1. Principles of EPR oxygen imaging In Vivo Oxygen Imaging Workshop University of Chicago June 25, 2012 Boris Epel

2. Outline Principles of EPR EPR spin probes EPR imaging principles Image registration and tumor localization Image visualization and statistics

3. What is spin – fundamental property of electron, like electrical charge or mass. – the rotation of a particle around some axis – Characterized by a spin quantum number, S – electron have spin ½ – In EPR, it is unpaired spins that are of importance.

4. Angular and Magnetic Moments Electron is a moving charge –it gives rise to a magnetic moment, µ Electron can be described as a magnetic dipole – bar magnet S N e-e-

5. Design of EPR experiment S N S N S N Constant field B 0 Radio frequency, Magnet will return back in some time: longitudinal relaxation EPR is the resonant absorption of radio frequency radiation by paramagnetic systems in the presence of an applied constant magnetic field Electron magnetic moment isn’t free to adopt an arbitrary orientation. There is a discrete set of orientations possible.

6. EPR spin probes Endogenous paramagnetic species found in mammalian bodies have very short live times, broad lines, or very low concentrations. At present, exogenous spin probes are the only practical reporters, and appropriate spin probes are the key to successful imaging. At present, iv injections are used for the delivery of spin probe. The development of other means of spin probe delivery (arterial and direct injection) is under way.

7. EPR oxymetry probes Soluble probes A Nitroxides B Trityl radicals Concentration (μM) of dissolved oxygen in the bulk volume Resolution 1 mmHg Particulate (Solid) probes C Lithium phthalocyanine and its derivatives

8. What EPR can measure Oxygen, pO 2 Redox status Acidosis, pH Thiols (GSH) Cell viability Viscosity Tissue perfusion Molecular motion Oxygen, pO 2

9. Operational frequency What is the optimum frequency? - depends on sample size Frequency~250 MHz~750 MHz1-2 GHz Penetration> 10 cm6-8 cm1-1.5 cm ObjectMouse, rat, rabbit Mouse, full body Mouse part Biological samples contain large proportion of water. They are aqueous and highly dielectric. Conventional EPR spectrometers operate at X-band ~9 GHz frequencies, which result in (i) ‘non-resonant’ absorption of energy (sample heating) and (ii) poor penetration of samples. Hence the frequency of the instrumentation needs to be reduced.

10. EPR vs MRI MRIEPR Magnetic field at 250 MHz5.9 T9 mT Radiofrequency pulse widthμsec – msec10 – 100 nsec Relaxation ratesmsec – secnsec - μsec Endogenous probesWater protons- Exogenous probes-Nitroxides, trityl Concentration>60 M< 1 mM StabilityStableMinutes Line widthHz – kHz100 kHz - MHz

11. In Vivo EPR Oxygen Imaging Trityl iv line Mouse cradle Resonator Fiducials Bladder flushing line Cutaneous thermocouple Gas anesthe- sia mask Tumor in the cast

12. Spectroscopy vs Parametric Imaging Inhomogeneous distribution High O 2 Low O 2 Slow relax. Fast relax.

13. One dimensional Two dimensional Three-dimensional Image Dimensionality

14. Imaging Principles Application of the linear magnetic field gradient  B

15. Magnetic Field Gradient Please do not leave metal objects close to the imager Homogeneous field, B 0 Linear gradient, 0 0 Linear gradient, 45 0 ‘projection’

16. Imaging Principles Application of the linear magnetic field gradient Obtaining multiple projections by use of different gradients orientations  B

17. Imaging Principles Application of the linear magnetic field gradient Obtaining multiple projections by use of different gradients orientations Image reconstruction (filtered backprojection)  B

18. Imager magnet

19. Electron Spin Echo Oxygen Imaging T 1 or T 2 [  s] pO 2 [torr] Spin-probe concentration [mM] Amplitude [a.u.] Deoxygenated OXO63 spin probe pO 2 =  (R – R (0 torr, 0 mM) -  C) R = 1/2  T 1 or 2 [mG]

20. Image resolution spatial1.2 mm temporal10 min (2.5 min rapid protocol) pO 2 1 torr

21. Pulse EPR: imaging sequences Electron Spin Echo (ESE) – T 2 imaging    T Inversion recovery (IRESE) – T 1 imaging

22. Concentration Dependence in Vivo T 1 shows only weak dependence on spin probe concentration T 1 – based EPR imaging is the perfect method for precise oxygen imaging

23. Imaging Procedures Prepare an animal Install animal in the resonator cradle Install resonator into imager Inject spin probe Image an animal Observe pO 2 image Acquire pO 2 statistics Determine area of interest Acquire MRI image Register EPRI and MRI Acquire CT/PET image Register CT/PET and EPR Optional ROI

24. ESE and MR Image Registration EPRI– 3mM deuterated FINLAND fiducials (~ 0.5 mm resolution) MRI– water fiducials Haney C. et al., Concepts in Magnetic Resonance B (2008), 33, 138-144. Mouse leg in the resonator. Polysiloxane half-cast with inserted fiducials 3D view of MRI and amplitude ESEI image registration. Fiducials are used to establish the coordinate transformation from MRI into ESE coordinate system 24

25. ESE and MRI Image Registration Fiducials MRI EPROI pO 2

26. Multimodality Rat Imaging A B C D Multi-B ESE 18 F-FDG PET T 2 -weighted MRI C pO 2

27. EPR oxygen image visualization Region of interest in this case area of the tumor from a registered MRI image ‘Three orthogonal slices’ view ROI and general statistics Colormap and view adjustment Cursor statistics Cursor

28. Summary In vivo EPR spectroscopy and imaging methods enable noninvasive measurement and mapping of tissue pO 2. Image resolution spatial1.2 mm temporal10 min (2.5 min rapid protocol) pO 2 1 torr

30. Direct injection of spin probe into artery 51 mm diameter loop-gap resonator 4 cm VX2 carcinoma Spin probe was continuously injected directly into the artery feeding the leg. This allowed us to use only 1/4 of the calculated injection dose B. Epel et al. Medical Physics 37 (2010) 2553-2559.

31. Imaging of Cycling Hypoxia Matsumoto, S., H. Yasui, et al. (2010). "Imaging Cycling Tumor Hypoxia." Cancer Research 70(24): 10019-10023. Yasui, H., S. Matsumoto, et al. (2010). "Low-Field Magnetic Resonance Imaging to Visualize Chronic and Cycling Hypoxia in Tumor-Bearing Mice." Cancer Research 70(16): 6427-6436. EPR Single Point Imaging Image duration 3 minutes. Magat, J., B. F. Jordan, et al. (2010). "Noninvasive mapping of spontaneous fluctuations in tumor oxygenation using F-19 MRI." Medical Physics 37(10): 5434-5441. 19 F MRI Image duration 3 minutes.

32. Rapid ESE Oxygen Imaging – 1 min Resolution 1 3 2 1 2 3 pO 2 [torr] T 2 – based pO 2 imaging Spontaneous fluctuations of pO 2 in tissues 60 0

33. Carbogen Challenge Experiment (2.5 min Images) The breathing gas is switched periodically between air and carbogen (95% O 2 and 5% CO 2 ) 1 cm 10 20 30 40 50 60 012.52537.55062.575 minutes 21 % 95 % 0 10 20 012.52537.55062.575 minutes pO 2 [torr] O2O2 1 2 1 2 pO 2 T 1 +T 2 pO 2 imaging