Biomedical EPR Part-B Methodology Instrumentation and Dynamics - Sandra R. Eaton

424 JAMES S. HYDE

services based on EPR analysis of the free radical content in alanine pellets exposed to high doses of ionizing radiation, including calibration services. Bruker offers an automated spectrometer, the e-scan, specially configured for this measurement. In 2002, a news release issued jointly by Kodak and Bruker describes a system for alanine dosimetry based on coating of microcrystalline alanine on a film, which is sold under the trademark Biomax. The system includes a bar code and alphanumeric identifier for sample tracking. The news release describes the system as an unmatched method for high-volume verification of radiation in processes ranging from the sterilization of medical devices to decontamination of food.

Another industrial application of EPR was introduced by Uchida and Ono in 1996 (Uchida and Ono, 1996). They employed a spin trapping strategy to measure the quantity of anti-oxidants in beer (Uchida et al., 1996; Uchida and Ono, 2000). This measurement correlated with shelf-life of the product. A patent was issued in 1998 (Ono and Uchida, 1998) for the process. Bruker has also configured the e-scan spectrometer for this application.

Modern approaches to EPR spectrometer design can be expected to result in enhanced performance and reduced cost for commercial applications of EPR, providing that the markets are sufficiently large. Using finite element modeling of microwave fields, resonator design and sample geometry can be optimized efficiently for each application. In addition, microwave circuits can be miniaturized and optimized by computer design. It is highly desirable to use the smallest magnet gap possible. In a patent entitled “Narrow Cavity Low Cost EPR Spectrometer” (Hyde, 1987), the author used a or cavity just 2 mm wide in order to reduce the mass of the magnet. Loop gap resonators provide ample opportunity to reduce magnet size in process-control or medical diagnosis applications. Modern PCs plus digital detection permit the equivalent of phase sensitive detection in the computer (ch. 7, this volume), manipulation of the gain and bandwidth of automatic frequency control circuits, and incorporation of quite sophisticated signal processing software.

Process control applications of EPR have been slow to develop, but the success that Bruker has been experiencing in recent years indicates progress. Newer computer-based engineering design tools, the introduction of digital detection, and modern computer-based signal analysis tools can be expected to provide the technological basis for further progress.

Development of medical diagnosis applications of EPR that are widely used has been a long-standing goal, but has remained elusive. It has become clear that success can only be achieved if there is a sound scientific rationale for EPR spectroscopy in comparison with other more familiar spectroscopic methods. Spin trapping in a context of free radical chemistry offers one

opportunity, since it can be argued that EPR is generally a preferred spectroscopy when free radicals are involved.

6.DISCUSSION

From the author’s own laboratory, several “mini-trends” that may flourish in the years ahead can be identified:

1) Optimization of resonators for ultra-small samples, i.e., submicroliter. In a context of loop gap resonators, electric discharge machining (EDM) permits cutting of very small gaps. In a poster (Camenisch et al., 2002), a Q-band resonator with a sample volume as small as 30 nl containing 1 picomole of protein was successfully used in a multiquantum experiment.

2) Step recovery pulse EPR. The concept is to generalize the step of incident microwave power used in saturation recovery to include other steps in experimental conditions including light level, temperature, pH value, stop flow mixing of reacting species, applied electric field, nuclear irradiation level in ENDOR, pressure, etc., and observe the time-dependent evolution to a new equilibrium condition. Observation of the transient response using either spin echo methods or CW methods is appropriate. The technological challenge lies in appropriate delivery of the step. Change in sample composition or temperature can result in changes in microwave resonance frequency or coupling, that require dynamic adjustment. Changes in the EPR field/frequency relationship during approach to a new equilibrium may require attention.

3)Blurring of the distinction between CW EPR and pulse EPR. High frequency field modulation is a form of pulse EPR, since it involves inversion of the magnetization during each sweep of the instantaneous modulation field through resonance. Multiquantum EPR is a temporal modulation of the saturation conditions that can be described equally well in the time domain or the frequency domain. MQ in the presence of high frequency field modulation has been demonstrated in unpublished work in the author’s laboratory.

4)Increased use of ELDOR. Intense saturation transfer mechanisms are one of the distinguishing characteristics of EPR spectroscopy. The technological challenge in exploiting this opportunity lies largely in resonator development. Frequency sweep of the pumping microwaves is highly desirable, but technically challenging. These methods were pioneered by the author in the 1960s. The hypothesis can be made that modern technology creates opportunities for improved bimodal resonators

426 JAMES S. HYDE

with frequency sweep of the pumping mode. ELDOR is a powerful method that remains underutilized because of technological complexities.

The material in this chapter has been largely derived from the author’s experience and must be considered to be a personal view of technological trends in EPR. Several significant instrumental trends, including developments in pulse spin echo EPR, in vivo EPR and ultra-high frequency EPR, are not discussed. However, the themes of this article, viz, resonator enhancement by electromagnetic field finite element modeling, analysis of noise, digital detection and acquisition of data at multiple microwave frequencies, are relevant to pulse, in vivo and ultra-high frequency EPR.

The chapter by Freed and Peisach in this volume gives additional information on pulse EPR and high frequency EPR, and the chapters by Swartz (ch.10) Halpern (ch. 11), and Krishna (ch. 12) in volume 23 provides an overview of in vivo EPR. The recent monograph on pulse EPR by Schwieger and Jeschke (2001) provides a foundation for future progress in that field.

7.REFERENCES

Anderson, J.R., Mett, R.R. and Hyde, J.S. (2002). Cavities With Axially Uniform Fields For Use In Electron Paramagnetic Resonance. II. Free Space Generalization. Rev. Sci. Instrum. 73, 3027-3037.

Brauer, J.R. (1997). IEEE Trans. Microwave Theory Tech., 45, 810.

Camenisch, T.G., Klug, C.R., Ratke, J.J., Hubbell, W.L. and Hyde, J.S. (2002). Multiquantum EPR of Arrestin K267C-MT SL at 35 GHz. Proc. Rocky Mountain Conf. on Analytical Chemistry, p. 43.

Christidis, T.C., Froncisz, W., Oles, T. and Hyde, J.S. (1994). Probehead with Interchangeable Loop-Gap Resonators and RF Coils for Multifrequency EPR/ENDOR. Rev. Sci. Instrum. 65, 63-67.

Christidis, T.C., Mchaourab, H.S. and Hyde, J.S. (1996). Hyperfine Selectivity Using Multiquantum Electron-Nuclear-Electron Triple Resonance. J. Chem. Phys. 104, 96449646.

Forrer, J., Pfenninger, S., Sierra, G. Jeschke, G. and Schweiger, A., Wagner, B. and Weiland, T. (1996). Probeheads And Instrumentation For Pulse EPR And ENDOR Spectroscopy With Chirped Radio Frequency Pulses And Magnetic Field Steps. Appl. Magn. Reson. 10, 263-279.

Froncisz, W. and Hyde, J.S. (1982). The Loop-Gap Resonator: A New Microwave Lumped Circuit ESR Sample Structure. J. Magn. Reson. 47, 515-521.

Froncisz, W., Oles, T. and Hyde, J.S. (1986) Q-Band Loop-Gap Resonator. Rev. Sci. Instrum. 57, 1095-1099.

Hwang, P.T.R. and Pusey, W.C. (1973) U.S. Patent 3,740,641.

Hyde, J.S. (1995). Electron Paramagnetic Resonance in Handbook of Microwave Technology, Volume 2, Ishii, T. K., ed., pp. 365-402, Academic Press, New York.

Hyde, J.S. (1965a). Gyromagnetic Resonance Spectroscopy, U.S. Patent 3,197,692. Hyde, J.S. (1965b). ENDOR Of Free Radicals In Solution. J. Chem Phys. 43, 1806-1818.

Hyde, J.S. (1975). EPR Spectrometer Resonant Cavity, U.S. Patent 3,878,454.

Hyde, J.S. (1987). Narrow Cavity Low Cost EPR Spectrometer, U.S. Patent. 3,931,569. Hyde, J.S. (1998a). Multiquantum EPR. in Foundations of Modern EPR, G.R. Eaton, S.S.

Eaton and K.M. Salikhov, eds., pp. 741-757, World Scientific, New York.

Hyde, J.S. (1998b). EPR at Varian: 1954-1974. in Foundations of Modern EPR, G.R. Eaton, S.S. Eaton and K.M. Salikhov, eds., pp. 695-716, World Scientific, New York.

Hyde, J.S. and Froncisz, W. (1989). Loop Gap Resonators. In Advanced EPR: Applications in Biology and Biochemistry, A.J. Hoff, ed., pp. 277-306, Elsevier, Amsterdam.

Hyde, J.S., Froncisz, W. and Kusumi, A. (1982). Dispersion Electron Spin Resonance with the Loop-Gap Resonator. Rev. Sci. Instrum. 53, 1934-1937.

Hyde, J.S., Newton, M.A., Strangeway, R.A., Camenisch, T.G. and Froncisz, W. ( 1991). Electron Paramagnetic Resonance Q-Band Bridge with GaAs Field-Effect Transistor Signal Amplifier and Low-Noise Gunn Diode Oscillator. Rev. Sci. Instrum. 62, 29692975.

Hyde, J.S., Mett R.R. and Anderson, J.R. (2002). Cavities With Axially Uniform Fields For Use In Electron Paramagnetic Resonance. III. Re-Entrant Geometries. Rev. Sci. Instrum. 73, pp. 4003-4009.

Korber, MR., Teuthorn, D. and Lockie, D. (2002). YIGs Tune High-Speed Millimeter-Wave Radios. Microwaves & RF, pp. 92-100 (July).

Lesniewski, P. and Hyde, J.S. (1990). Phase Noise Reduction of a 19 GHz Varactor-Tuned Gunn Oscillator for Electron Paramagnetic Resonance Spectroscopy. Rev. Sci. Instrum. 61, 2248-2250.

Mchaourab, H.S., Christidis, T.C., Froncisz, W., Sczaniecki, P.B. and Hyde, J.S. (1991). Multiple-Quantum Electron-Electron Double Resonance. J. Magn. Reson. 92, 429-433.

Mchaourab, H.S., Christidis, T.C. and Hyde, J.S. (1993). Continuous Wave Multiquantum Electron Paramagnetic Resonance Spectroscopy. IV. Multiquantum Electron-Nuclear Double Resonance. J. Chem. Phys. 99, 4975-4985.

Mett, R.R., Froncisz, W. and Hyde, J.S. (2001). Axially Uniform Resonant Cavity Modes For Potential Use In Electron Paramagnetic Resonance Spectroscopy. Rev. Sci. Instrum. 72, 4188-4200.

Nelson, F.A. and Baker, G.A. (1967), Gyromagnetic Resonance Apparatus Utilizing Two Sample Signal Comparison, U.S. Patent 3,348,136.

Newton, M.E. and Hyde, J.S. (1991). ENDOR at S-Band (2-4 GHz) Microwave Frequencies.

J. Magn. Reson., 95, 80-87.

Ono, M. and Uchida, M. (1998). Analytical Method For Evaluating Flavor Stability Of Fermented Alcoholic Beverages Using Electron Spin Resonance, U.S. Patent 5,811,305.

Pace, M.D., Christidis, T.C. and Hyde, J.S. (1993). S-Band ENDOR of Hyperfine

Resonant Structure For Pulsed ESR Transparent To High-Frequency Radiation. Rev. Sci. Instrum. 59, 752-760.

Pfenninger, S., Froncisz, W. and Hyde, J.S. (1995). Noise Analysis of EPR Spectrometers with Cryogenic Microwave Preamplifiers. J. Magn. Reson. 113A, 32-39.

Piasecki, W., Froncisz, W. and Hyde, J.S. (1996). Bimodal Loop-Gap Resonator. Rev. Sci. Instrum. 67, 1896-1904.

Piette, L.H., Ludwig, P., Adams, R.N. (1962). EPR And Electrochemistry. Studies Of Electrochemically Generated Radical Ions In Aqueous Solution, Anal. Chem., 34, 916.

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Pusey III, W.C. (1973). How To Evaluate Potential Gas And Oil Source Rocks. World Oil, April

Rempel, R.C., Ward, C.E., Sullivan, R.T., St. Clair, M.W., Weaver, H.E. (1964). Gyromagnetic Resonance Method and Apparatus, U.S. Patent 3,122,703.

Rinard, G.A., Quine, R.W., Song, R., Eaton, G.R., Eaton, S.S. (1999). Absolute EPR Spin Echo and Noise Intensities. J. Magn. Reson. 140, 69-83.

Robins, R.J. (1982). Phase Noise in Signal Sources, Peregrinus Ltd. on behalf of the Institute of Electrical Engineers, Herts, England.

Saraceno, A.J., Fanale, D.I. and Coggeshall, N.D. (1961). An Electron Paramagnetic Resonance Investigation of Vanadium in Petroleum Oils. Anal. Chem 33, 500.

Saraceno, A.J. (1963). Determination of Vanadium Content of Hydrocarbon Oils by Electron Paramagnetic Resonance Spectrometry, U.S. Patent 3,087,888 (reissued 1967).

Schwieger, A. and Jeschke, G. (2001). Principles Of Pulse Electron Paramagnetic Resonance, Oxford University Press, New York.

Strangeway, R.A., Mchaourab, H.S., Luglio, J., Froncisz, W. and Hyde, J.S. (1995). A General Purpose Multiquantum Electron Paramagnetic Resonance Spectrometer. Rev. Sci. Instrum. 66, 4516-4528.

Symons, M.C.R. (1995). Whole Body Electron Spin Resonance Imaging Spectrometer, In Bioradicals Detected By ESR Spectroscopy, H. Ohya-Nishiguchi and L. Packer, eds., pp. 93-102, Birkhäuser Verlag, Basel, Switzerland.

Uchida, M. and Ono, M. (1996). Improvement For Oxidative Flavor Stability Of Beer – Role Of OH-Radical In Beer. J. Am. Soc. Brew. Chem 54, 198-204.

Uchida, M., Suga, S. and Ono, M. (1996). Improvement For Oxidative Flavour Stability Of Beer – Rapid Prediction Method For Beer Flavour Stability By Electron Spin Resonance Spectroscopy. J. Am. Soc. Brew. Chem 54, 205-211.

Uchida, M. and Ono, M. (2000). Technological Approach To Improve Beer Flavor Stability: Analysis Of The Effect Of Brewing Processes On Beer Flavor Stability By The Electron Spin Resonance Method. J. Am. Soc. Brew. Chem. 58, 8-13.

Varian Associates Technical Information Bulletin: Signal Amplitudes in Electron Paramagnetic Resonance, Fall 1965, pp. 10-13.

The environmental factors that influence the spins from one perspective are problems to be controlled, but also each becomes a topic that can be studied via the spins. These include temperature, viscosity, and other paramagnetic species, electrochemical potential, pH, time (kinetics) and so on. Many of these topics are discussed in the chapters in these two books, and new applications converting problems to insights can be expected in the near future.

The table lists a few of the over 100 defined EPR measurements. One such list is in a 1997 issue of the EPR Newsletter (Eaton and Eaton), and on the authors’ web site. Were one to put a check in each box for which there is at least one published experiment, there would still be many empty boxes. It is still true that at least 99% of all EPR spectra, whether for routine analysis or for research, in materials or in biomedical areas, are linear, CW X-band spectra, and most use a rectangular cavity resonator. However, for many problems one will achieve better insight into the sample using pulsed, time-domain, or non-linear CW EPR spectroscopy, and often frequencies other than X-band will be optimum, and we predict trends in this direction. Each type of experiment may become the crucial way to look at a particular problem.

Combining EPR with other physical methods provides yet a further dimension from which to view biological systems. In this concept we mean more than just hyphenated techniques such as liquid-chromatography-EPR or laser-excitation-EPR, as important as these are. We mean conceptually (and maybe graphically) overlaying MRI images and EPR images, or comparing molecular dynamics with macroscopic flow patterns. Analogous extensions might combine first-principles calculations and multidimensional EPR parameter measurements.

National EPR Centers, funded by the National Institutes of Health in the USA, and comparable Centers in Europe, provide access to specialized equipment and cutting-edge methodology, especially for biomedical applications. In late-2003, the U.S. Centers include the National Biomedical ESR Center in Milwaukee, which was discussed in the introduction to these volumes, the EPR Center for the Study of Viable Systems (Dartmouth), the Center for Imaging In Vivo Physiology (Chicago, Denver, and Baltimore), and the Advanced ESR Technology Center (ACERT) (Cornell). These Centers are funded to develop the technological advances that will define the future of biomedical ESR, and to make these tools available to researchers. Major EPR labs in the USA and other countries also help with specialized measurements. For example, the National High Magnetic Field Laboratory in Tallahassee, Florida, provides access to EPR at much higher magnetic fields and frequencies than are available elsewhere in the USA, and thus makes it possible to explore properties of spin systems hitherto unattainable. The most useful instrumentation and methodology developed in research labs will find its way into future generations of commercial EPR spectrometers. At present, the primary constraint on expansion of commercial EPR instrumentation is the limited size of the market. As the research community recognizes the importance of new techniques the capability will be come available. This process is well illustrated by the recent availability of pulsed ELDOR instrumentation and software to implement the DEER method for measuring distances between electron spins (see Biol. Magn. Reson. 19, 2000). One can anticipate the commercial availability of application-specific modules as accessories to EPR spectrometers, and the development of specialized spectrometers for measurements not supported by the standard spectrometers, such as the recently-released transient response spectrometer (Bruker E500-T).

The chapters in these two volumes already give a glimpse of the future, since they are written by researchers working to create better ways of learning about biological systems with EPR. There will be increasing use of multiple microwave frequencies, especially much higher and much lower than X-band, and of two-frequency (ELDOR) methods. The slow-scan, analog filter, lock-in detection methods that are almost universal in CW EPR

will be supplanted by many kinds of direct detection, direct digitization, and post-processing methods. Increasing ability to study aqueous samples will bring EPR methods closer to physiological conditions. Faster electronic components will reduce the dead time in pulse experiments and increase the digitization rates in CW and pulse experiments.

Resonators are the heart of the EPR spectrometer. The development of the loop-gap-resonator (LGR) was important in its own right, but arguably its most important contribution was to free us from the constraints of thinking about cavities. Now, a wide variety of resonant structures are being developed to optimize S/N for special samples, ranging from flat radiation dosimeter film to animals. Anything that the electrical engineer can design to resonate at the appropriate frequency potentially could be an EPR resonator for some application. Computer programs such as the High Frequency Structure Simulator (HFSS, Ansoft Corporation) will help refine intuitive approaches to nearly optimized resonator designs. As Jim Hyde pointed out in Volume 24 chapter 7, there are many types of resonators available for experiments at X-band, but no where near that variety at lower or higher frequencies. This is a challenge and an opportunity. Multifrequency EPR will grow in proportion to the availability of new resonators designed to facilitate special experiments.

Almost all EPR investigations, especially those of biological systems, ultimately are limited by signal-to-noise of available instrumentation, because the spin concentrations are inherently low, or the exploration of parameters requires a study of concentration dependence extending to very low spin concentrations. Some ways to approach the thermal noise limit of spectrometers are being explored. For example, using a low-noise preamplifier cooled to the same temperature as the sample helps reduce noise by the predictable amount (Rinard et al., 1999). A crossed-loop resonator makes it possible to place an amplifier right at the signal source even in pulsed EPR (Rinard et al. 1996a,b). The importance of minimizing noise as well as maximizing signal will result in the design of improved lownoise microwave sources, resonators specific to the sample type, as described by Hyde in this volume, and optimization of the location of the first stage amplifier as mentioned above. The result, which we term an “application-specific module” will not have the general applicability of the rectangular X-band cavity that has so dominated EPR in the past. However, building tools for specific problems will open new horizons for EPR.

Even magnets may not look the same in the future. At high microwave frequencies, superconducting magnets are required because iron-core electromagnets cannot achieve the required high magnetic field. Development effort for high-field EPR is mostly in national magnet

development facilities, such as in France (e.g., Isber et al., 2001), Japan (e.g., Motokawa 2000), and the USA (National High Magnetic Field Laboratory, Tallahassee, Florida) (e.g., Hassan et al., 2000). EPR at frequencies lower than L-band can use air-core magnets (e.g., Rinard et al., 2002). Some potentially useful resonators can be small enough that application-specific spectrometer systems can be envisioned with very small, easily portable, permanent magnets or electromagnets.

There are numerous vendors of accessories and supplies essential for various aspects of EPR measurements, including Oxford Instruments (cryostats), Cryo Industries (cryostats), Wilmad (sample tubes, quartz Dewars, standard samples), Oxis (spin traps), Molecular Specialties (loop gap resonators), Research Specialties (accessories and service), Summit Technology (small spectrometers, accessories), Scientific Software Services (data acquisition), Cambridge Isotope Labs (labeled compounds), and CPI (klystrons). Addresses and up-to-date information can be accessed via the EPR Newsletter.

Few EPR spectrometers have the degree of automation that has become common in other spectroscopy areas. Bruker has a dosimetry spectrometer with an automatic sample changer, and software to prepare reports. The latest pulsed EPR spectrometers are able to execute long (including overnight) multipulse experiments without operator intervention. The most recent cryogenic temperature control systems also can be programmed and can control the cryogen valve in addition to the heater. There is one research lab that has highly automated ENDOR spectrometers. Except for these examples, EPR has not been automated to a very significant degree. In the future, one can anticipate being able to run EPR spectra from a remote site if someone puts the sample into the spectrometer and sets up what ever environmental control (e.g., temperature, atmosphere over the sample) is needed for the experiment. Some applications of EPR will see broad use only if multiple samples can be studied with sufficient automation that high throughput can be attained.

Obtaining an EPR spectrum may be the easy part of EPR spectroscopy. Interpretation of the results often is the hard part. By this we mean not just the interpretation in terms of what species is present, or what the line shape or means about this species, but rather what the EPR reveals about the biological system. One aspect of this is to educate scientists who have expertise in both the spectroscopy and the biology, so that each can be interpreted without falling into traps. It is toward this end that these books have tried to give an overview, with references to other books and reviews to help researchers make the transition to modern biomedical ESR.