998.
Kuppusamy, P., Shankar, R. A., and Zweier, J. L. In vivo measurement of arterial and venous oxygenation in the rat using 3D spectral-spatial electron paramagnetic resonance imaging. Phys Med Biol, 43: 1837-1844, 1998.
Velan, S. S., Spencer, R. G., Zweier, J. L., and Kuppusamy, P. Electron paramagnetic resonance oxygen mapping (EPROM): direct visualization of oxygen concentration in tissue. Magn Reson Med, 43: 804-809, 2000.
Kuppusamy, P., Shankar, R. A., Roubaud, V. M., and Zweier, J. L. Whole body detection and imaging of nitric oxide generation in mice following cardiopulmonary arrest: detection of intrinsic nitrosoheme complexes. Magn Reson Med, 45: 700-707, 2001.
Ilangovan, G., Li, H., Zweier, J. L., Krishna, M. C., Mitchell, J. B., and Kuppusamy, P. In vivo measurement of regional oxygenation and imaging of redox status in RIF-1 murine tumor: Effect of carbogen-breathing. Magn Reson Med, 48: 723-730, 2002.
Kuppusamy, P., Li, H., Ilangovan, G., Cardounel, A. J., Zweier, J. L., Yamada, K., Krishna, M. C., and Mitchell, J. B. Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels. Cancer Res, 62: 307-312, 2002.
Yamada, K. I., Kuppusamy, P., English, S., Yoo, J., Irie, A., Subramanian, S., Mitchell, J. B., and Krishna, M. C. Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging. Acta Radiol, 43: 433-440, 2002.
WEB SITESThe Illinois EPR Research Center: http://ierc.scs.uiuc.edu/The National Biomedical EPR Center at the Medical College of Wisconsin : http://www.biophysics.mcw.
edu/bri-epr/bri-epr.htmlNIEHS - Spin Trap Database: http://epr.niehs.nih.gov/stdb.html