1. G.R.Eaton, S.S.Eaton, K.Ohno, EPR imaging and In vivo EPR
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

2. 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

3. NMR – EPR comparison of energies
Radio wave in the range : 90 – 700 MHz
Field value : T
Relaxation time
: 10-3 to 10 sec
EPR
Microwave in the range : 1.2 GHz – 100 GHz
Field : – 0.3 T
Relaxation time
: 10-9 – 10-6 sec
“Additional problems with biological EPR spectroscopy is the microwave absorption H2O in biological objects.”

4. A serious limitation for FT-EPR spectroscopy
Dead Time

5. Principle of EPR spectroscopy
Relaxation
T1 – Spin lattice relaxation
E = g(B0+B1)
T2 – Spin-spin relaxation
T2* – Spin-spin relaxation  B0
Expt. Obtained spectrum
Absorption spectrum

6. Oscillating Magnetic field
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

7. Phase Sensitive Detection in EPR3 2
Max 1 3 4 2 5
-Max 1 4 5
Field
Field

8. Nuclear magnetic coupling – “Hyperfine splitting”-1 +1 - 1 2 + N O.
S = 1 for 14N
2S+1 = 3

9. Secondary Hyperfine Splittings- 1 2 + - 1 2 +1 -1 + N O. H
Expected
Experimentally measured

10. 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)

11. Superoxide trapping: Example 1 Xanthine / Xanthine oxidase
DEPMPO
DEPMPO-OOH
Xanthine
Hypoxanthine + xo
EPR spect. of DMPO-OH

12. 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

13. Fe(MGD)
Fe(MGD)-NO

14. Superoxide trapping: Example 1 Nitric oxide synthase (NOS)
Fe-MGD
DMPO-OO-

15. EPR Imaging

16. EPR Imaging – Concept of gradient Field1 2
MAGNET
MAGNET 3 4 Bo
Field is being uniform (g(B0+B1)) all the four spin pockets come to resonance frequency at a time

17. Principle of cw EPR Imaging
Gradient Direction
Projection
2D image
Re-construction
Projections
Gradient
generation Bo 1 2 3 4
1- 4 N S Bo 1 2 3 4
(x+Bo)
(x-Bo) N S Bo
1, 3
2, 4 Bo 1 2 3 4
x+Bo
x-Bo N S 3
1,4 2

18. 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

19. NORMAL TISSUE RIF-1 TUMOR 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0 16.5
RESONATOR
NORMAL TISSUE
RIF-1 TUMOR
3.0
4.5
6.0
7.5
9.0
10.5
12.0
13.5
15.0
16.5
Time (min)
256
Kuppusamy et al, Canc. Res, 1998, 58, 1562

20. Breathing Mouse (pO2=2.5 mmHg) Carbogen Breathing Mouse (pO2= 95 mmHg)
Pharmacokinetics of Nitroxides at different Oxygenation of RIF-1 Tumor
Room air
Breathing Mouse (pO2=2.5 mmHg)
Carbogen Breathing Mouse (pO2= 95 mmHg)
15N-TPL and LiPc
0.5 min
Nitroxide intensity ->
10 min
Nitroxide intensity ->
100
Frequency 10 20 30
0.05
0.10
Rate constant (min-1)
0.15
0.0 40
3-CP
room air 60
3-CP
Carbogen 40 10
Frequency
I/I0 x 100
15N-TPL
room air 20
15N-TPL
Carbogen
0.0
0.05
0.10
0.15 1
Rate constant (min-1) 10 20 30 40
Time (minutes)
Ilangovan, G. et al Mol. Cell. Biochem., 2002, 234, 393

21. 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