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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.

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Presentation on theme: "Introduction to EPR/ESR Spectroscopy and Imaging Suggested reading: C.P.Poole, Electron Spin Resonance, A comprehensive Treatise on Experimental Techniques."— Presentation transcript:

1 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  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 NN

3 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 : T “Additional problems with biological EPR spectroscopy is the microwave absorption H 2 O in biological objects.” NMR – EPR comparison of energies Relaxation time: to 10 sec Relaxation time: – sec

4 A serious limitation for FT-EPR spectroscopy Dead Time

5 B0  E = g  (B0+B1) Principle of EPR spectroscopy Absorption spectrum Expt. Obtained spectrum Relaxation T 1 – Spin lattice relaxation T 2 – Spin-spin relaxation T 2 * – Spin-spin relaxation  

6 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 UnmodulatedModulated

7 0 -Max Max Phase Sensitive Detection in EPR Field

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

9 N O. H Expected Experimentally measured Secondary Hyperfine Splittings

10 EPR 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 trapUnstable radicalStable radical (?)+ (No EPR signal)  Superoxide radical (O 2.- )  Hydroxyl radical (OH. )  Nitric oxide (NO:) (No EPR signal)(EPR signal)

11 O2O2 O 2 -. DEPMPO DEPMPO-OOH Xanthine Hypoxanthine + xo EPR spect. of DMPO-OH Superoxide trapping: Example 1 Xanthine / Xanthine oxidase

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 O 2. 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 Bo EPR Imaging – Concept of gradient Field MAGNET 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 Bo Bo 1, 3 2, 4 3 1,4 2 2D image Re-construction Projections Gradient generation Gradient Direction Projection NS Bo (x +Bo) (x -Bo) NS Bo x +Bo x -Bo NS

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

19 RESONATOR Time (min) NORMAL TISSUE RIF-1 TUMOR Kuppusamy et al, Canc. Res, 1998, 58, 1562

20 Nitroxide intensity -> Room air Breathing Mouse (pO2=2.5 mmHg) Carbogen Breathing Mouse (pO2= 95 mmHg) Nitroxide intensity -> Frequency Rate constant (min -1 ) Frequency Rate constant (min -1 ) Time (minutes) I/I 0 x N-TPL and LiPc 0.5 min 10 min 3-CP room air 3-CP Carbogen 15 N-TPL room air 15 N-TPL Carbogen Pharmacokinetics of Nitroxides at different Oxygenation of RIF-1 Tumor Ilangovan, G. et al Mol. Cell. Biochem., 2002, 234, 393

21 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 Fe-MGD-NO Strong EPR signal

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