Introduction to EPR/ESR Spectroscopy and Imaging

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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
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Magnetic momentum of an add electron
?s g?S
?N
?L g?L
? ?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
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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 1.2 GHz 100 GHz
Field 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.
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A serious limitation for FT-EPR spectroscopy
Dead Time
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Principle of EPR spectroscopy
?
Relaxation
T1 Spin lattice relaxation
?E g?(B0B1)
T2 Spin-spin relaxation
T2 Spin-spin relaxation
?
B0
Expt. Obtained spectrum
Absorption spectrum
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Field (B1) modulation in EPR
Why
Absorption signal is weak, compared NMR, and
buried under equally amplified noise.
Modulation frequency
Modulation amplitude
Oscillating Magnetic field
B1
Unmodulated
Modulated
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Phase Sensitive Detection in EPR
Max
0
-Max
Field
Field
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Nuclear magnetic coupling Hyperfine splitting
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Secondary Hyperfine Splittings
H
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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
Unstable radical
Stable radical (?)

(No EPR signal)
(No EPR signal)
(EPR signal)
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Superoxide trapping Example 1 Xanthine /
Xanthine oxidase
EPR spect. of DMPO-OH
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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
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Fe(MGD)
Fe(MGD)-NO
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Superoxide trapping Example 1 Nitric oxide
synthase (NOS)
Fe-MGD
DMPO-OO-
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EPR Imaging
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EPR Imaging Concept of gradient Field
1
2
MAGNET
MAGNET
4
3
Bo
Field is being uniform (g?(B0B1)) all the four
spin pockets come to resonance frequency at a time
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Principle of cw EPR Imaging
Projection
Gradient Direction
N
S
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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

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NORMAL TISSUE
RIF-1 TUMOR
Time (min)
Kuppusamy et al, Canc. Res, 1998, 58, 1562
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Pharmacokinetics of Nitroxides at different
Oxygenation of RIF-1 Tumor
Carbogen Breathing Mouse (pO2 95 mmHg)
Room air Breathing Mouse (pO22.5 mmHg)
15N-TPL and LiPc
0.5 min
10 min
Nitroxide intensity -gt
100
60
40
Frequency
10
I/I0 x 100
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0
0.05
0.10
0.15
0.0
1
Rate constant (min-1)
30
0
10
20
40
Time (minutes)
Ilangovan, G. et al Mol. Cell. Biochem., 2002,
234, 393
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Example 1 In vivo Imaging of NO generation
Fe-MGD NO
Fe-MGD-NO
No EPR signal
No EPR signal
Strong EPR signal
NO generated in the thoracic region of a mouse,
subjected to cardiopulmonary arrest
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