EPR [wiki] is a magnetic resonance technique which detects the resonance transitions between energy states of unpaired electrons in an applied magnetic field. The electron has spin, which gives it a magnetic moment. The magnetic moment makes the electron behave like a tiny bar magnet. When we apply an external magnetic field, the paramagnetic electrons can either orient in a direction parallel or antiparallel to the direction of the magnetic field. This creates two states with different energies ±½gµBB0 . g is the g-factor, which is a proportionality constant equal to 2.0023 for free electron, but strongly dependent on electronic envionment of the species. µB is the Bohr magneton [wiki], which is the natural unit of electronic magnetic moment. The splitting of the energy levels by applied magnetic field is called the Zeeman effect [wiki]. The difference between the spin state energies can be probed by application of the additional magnetic field oscillating with the frequency ν, such that ΔE = hν. Here ΔE is energy difference between levels and h is Planck constant. This field will drive the electron between the states only if the resonance condition ΔE = hν = gµBB0 is satisfied.
In Vivo systems contain very small concentrationt of paramagnetic species. Many of the species are also short lived intermediates of various biochemical reactions. taken together these factors limit direct observaton of in vivo paramagnetic species using conventional EPR. One EPR technique to observe these intermediates is called spin trapping. The exogenous spin probe that forms paramagnetic adduct in the presence of the species of interest that allows us to quantify and localize them.
Another application of EPR is measurement of environmental parameters of exogenous spin probe such as partial oxygen pressure, pH, viscosity, thiol concentration and temperature. These parameters affect the EPR resonance frequencies by shifting them or altering spin probe relaxation. The careful choice of the spin probe allows specific measurement of only the parameter of interest, for the elucidation of the In vivo microenvironment.
The three-dimensional EPR imaging allows measurement of spin probe distribution in the animal body. The imaging modalities that measure some other parameter in addition to the distribution are typically referred to as 4D imaging. If EPR spectrum is obtained in every point of space the imaging modality is called spectral-spatial. Prior to imaging the spin probe is injected into blood stream of an animal. In case of mice the tail vein is cannulated for this purpose. For larger animals more localized delivery is practiced. Then the animal is placed into an EPR imager that has a static magnetic field electromagnet, three dimensional gradient system, resonator and radio frequency bridge. Similar to MRI, EPR in vivo imaging uses frequencies in the range from 250 MHz to 1 GHz. This choice of frequency ensure good penetration of RF energy through the body. The magnetic fields required for resonance are much lower than the ones used for MRI and are about 100-400 Gauss. Continuous Wave, Rapid Scan, and Pulsed Imaging techniques are used.
Although the basic principles of in vivo EPR imaging are similar to the principles of Nuclear Magnetic Resonance Imaging (MRI), the methodology of EPRI is considerably different from the conventional MRI due to the five to six order of magnitude reduction in phase (T2e) and spin-lattice (T1e) relaxation times of the spin probes. This complicates the application of pulsed magnetic field gradients. For oxymetry we employ electron spin echo pulse sequence shown above. The location of spin probe in a sample is encoded using static magnetic field gradients. Tomographic methods that employed by the Center use gradients sampled in the polar coordinate system as shown in the Figure. The image is reconstructed from projections using inverse Radon transformation. Typical EPR images have spatial resolution of ~1 mm and time resolution of 1 minute. EPR oxygen images have greater than 1 torr absolute accuracy.
EPR p02 images are predictive of tumor resistance to radiation and also to recurrence after treatment. We are currently developing methodologies to target hypoxia based on our p02 images in order to deliver an extra dose of radiation with the goal of deascreasing tumor recurrence and extending patient survival. Development of this technique will allow clinicians to focus radiation on resistant locations within the tumor and spare vital tissues, ensuring higher post-treatment quality of life for the patient. The figure at the left shows data illustrating the difference between highly hypoxic individuals and normoxic ones.
In recent years, major insight has been generated by exploration of the molecular behavior of diseased tissue. However, much is lost by ignoring the micro-environmental complexity of the living animal. To fully understand biological pathways and their component interactions, it is crucial that extensive, simultaneous physiologic information be obtained from various well-resolved locations in living animals. Only armed with this information can mechanisms that govern complex physiologic system interactions be put in proper context. Physiologic EPR images provide this information. Although the initial emphasis of our Center is cancer treatment, the EPR technology can be applied to crucial questions in stroke, peripheral vascular disease, and heart attack.