Introduction to EPR/ESR Spectroscopy and Imaging
Suggested reading:
C.P.Poole, Electron Spin Resonance, A comprehensive Treatise on Experimental Techniques
J.A.Weil, J.R.Bolton, J.E.Wertz, Electron Paramagnetic Resonance: Elementary
Theory and Practical Applications
G.R.Eaton, S.S.Eaton, K.Ohno, EPR imaging and In vivo EPR
Magnetic momentum of an add electron
s = gS
L = gL

N
N
= 1838
This is the ratio of rest mass of
proton to the rest mass m of
electron
Thus EPR energies are generally about 2000 times as big as NMR energies
NMR – EPR comparison of energies
NMR
Radio wave in the range : 90 – 700 MHz
Field value : 2 - 14 T
Relaxation time
: 10-3 to 10 sec
EPR
Microwave in the range
Field
: 1.2 GHz – 100 GHz
: 0.03 – 0.3 T
Relaxation time : 10-9 – 10-6 sec
“Additional problems with biological EPR spectroscopy is the
microwave absorption H2O in biological objects.”
Dead Time
A serious limitation for FT-EPR spectroscopy
Principle of EPR spectroscopy

Relaxation
T1 – Spin lattice relaxation
E = g(B0+B1)
T2 – Spin-spin relaxation
T2* – Spin-spin relaxation

B0
Absorption spectrum
Expt. Obtained spectrum
Field (B1) modulation in EPR
Why:
Absorption signal is weak, compared NMR, and buried
under equally amplified noise.
Modulation frequency
Modulation amplitude
B1
Oscillating Magnetic field
Unmodulated
Modulated
Phase Sensitive Detection in EPR
3
2
Max
1
2
4
3
0
5
-Max
1
5
4
Field
Field
Nuclear magnetic coupling – “Hyperfine splitting”
-1
+
1
0
2
+1
-
1
2
-1
0
+1
N
O.
S = 1 for 14N
2S+1 = 3
Secondary Hyperfine Splittings
+1
+
1
0
2
-1
+1
-
1
0
2
-1
Expected
1
2
1
1
+
2
2
1
1
+
2
2
1
2
+
1
2
1
1
+
2
2
1
1
+
2
2
1
2
+
Experimentally measured
H
N
O.
EPR spin trapping
Many free radicals, generated by enzymatic reactions are not stable
enough to detect by EPR spectroscopy.
 Superoxide radical (O2.-)
 Hydroxyl radical
(OH.)
 Nitric oxide (NO:)
They need to be stabilized to detect by EPR: “Spin trapping”
Spin trap
+
(No EPR signal)
Unstable radical
(No EPR signal)
Stable radical (?)
(EPR signal)
Superoxide trapping: Example 1 Xanthine / Xanthine oxidase
Xanthine
EPR spect. of DMPO-OH
xo
Hypoxanthine
O2
O2-.
+
DEPMPO
DEPMPO-OOH
Trapping Nitric Oxide
Although NO is paramagnetic, it is impossible to detect by EPR directly, because
being small, it relaxes very fast as in the case of O2. Thus special approaches
are required to restrict its motion to get reasonable spectrum.
Fe complexes of dithiocarbamate and its derivatives
Fe(MGD)
Fe(MGD)-NO
Superoxide trapping: Example 1 Nitric oxide synthase (NOS)
Fe-MGD
DMPO-OO-
EPR Imaging
1
2
3
4
Bo
Field is being uniform (g(B0+B1))
all the four spin pockets come to
resonance frequency at a time
MAGNET
MAGNET
EPR Imaging – Concept of gradient Field
Principle of cw EPR Imaging
Projection
Gradient Direction
1- 4
Gradient
generation
1 2
N
S
3 4
Bo
Bo
1
2
N
Projections
2, 4
1, 3
S
3
4
Bo
(x+Bo)
(x-Bo)
Bo
Re-construction
x-Bo
1,4
1
2
N
S
3
x+Bo
3
2
4
Bo
2D image
Pros and Cons of EPR imaging

Not adequate concentration of radicals available in biological systems

Needs exogenous infusion of stable radicals species in organs
or whole body imaging

Needs significant reduction of microwave frequency to avoid
microwave absorption. This significantly compromises the
sensitivity
But….

It is an unique technique to study redox status of tissues,
organs or in whole body, which cannot be achieved by
other techniques
RESONATOR
NORMAL TISSUE
RIF-1 TUMOR
3.0
0
Kuppusamy et al, Canc. Res, 1998, 58, 1562
4.5
6.0
7.5
9.0 10.5 12.0
Time (min)
13.5 15.0 16.5
256
Pharmacokinetics of Nitroxides at different Oxygenation of
RIF-1 Tumor
15N-TPL
Room air
Breathing Mouse
(pO2=2.5 mmHg)
and LiPc
Carbogen Breathing
Mouse (pO2= 95
mmHg)
0.5 min
10 min
Nitroxide intensity ->
100
3-CP
room air
10
20
10
0
0.0
15N-TPL
Carbogen
30
Frequency
room air
Frequency
15N-TPL
60
40
I/I0 x 100
3-CP
Carbogen
Nitroxide intensity ->
0.05
0.10
0.15
Rate constant (min-1)
1
0
10
20
30
40
40
20
0
0.0
0.05
0.10
0.15
Rate constant (min-1)
Time (minutes)
Ilangovan, G. et al Mol. Cell. Biochem., 2002, 234, 393
Example 1 In vivo Imaging of NO generation
Fe-MGD
No EPR signal
+
NO
No EPR signal
Fe-MGD-NO
Strong EPR signal
NO generated in the thoracic region of a mouse, subjected to
cardiopulmonary arrest