introduction to epr/esr spectroscopy and imaging suggested reading: c.p.poole, electron spin...
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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