Introduction to EPR/ESR Spectroscopy and ImagingIntroduction 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 = gS

L = gL

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

N

NMR

EPR

Microwave in the range : 1.2 GHz – 100 GHz

Field : 0.03 – 0.3 T

Radio wave in the range : 90 – 700 MHz

Field value : 2 - 14 T

“Additional problems with biological EPR spectroscopy is the microwave absorption H2O in biological objects.”

NMR – EPR comparison of energies

Relaxation time : 10-3 to 10 sec

Relaxation time : 10-9 – 10-6 sec

A serious limitation for FT-EPR spectroscopy

Dea

d T

ime

B0

E = g(B0+B1)

Principle of EPR spectroscopy

Absorption spectrum Expt. Obtained spectrum

Relaxation

T1 – Spin lattice relaxation

T2 – Spin-spin relaxation

T2* – Spin-spin relaxation

Modulation frequency

Modulation amplitude

Field (B1) modulation in EPR

Why: Absorption signal is weak, compared NMR, and buried under equally amplified noise.

B1 Oscillating Magnetic field

Unmodulated Modulated

0

-Max

Max

Phase Sensitive Detection in EPR

51

2

3

4

1

2

3

4

5

Field

Field

Nuclear magnetic coupling – “Hyperfine splitting”

N

O.

S = 1 for 14N

2S+1 = 3

-1

2

+1

2

-1

0

+1

-1

0

+1

N

O.

H

Expected Experimentally measured

Secondary Hyperfine Splittings

-1

2

+1

0

-1

+1

2

+1

0

-1

-1

2

-1

2+

1

2

+12

-1

2+

1

2

-1

2

-1

2+

1

2

+12

-1

2+

1

2

EPR spin trappingEPR spin trapping

Many free radicals, generated by enzymatic reactions are not stable enough to detect by EPR spectroscopy.

They need to be stabilized to detect by EPR: “Spin trapping”

Spin trap Unstable radical Stable radical (?)+

(No EPR signal)

Superoxide radical (O2.-)

Hydroxyl radical (OH.)

Nitric oxide (NO:)

(No EPR signal) (EPR signal)

O2 O2-.

DEPMPO

DEPMPO-OOH

Xanthine

Hypoxanthine

+

xoEPR spect. of DMPO-OH EPR spect. of DMPO-OH

Superoxide trapping: Example 1 Xanthine / Xanthine oxidase

Trapping Nitric Oxide 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 ImagingEPR Imaging

Bo

EPR Imaging – Concept of gradient Field

MA

GN

ET

MA

GN

ET1 2

3 4

Field is being uniform (g(B0+B1)) all the four spin pockets come to resonance frequency at a time

Principle of cw EPR Imaging

Bo

1 2

3 4

1- 4

Bo

Bo

1, 3 2, 4

3

1,4

2

2D image

Re-construction

Projections

Gradientgeneration

Gradient Direction Projection

N S

Bo

1 2

3 4

(x+Bo) (x-Bo)

N S

Bo

1 2

3 4

x+Bo

x-Bo

N S

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

It is an unique technique to study redox status of tissues, organs or in whole body, which cannot be achieved by other techniques

But….

RESONATOR

Time (min)0 256

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

Kuppusamy et al, Canc. Res, 1998, 58, 1562

Nitroxide intensity ->

Room air Breathing Mouse (pO2=2.5 mmHg)

Carbogen Breathing Mouse (pO2= 95

mmHg)

Nitroxide intensity ->

Fre

qu

en

cy

Rate constant (min-1)0.05 0.10

0

20

40

60

0.150.0

Fre

qu

en

cy

0

10

20

30

0.05 0.10Rate constant (min-1)

0.150.0

40

Time (minutes)

0 10 20 30 40

I/I

0 x

10

0

1

10

100

15N-TPL and LiPc

0.5 min

10 min

3-CProom air

3-CPCarbogen

15N-TPLroom air

15N-TPL Carbogen

Pharmacokinetics of Nitroxides at different Oxygenation of RIF-1 Tumor

Ilangovan, G. et al Mol. Cell. Biochem., 2002, 234, 393

NO generated in the thoracic region of a mouse, subjected to cardiopulmonary arrest

Example 1 In vivo Imaging of NO generation

Fe-MGD + NO

No EPR signal No EPR signal

Fe-MGD-NO

Strong EPR signal