EPR Research

A novel Electron Paramagnetic Resonance technique has been developed to image physiologic characteristics and toxic radicals in living tissues of animals and  humans. This information has, heretofore, been unavailable. Recent advances in spectroscopic technique and spin probe  development, as well as the understanding of the information that can be provided, poise this technique on the verge of breakthrough. The  technique uses very low frequency (100 to 300 MHZ) electron paramagnetic resonance  imaging. Very low frequency is necessary to allow the electromagnetic energy, which stimulates resonant absorption,  to penetrate deep into the tissues of animals. We develop Center facilities with an array of EPR imagers capable of providing real time information on tissue microenvironment. The Center functions within a major medical research facility. This offers adjacent, coordinated animal care, access to MRI, CT, PET and optical scanning and IMRT irradiators.

ManhattanPrj.jpg Change in pO2 for ASMase (+/+) blue and ASMase (-/-) red.

In human subjects an unexpected effectiveness, of radiation delivered in large doses (> 15 Gy/dose) is observed. It is possible that these radiation doses stimulate a signal cascade in the host vasculature that is initiated by the acid sphingomyelinase (ASMase) gene product ceramide, which causes rapid death of microvessel endothelial cells supplying oxygen to tumor cells.  This causes a rapid onset of hypoxia, which interferes with tumor cell repair of damage induced by the radiation.  This sensitizes the tumor cells to radiation by post-radiation hypoxia.  

We compare the change in pO2 15 min. after 20 Gy radiation in ASMase knockout and wild type mice.  pO2 change is plotted in Figure; the tumor pO2change in the wild-type animal is larger than in tumors in ASMase knockout mice (p=0.02).  This is a strong argument for the proposed hypothesis.


Apparent pO2 from R1 (0) and R2 (•) relaxation rates in a mouse as a function of spin probe concentration for different experiments as the spin probe is infused at different rates.

We discovered that the self-broadening of trityls is much larger in the presence of salt than had been measured in pure water. This effect introduces additional enhancement of R2 and diminishes the absolute pO2 resolution of the measurements. The rate of energy dissipation to the lattice of the detected magnetization (R1) is far less affected by spin exchange between spin probes because the energy is transferred to another member of the magnetization spin system.  Thus, T2 (or R2) related processes are affected far more by spin exchange processes than are T1 (or R1) processes.  We implemented R1imaging by using inversion recovery (IR), sampling the inversion recovery time with a fixed echo time ESE readout (IRESE). Figure shows the in vitro reduction of the concentration effect on R1 relative to R2. In this experiment, a mouse was imaged repetitively as the rate of trityl infusion was increased.  In vitro, the effect is a factor of 5 reduction of self-broadening, whereas in vivo, there is an order of magnitude reduction in self-broadening when R1-sensitive pulse sequences are used.  This establishes the R1 EPROI as virtually an absolute oxygen image.

time_resolved_imaging.jpg Time dependence of pO2 from different regions of mouse leg during FiO2 modulation experiment. Area 1 is located on the tumor periphery, while area 2 is inside the tumor. The effect of PCA on T1/R1 images and correlation image of FiO2 function and animal pO2 are shown. Time resolution 2.5 min.

Using the partially deuterated trityl OX063d24 we were able to reduce the acquisition time to up to 1 minute per image. In order to assess the sensitivity of the tissues pO2 to transient changes in hypoxia, we subjected mice to alternation in the fraction of inhaled O2 (FiO2) of the inhaled gas.  The driving FiO2 in the Figure is shown above on the right along with the sequence of pO2values from the 27 voxels from the hypoxic tumor region [2] and the better oxygenated region [1] in the tumor periphery indicated by red contour. 

This technology is the basis for the development of major tools for the analysis of the relationship between temporal pO2 variation of normal tissue and tumor tissue and the absolute levels of oxygenation.  These, in turn, must be analyzed relative to the response to therapy.  The temporal correlation needs to be investigated as to which of these patterns is the most significant determinant of tumor resistance to radiation and chemotherapy.


Scheme of Alderman-Grant / loop-gap bimodal resonator. Cross-wire surface coil.

Previously, most of the animal work was performed with reflection-type resonators. The refinement of more complex aspects of biology requires higher SNR of EPR measurements.  This is possible to achieve with bimodal resonators.  Bimodal resonators consist of two independent resonators, which induce mutually orthogonal magnetic fields in the same volume. The Center developed number of designs, the diagram of one of them, Alderman Grant/ Loop Gap animal resonator is shown on the left.

For the Center technology to be applied to human subjects, the surface coils are crucial. In collaboration with Life Services, LLC, Stillwater, MN we developed a 2'' crossed wire resonator.  At this early stage, isolation of nearly 40 dB have been achieved. 

Oxygen Imaging

  • Microvessel Density with high spectral and spatial resolution MRI
     Gregory S. Karczmar, PhD, Department of Radiology, University of Chicago
  • Image guidance and assessment of radiation induced gene therapy
     Charles A. Pelizzari, PhD, Department of Radiation and Cellular Oncology, University of Chicago
  • Radiation Biology of EPR Oxygen Images
     Howard J. Halpern, MD, PhD, Department of Radiation and Cellular Oncology, University of Chicago
  • Correlating 64Cu-ATSM positron emission tomography images of tumor hypoxia with oxygen tension images measured by electron paramagnetic resonance
    Hania Al Hallaq, PhD, Department of Radiation and Cellular Oncology, University of Chicago

   Temperature Imaging

  • Hyperthermia and perfusion effects in cancer therapy
    Mark Dewhirst, DVM, PhD, Department of Radiation Oncology,  Duke University, Durham, NC

   Tumor Physiology

  • Development of polymer-linked nitroxides for tumor imaging
    Gerald M. Rosen, PhD, JD, Department of Pharmaceutical Sciences, University of Maryland, Baltimore

   General Physiology

  • Novel nitroxides for In Vivo cellular imaging by  EPR
    Joseph P.Y. Kao, PhD, Medical Biotechnology Center,  University of Maryland Biotechnology Institute

   EPR Imaging of Cellular Signalling in Living Animals

  • In Vivo detection of tumor hypoxia signaling
    Howard J. Halpern, MD, PhD, Department of Radiation and Cellular Oncology, University of Chicago
  • In Vivo nitric oxide signaling in bacterial infection
    Gerald M. Rosen, PhD, JD, Department of Pharmaceutical Sciences, University of Maryland, Baltimore

   Physical Chemistry Relevant to Structural Biology at Radiofrequency

  • Interaction of spin labels with transition metals
    Gareth R. Eaton, PhD, Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado
  • Metal ion probes of membrane protein structure and function
    Michael K. Bowman (MINT Center, the University of Alabama, Tuscaloosa, AL)

   Technology Development

  • Novel technology for hybrid PET/MRI and PET/EPRI in cancer imaging
    Howard J. Halpern, MD, PhD, Department of Radiation  and Cellular Oncology, University of Chicago
    Alexandre Vaniachine, PhD, High Energy Physics Division, Argonne  National Laboratory
  • In Vivo EPR bioengineering research partnership
    Gareth R. Eaton, PhD, Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado
  • Algorithm development/optimization for EPRI oxygen imaging
    Xiaochuan Pan, PhD, Department of Radiology, University of Chicago
  • Evaluation of cylindrical TE011 re-entrant geometries for use with aqueous samples
    James S. Hyde, PhD, Department of Biophysics, Medical College of  Wisconsin