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