Transcript EPR Imaging
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 Magnetic momentum of an add electron s = gS L = gL 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 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 Field : 1.2 GHz – 100 GHz : 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.” Dead Time A serious limitation for FT-EPR spectroscopy Principle of EPR spectroscopy Relaxation T1 – Spin lattice relaxation E = g(B0+B1) T2 – Spin-spin relaxation T2* – Spin-spin relaxation B0 Absorption spectrum Expt. Obtained spectrum 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 Phase Sensitive Detection in EPR 3 2 Max 1 2 4 3 0 5 -Max 1 5 4 Field Field Nuclear magnetic coupling – “Hyperfine splitting” -1 + 1 0 2 +1 - 1 2 -1 0 +1 N O. S = 1 for 14N 2S+1 = 3 Secondary Hyperfine Splittings +1 + 1 0 2 -1 +1 - 1 0 2 -1 Expected 1 2 1 1 + 2 2 1 1 + 2 2 1 2 + 1 2 1 1 + 2 2 1 1 + 2 2 1 2 + Experimentally measured H N O. 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 + (No EPR signal) Unstable radical (No EPR signal) Stable radical (?) (EPR signal) Superoxide trapping: Example 1 Xanthine / Xanthine oxidase Xanthine EPR spect. of DMPO-OH xo Hypoxanthine O2 O2-. + DEPMPO DEPMPO-OOH 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 Imaging 1 2 3 4 Bo Field is being uniform (g(B0+B1)) all the four spin pockets come to resonance frequency at a time MAGNET MAGNET EPR Imaging – Concept of gradient Field Principle of cw EPR Imaging Projection Gradient Direction 1- 4 Gradient generation 1 2 N S 3 4 Bo Bo 1 2 N Projections 2, 4 1, 3 S 3 4 Bo (x+Bo) (x-Bo) Bo Re-construction x-Bo 1,4 1 2 N S 3 x+Bo 3 2 4 Bo 2D image 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 RESONATOR NORMAL TISSUE RIF-1 TUMOR 3.0 0 Kuppusamy et al, Canc. Res, 1998, 58, 1562 4.5 6.0 7.5 9.0 10.5 12.0 Time (min) 13.5 15.0 16.5 256 Pharmacokinetics of Nitroxides at different Oxygenation of RIF-1 Tumor 15N-TPL Room air Breathing Mouse (pO2=2.5 mmHg) and LiPc Carbogen Breathing Mouse (pO2= 95 mmHg) 0.5 min 10 min Nitroxide intensity -> 100 3-CP room air 10 20 10 0 0.0 15N-TPL Carbogen 30 Frequency room air Frequency 15N-TPL 60 40 I/I0 x 100 3-CP Carbogen Nitroxide intensity -> 0.05 0.10 0.15 Rate constant (min-1) 1 0 10 20 30 40 40 20 0 0.0 0.05 0.10 0.15 Rate constant (min-1) Time (minutes) Ilangovan, G. et al Mol. Cell. Biochem., 2002, 234, 393 Example 1 In vivo Imaging of NO generation Fe-MGD No EPR signal + NO No EPR signal Fe-MGD-NO Strong EPR signal NO generated in the thoracic region of a mouse, subjected to cardiopulmonary arrest