0020-711 X/92 $5.00 + 0.00 Copyright 0 1991 Pergamon Press plc

Inf. _I. Biochem. Vol. 24, No. 2, pp. 205-214, 1992 Printed in Great Britain. All rights reserved

IN VW0

ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY/IMAGING: FIRST EXPERIENCES, PROBLEMS, AND PERSPECTIVES SILVIA COLACICCHI,

Dipartimento

MARCO

FERRARI

and

ANTONELLO

SOTGIU

di Scienze e Tecnologie Biomediche e Biometria, Universita’ dell’Aquila, Collemaggio, 671O@L,‘Aquila, Italy [Tel. 39-862-433496; Fax 39-862-4334331 {Received 26 February 1991)

INTRODUCTION

Electron paramagnetic resonance (E.P.R.) spectroscopy permits the observation of species with one or more unpaired electrons. It has been applied in many fields of physics, chemistry, and biology, allowing the possibility of obtaining structural and dynamic information on paramagnetic species and their chemical environment. The use of X- or Q-band frequencies (9.5 and 35 GHz, respectively) has limited the observation to small samples, 1 or 2mm in size. Observation of larger samples has been hampered by losses in the electric and magnetic fields associated to the microwave radiation. In dipolar liquids, such as aqueous solutions, losses at X- and Q-bands are mainly due to the interaction between the electric field and the molecular dipoles. At lower frequencies power dissipation occurs through ionic currents set up by the magnetic field. Losses depend on the physical and geometrical characteristics of the sample, on the operating frequency and on the design of the cavity resonator. The first experiment of in vivo E.P.R. goes back to 1975 (Feidmann et al.). By chronically implanting an X-band transmitter in the form of a metallic helix into the rat liver, it was possible to collect the signal of an injected nitroxide spin label. The metabolic reduction caused a rapid decrease of the observed signal. Due to the invasivity and limited applicability of this procedure, no further in viuo experiments have been performed for more than a decade. The possibility of observing large biological samples was provided by the development of low frequency spectrometers and lumped parameter resonators (Froncisz and Hyde, 1982; Sotgiu, 1985). In these resonators the electric field is mostly confined in gaps or re-entrances and the sample region has the shape of a cylinder whose size is, to some extent, under designer control. This design is effective in reducing the losses in electric field, thus minimizing total power dissipation. Losses in the magnetic field cannot be avoided and the only way to reduce their effect is to lower the operating frequency. The major technological breakthrough has been, in fact, the adoption of frequencies EC 24,*--f

lower than 1 GHz permitting in vivo E.P.R. experiments. Few experiments have been reported at frequencies around 1.5 GHz, where the size of the samples were limited to the rat rail (Berliner and Wan, 1989) or whole mice weighing 15-20 g (Lukiewicz and Lukiewicz, 1984). Imaging techniques can add extremely useful information in the study of living systems and the development of in viva and imaging techniques are strongly interleaved. The easiest way to obtain spatial resolution is by adding one or more sets of gradient coils to an E.P.R. spectrometer. The spectra collected in the presence of magnetic field gradient contain information about the sample projections, and are used to perform image reconstruction. At frequencies below 1 GHz, i.e. in the radio frequency range, new characteristics are needed for some spectrometer components and the requirements of E.P.R. imaging could possibly push toward new technical solutions.

ELECTRONIC

VS NUCLEAR

IMAGING

The development of E.P.R. imaging occurs when the clinical applications of nuciear magnetic resonance (N.M.R.) imaging is able to give superb anatomic detail. Therefore some kind of comparison between the two imaging techniques is required. Up to now in vivo E.P.R. can only detect paramagnetic agents injected into animals up to a tissue concentration of close to 0.1 mM. This concentration is a factor lo6 lower than the molarity of the protons observed in N.M.R., thus affecting the intensity of the E.P.R. signal. Moreover, the linewidth of the studied paramagnetic radicals is, in the best case, lo3 times larger than the proton linewidth. Therefore, at parity of field gradients, N.M.R. experiments can more accurately monitor the spatial spin density. The larger linewidth of E.P.R. can be balanced by an increase of the values of the gradients necessary for imaging, but larger gradients are responsible for a further lowering of the sensitivity due to the spread of the resonance on the range of field values inside the sample volume. These drawbacks are partially compensated by the higher observation frequency of E.P.R. transitions, 205

206

SILWA COLACICCHI

the sensitivity being proportional to the square of the frequency, and by the shorter longitudinal relaxation time r,, because it permits an exchange of a larger amount of power between the microwave field and the sample. To have some insight on the relative sensitivity of the two techniques we can use the treatment developed by Abragam (1962). The signal to noise ratio (S/N) at the resonator’s ends can be written in the form

al.

which modify proton relaxation times. The trend is, in fact, toward the development of contrast agents which are specific to particular organs or tissues. On the other hand, nitroxide free radicals used for E.P.R. spectroscopy and imaging are in themselves very specific and a large amount of literature exists on their applications (see Piette and Hsia, 1979, for a review). Examples are the selectivity for tumoral tissue or the sensitivity to some local parameters such as pH or oxygen content (Swartz et af., 1986a). Moreover, E.P.R. spectra, being sensitive to molecular motion and chemical environment, give the possibility to extract information from their lineshape. In conclusion, E.P.R. spectroscopy/imaging might be able to distinguish more physical details than corresponding N.M.R. techniques. The existing literature, which will be discussed in the following paragraph, comprises of more than 10 papers on in vivo experiments and apparatus, and some papers on ex uiuo experiments such as isolated perfused heart, which constitutes a very useful experimental model. In addition, a large amount of literature exists on in vitro experiments, investigating biological and metabolic behavior of nitroxides and other free radicals in cells (Swartz et al., 1986b; Swartz, 1990).

where M represents either the nuclear or electronic magnetization and is proportional to the number of spins in the sample, 9 is the filling factor, Q the merit figure of the resonator, w the operating frequency, V, the resonator volume, p, the magnetic permeability of the empty space, k the Boltzman constant, T the absolute temperature, and df the observation bandwidth. Applying this formula to the experimental conditions of in vivo E.P.R. (sample volume = 45 cm3, Q 2 30, o = 300 MHz, d, = 1 Hz), a minimum detectable number of spins Nmin equal to 10” is obtained. This corresponds to a minimum detectable concentration of the order of lo-’ M. The observed concentrations reported in literature are in the range of 10e6 M. This can probably be explained by the operating conditions of low frequency E.P.R. spectrometers and will be discussed in the following paragraph. Due to the much higher proton concentration N.M.R. reaches the same sensitivity at a frequency of the order of a few hundred kHz. A different kind of comparison can be made on the basis of the specificity of the technique. N.M.R. imaging is, in fact. sensitive to water concentration, thus providing mainly morphologicai images. The diagnostic capabilities of the N.M.R. technique can be improved by the use of paramagnetic agents,

OR

et

EXPERIMENTAL APPARATUS

Microwave section

A traditional X-band E.P.R. spectrometer is composed of a microwave section, which includes a microwave source (usually a klystron), an attenuator, a circulator, a resonant cavity, and a diode detector (Fig. 1). Due to the field modulation, the receiving system is a lock-in amplifier, which acts as a narrow band filter. The design and characteristics of E.P.R. spectrometers are well documented, and the frequency scaling down necessary to perform in vivo or imaging experiments, at present, has not modified

HIBRID T

DETECTOR

b

I

I MICROWAVE SOURCE magnet

MODULATIDN

Fig. 1. Block

scheme of the E.P.R.

s~troscopy/ima~ing

apparatus.

fn viva E.P.R. spectroscopy this design. For frequencies in the range of 0.2-2 GHz the microwave sources are usually solid state oscillators and the sample cavities are lumped parameter resonators.

207

three-arm resonator of Fig. 2 the flux in the central arm is, in fact, the sum of the flux in the two lateral arms and q can be written as HfdV

Microwaue resonators

The sensitivity of an E.P.R. spectrometer is linearly dependent on the product ~IQ, where Q is the quality factor and q the filling factor of the resonator. For a homogeneous field distribution inside the sample region, 1 is proportiona to the ratio between sample and cavity volume. A conventional rectangular cavity operating at X-band, containing an aqueous sample, has a value of VQ product near 0.5. Lumped parameter resonators offer the feature of a great improvement in the filling factor, with a little decrease of Q, and can be designed to have an VQ product around 30. In X-band applications these resonators need a much lower microwave power to achieve a given field intensity. The lower Q, moreover, does not permit the demodulation of the phase noise of the generator, thus making easier the automatic frequency control which locks the oscillator to the cavity. Lumped parameter resonators can be broadly classified as loop-gap (Froncisz and Hyde, 1982), and re-entrant resonators (Sotgiu, 1985; Sotgiu and Gualtieri, 1985). Examples are shown in Fig. 2. The loop-gap type is composed of a cylindrical surface of conducting material with one or more gaps. The whole inner volume of the cylinder is available for the sample. This internal structure is surrounded by a metal shield which prevents irradiation; the coupling between the cavity and the bridge is obtained by a loop. In re-entrant cavity the electric field is almost completely confined in the space between the reentrances and the magnetic field in the surrounding arms. The centrat arm is used for the sample and one of the two lateral arms for coupling the cavity with the bridge. An important feature of this resonator is that by changing the size of the diameter b of the central arm with respect to that of the lateral arms a, it is possible to improve the filling factor. In the

(4

gap

2aZ

H2dV = 62 J cav

I

where H, is the microwave magnetic field and the integrals are calculated within the sample and cavity votumes. In case of arms of identical sections and for a sample size equal to the volume of the central arm, the filling factor is near 0.6 which is much higher than the filling factors obtained by traditional resonators. By increasing the ratio between the two diameters a and b, it is possible to achieve a filling factor near to 1. Losses Microwave energy has an electrical and a magnetic component, and can be dissipated by the samples through two different mechanisms: losses in the electric field due to the dielectric properties of the sample, and losses in the magnetic field which sets up ionic currents. The relative intensities of these two effects depends on the frequency and on the sample conductivity. At the frequency used in medical N.M.R. (5-20 MHz) the magnetic field is mainly responsible for losses, while in X-band E.P.R. spectroscopy (9.5 GHz) losses are always associated with the electric field. The maximum in the electric field losses occurs when the applied radio frequency has the same frequency as the natural mobility of molecules. For water, this maximum is very broad extending from 1 to 10 GHz, where rotations of dipolar molecules are excited (Hasted, 1973). As previously seen, the design of lumped parameter resonators has reduced these losses to a minimum by confining the electric field to gaps or re-entrances. A more serious problem is represented by the losses in the magnetic field. Electron spin resonance, in fact, is based on transitions induced by the magnetic

03

Fig. 2. Loop-gap resonator (A) and re-entrant cavity (B). a: diameter of the lateral arms; b: diameter of the central arm where the sample is positioned.

208

SILVIACOLACICCHI et al.

component of the radio frequency field. The ionic currents generated by the magnetic field, which dissipate power on the sample resistance, cannot be avoided. It has been shown (Gadian, 1979; Hoult and Lauterbur, 1979) that the power absorbed by a conductive sample of spherical shape is proportional to the 5th power of the radius and to the square of the operating frequency. Therefore, once the size of the sample is fixed, the only way to reduce losses is to lower the operating frequency. The lower limit is found at the frequency value where the sample dissipation is the same as that of the other circuit elements (mainly the cavity walls). By combining the frequency dependence of the losses with the general dependence of sensitivity on the square of operating frequency, one obtains that in a broad frequency range between 200 and 800 MHz the sensitivity is almost constant. The choice of the operating frequency in this range becomes indifferent, and must be done on the basis of other considerations, such as sample dimensions. Magnetic fields and gradients The magnetic field necessary to obtain electron paramagnetic resonance at frequencies between 250 and 800 MHz is of the order of lo-30 mT and could be obtained by air core solenoids. Some complexity is involved in the design of the coil system necessary to generate the three linear gradients of the main field B: cYBJ&, a&jay, dB,/az. A complete gradient assembly consists of three sets of coils, each dedicated to a single component of the field gradient. The component of the gradient along the z direction of the main field is obtained by two coils in Helmholtz configuration and reversed direction of the current. The two perpendicular components are each obtained by four coils of rectangular shape (Andersen, 1961). The intensity of the gradients, which can be obtained on large volumes using these coils, is in the order of 50 mT/m. These sets of coils have been adopted for the generation of gradients in small volumes. An increase of the sample dimensions requires a different geometry. Multipolar magnets (Sotgiu, 1986) give a completely different approach. They have a circular structure, with the poles lying inside a circumference and permit to generate both the main field and two of the gradients necessary for image reconstruction. By changing the currents on the individual poles it is possible to rotate the field direction around the axis of the magnet and at the same time to superimpose a gradient to the main field. Gradient intensities of the order of 200mT/m can be easily obtained. The first prototype of this magnet (Alecci, 1991), designed for low frequency E.P.R., has an internal diameter of 27 cm and can generate fields up to a value of 0.03 T. Reconstruction technique The use of the magnetic field gradients is needed to generate a linear distribution of magnetic field values in the sample. This causes a spread of the Larmor frequencies which are dependent on the spatial position in the sample. During a field sweep, the different locations give their contribution to the E.P.R. spectrum sequentially. The result is the projection of the

spatial spin density along the gradient direction. If only one gradient direction is used, the mono-dimensional image obtained represents the projection of the spin density along the gradient direction. More realistic images need the use of gradients in two or three spatial directions. The procedure for image data acquisition is the following: a fixed gradient vector is selected by adjusting the coil currents, and it is superimposed to the main field. A sequence of spectra is collected changing the direction of the gradient vector each time, according to a raster in polar coordinates. For a two-dimensional (2D) image all the gradient directions are laying on a plane, and for a three-dimensional (3D) image in the three spatial dimensions. The last process (3D) thus requires a larger number of data, but it is the only way to reconstruct the real spin density. In fact, with this method the 2D images are merely projections on a plane of the object density function. Once the process of collecting data is accomplished, each projection is deconvoluted with the zero gradient line. This step is necessary because E.P.R. lines are very broad and the spatial reconstruction without this step, as already seen, would require very high gradient values with a further lowering of the sensitivity. The deconvolution process is also necessary to avoid overlapping distortion caused by spectra hyperfine structure. In fact, the nitroxides commonly used in E.P.R. imaging show a typical triplet structure. The ideal result of this process would be that of restoring the true projections of the sample by the elimination of all the contributions from the natural lineshape. The imaging method, which fits this field generation technique, is the reconstruction from projections, called the Fourier reconstruction method. This algorithm is based on the fact that the 3D Fourier transform of the unknown spin density and the set of projections are Fourier pairs (Mersereau and Oppenheimer, 1974). Some examples of E.P.R. images will be introduced later. IN VW0

EXPERIMENTS

Spin probes Many paramagnetic species are naturally present in living organisms at very low concentrations. Most of them cannot be observed by presently available low frequency E.P.R. instrumentation, which is capable of detecting only substances with an in situ concentration around 10-4-10~5 M. Among the physiological paramagnetic substances, melanin has long been known to have a stable radical which yields a strong E.P.R. signal. This radical has been detected by a surface coil in a mouse experimen-, tal tumor (Lukiewicz and Lukiewicz, 1984). Interest has been focused on melanin because it can undergo redox reactions with many molecules such as transition metal ions and molecular oxygen. A study of the melanin relaxation times should be able to provide information on oxygen tension making this radical a useful probe for measuring tissue oxygenation. Recently, melanin E.P.R. imaging was per-

In uivo E.P.R. spectroscopy formed on a pigmented amphibian skin preparation during hypoxia (Vahidi et al., 1989). The development of in vivo E.P.R., however, has been strictly related to the use of nitroxide free radicals and to the knowledge of nitroxide metabolism (Swartz, 1990). The most commonly employed spin labels are reported in Fig. 3. In vitro measurements have been performed on single cells and homogenates. An impressive series of ex uivo experiments have been performed to observe signals on fragments of tissue, blood, or in the perfusion medium of isolated perfused organs or in the perfused organ itself such as Langendorff perfused heart (Zweier and Kuppusamy, 1988; Monti et al., 1990). These experiments suggest that the possibility to detect free radicals and/or spin traps in situ might give a relevant contribution to many open questions of organ and system pathophysiology. Nitroxides have been used as probes of oxygen concentration and as active agents in E.P.R. and N.M.R. spectroscopy/imaging. E.P.R. oximetry can be obtained in two different ways. The more direct is the measure of the relaxation times of the paramagnetic probe induced by the presence of the oxygen magnetic moment. Alternatively, the catabolic reduction rate of several nitroxides has shown a relation with oxygen concentration (Swartz et al., 1986a). Many studies have been performed to understand nitroxide pharmacokinetics on isolated cells; their indication, however, should be confirmed by in vivo experiments. In vivo most of the extracellular reduction of nitroxides is due to ascorbic acid. Intracellularly, nitroxide reduction is an enzyme-associated mechanism which depends on their structural properties (Swartz et al., 1986b; Chen et al., 1988). In isolated cell culture, the main sites of reduction should occur at the level of mitochondria and microsomes. Ascorbate free cytoplasm, in fact. has a very low reducing activity for nitroxides.

209

Liposomes have been extensively employed as in vivo delivery vehicles for their stability in the circulating blood. Nitroxide liposome systems have been explored in order to avoid the rapid reduction of nitroxide in the extracellular space and the diffusion of free nitroxides from the monitored organ. Liposomes should protect nitroxides from reduction and provide more stable E.P.R. signals. Recent studies suggested that temperature also strongly affects nitroxides biological half-lives (Lukiewicz et al., 1989). The reduction rate of some nitroxides was measured in different types of cells (Iannone et al., 1989). The reduction depends on the chemical structure and the oxygen concentration. A reduction rate of 6-fold higher was found in the absence of oxygen. The six-membered piperidine ring is reduced faster than the corresponding five-membered pyrroline ring. In addition, the difference in the location of the nitroxide functional group on the molecule and the degree of lipophilicity affect their biodistribution and reduction rate. By monitoring the spin label catabolism of suitable nitroxides an accurate measurement of in situ oxygen concentration could be obtained. The use of these properties to obtain a quantitative oximetry on living tissues should be simplified by a linear relation between the reduction rate of the probe in the expected physiological range of oxygen concentration. Hydroxylamines are the main products of nitroxide metabolism, but both the species can quickly exchange electrons oxidizing the hydroxylamines and reducing nitroxides to hydroxylamines. The mechanism of oxidation of hydroxylamines probably involves cytochrome-c-oxidase and the rate of oxidation increases with increasing concentrations of oxygen (Chen et al., 1989). It is then clear that, in Go, the nitroxide/hydroxylamine equilibrium is different for different types of cells and tissues and depends on the pathophysiological condition of the living animal CHo

CAT1

TEMPO

OH

CHs

Al .,

TEMPOL

COOH

PCA

CHs CHs

CHo

0

TEMPONE CHo CHa Fig. 3. Most commonly employed spin labels.

CONHa

CTPO

210

SILVIACOLACICCHI et al.

An attempt in the direction of discriminating tissues with different degrees of oxygenation is the in vivo imaging of a mouse injected with an oxygen sensitive spin label recently performed by Halpern et al. (1989a,b) at very low frequency. For the imaging application, deuterated spin labels have been proposed as a tool to improve sensitivity and resolution. The effect of using perdeuterated labels is that of reducing the separation of the proton’s unresolved superhyperfine structure of a factor proportional to the ratio of their gyromagnetic ratio. This translates to a linewidth reduction of about a factor 6 with a corresponding increase in sensitivity. Many other nitroxides for selective localization inside cells have been recently developed (Hu et al., 1989) and two new classes of paramagnetic materials: fusinite and phathalocyanine have been recently proposed (Clarkson et al., 1990; Swartz er al., 1990) for their high sensitivity to low oxygen concentration. In vivo spectroscopy/imaging Feldman first recorded an in vivo spectrum of TEMPOL nitroxide using traveling wave helices implanted in the left lobe of the rat liver (Feldman et al., 1975). More recently, another in vivo experiment using an invasive technique was reported by Subczynski et al. (1986). An oxygen permeable capsule containing a deuterated spin label was implanted in the mouse peritoneal cavity. E.P.R. spectra recorded from the capsule, using a loop-gap resonator resonating at 1.1 GHz, were found sensitive to changes of inspired oxygen tension. More recently, E.P.R. spectra were recorded in vivo without any surgical intervention. Reliable spectroscopic and kinetic data were obtained from mouse liver using a surface coil operating at 1.1 GHz with sufficient sensitivity and stability. The kinetics of Tempone. Catl, and PCA nitroxides were measured and their different half-lives were determined (Bacic et al., 1989). By inserting different parts of the mice in the active region of a re-entrant cavity Ferrari et al. (1990) were able to obtain the uptake, distribution, and reduction of the oxygen sensitive nitroxide PCA. Data was obtained from the liver and the head of pentobarbital anesthetized mice during different circulatory and ventilatory conditions. Figure 4 shows a typical clearance of the PCA from the heads of the mice. In this study it was impossible to find any significant variation in the reduction rate during moderate hypoxia. Dynamic studies of CTPO and TEMPOL in rat heads were reported by Ishida et al. (1989) using a loop-gap resonator having an inner shield in the loop operating at 760-820 MHz. CTPO clearance was measured also on rat tail using a rectangular twoarm, one re-entrance cavity resonator operating at 1.4GHz (Berliner et al., 1989). Rat tail and CTPO were used by Alecci ef al. (1990) to report first a 3D E.P.R. image at 1.2 GHz. The reconstruction procedure for 32 x 32 x 32 tail slices required 25 min and was capable to resolve the tail vascular and anatomical structures. The right panel of Fig. 5 shows a 2D image of the rat tail when the CTPO radical was diffused through all the tail tissue. An annular ring of similar size, containing 10m4M

CTPO, is shown in the left panel. Both images clearly indicate the presence of reconstruction artifacts in the background. This kind of mathematical noise is probably due to the deconvolution and reconstruction algorithms, required by the E.P.R. data, and should be improved by further software development. A less accurate E.P.R. imaging experiment was reported previously by Berliner et al. (1987). A melanoma tumor implanted into the mouse tail was studied using a flat loop single turn coil resonating at 1.55 GHz. CTPO was administered for several hours and four cross-sectional projections were performed in 10 min. These data suggest that E.P.R. might be a useful tool to study in vivo biological properties of experimental tumors. The reduction rates of several nitroxides were compared on tumors grown in mouse muscle using a 1.1 GHz spectrometer equipped with a loop-gap surface coil (Glockner et al., 1989). An S-band (24GHz) spectrometer adapted with a loop-gap resonator was used to monitor TEMPOL concentration on B- 16 murine melanoma implanted into the mouse tail and treated with hypertermia (Lukiewicz et al., 1989). The biological half-life of the spin label increased by about 250% between 21 and 44°C and increased 200% when the mouse was breathing pure oxygen. In general, a faster reduction rate in respect of normal cells was found in tumors, probably because tumor cells are hypoxic. Halpern et al. (1989b) first were able to perform a 2D image from a whole mouse using a spectralspatial back projection algorithm. The tetraperdeuterated spin probe 4-hydro-3-carbamoyl-2,3,5,5tetraperdeuteromethyl pyrrolidin- 1-yloxy (MhCTPO) was injected intraperitoneally by bolus followed by a continuous infusion. An image could be obtained by 32 projections in about 60min. In spite of the importance of these experiments, preliminary studies on simpler models, as isolated organs, permit a more accurate evaluation of spin label kinetics. Zweier and Kappusami (1988) first applied an L-band E.P.R. instrument to measure free radicals in the beating rat heart. The spectrometer consisted of a l-2 GHz microwave bridge with a source locked to the resonance frequency of a loopgap resonator and permitted to observe a minimum radical concentration of 0.4 p M in aqueous solution. The kinetic of TEMPOL free radical uptake and metabolism was studied in normally perfused and globally ischemic hearts. Moreover, by monitoring the changes in the width of the observed E.P.R. signal, which depends on paramagnetic broadening due to oxygen, they were able to determine the myocardial oxygen concentration. Similar work was performed by Rosen et a/. (1988), Baker et al. (1990) and by ourselves (Gallo et al., 1990). Developmental trends The present applications of low frequency E.P.R. spectroscopy and imaging have shown that these are the bases to address a large variety of medical and biological problems. In our opinion the main technical development that must be done to increase sensitivity, particularly for imaging applications, is the adoption of pulsed techniques. They should permit to reduce the acquisition time of at least an order of

Fig. 4. Clearance curves of the PCA triplet central line from mouse head after 0.33 mM intravenous injection. The 18 curves are taken consecutively every 150 sec. Scanning time was 26 set and time constant 125 msec. The inset shows the typical PCA triplet. The square indicates the central peak selected for clearance studies.

min_

SILVIA COLACICCHI et al.

212

rat tail 10 min after

1 mM CTPO

intravenous

infusion

beginning.

Collection

time about

2 min.

In vioo E.P.R. spectroscopy magnitude. In this way E.P.R spectroscopy and imaging might give a relevant contribution to the study of tissue oxygenation and free radicals in situ detection in many medical areas. In addition, other different areas might be explored, such as drug distribution and biological effects of hypertermia. It has been shown how the major development in viva E.P.R. spectroscopy and imaging has been the adoption of frequencies well below 1 GHz. It is now clear that the ranges of low frequency E.P.R. imaging and that of high frequency N.M.R. (which is in the range of 200-400 MHz) are now overIapping. The adoption of pulsed techniques would make the development of E.P.R. spectroscopy similar to that of N.M.R. The reason for which pulsed techniques have not yet been adopted is that the relaxation times of the paramagnetic species of biological relevance are very short and require extremely fast acquisition rates and a high pulsed power that can be delivered by a travelling wave tube (TWT) microwave amplifier. The only commercial unit for pulsed E.P.R. presently available has acquisition rates of the order of 100 megasamples/sec and a 1 kW pulsed power. This level of power can be obtained in a much easier way and with more reliable solid state technology at lower frequencies. Therefore, a pulsed technotogy could be developed in an easier way at frequencies below 1 GHz. In a large variety of scientific and commercial applications, moreover, the technology pushes toward a very fast acquisition rate so that one of the difhculties of the pulsed technology should be easily overcome. Different developments can come from the adoption of double resonances. Proton electron double resonance imaging (PEDRI) has been used for imaging spin labels in uivo in the rat (Nicholson et al., 1990). Images of the nitroxide free radical PCA clearance have been obtained from a rat kidney by a whole body N.M.R. imager operating at both E.P.R. and N.M.R. frequencies (Grucker, 1990). The importance of this technique is that it could be capable of providing evidence of the presence of paramagnetic species, even if these could not be observed by E.P.R. An example is given by paramagnetic ions such as gadolinium, which has been adopted as contrast agents in N.M.R., but cannot be observed at room temperature by E.P.R. techniques. Acknowledgements-This

research was supported in part by

CNR connibution 89.0256504, by grants from ‘INFN, INFM-GNSM. and bv a contribution of Bracco. Industria Chimica. This research has been performed in the framework of the “Centro di Ricerca Interuniversitario Studio dei meccanismi molecolari coinvolti nel danno tissutale da ipossia e iperossia e di molecule the modificano tali lesioni”. REFERENCES

Abragam A. (1962) The Principies of Nuclear Magnetism. Oxford Univ. Press. Alecci M., Gualtieri G., Sotgiu A., Testa L. and Varoli A. (1991) Multipolar magnet for low-frequency ESR imaging. Meas. Sci. Technoi. 2, 32-31. Alecci M., Colacicchi S., Indovina P. L., Momo F., Pavone P. and Sotgiu A. (1990) Three-dimensional in viuo ERS

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Andersen W. A. (1961) Electrical current shims for correcting magnetic fields. Rev. Sci. Instrum. 32, 241-250. Bacic G., Nilges M. J., Magin R. L., Walczak T. and Swartz H. M. (1989) in km localized SRS spectroscopy reflecting metabolism. Magn. Resan. Med. 10, 266272. Baker J. E., Froncisz W. and Kalyanaraman B. (1990) Direct measurement of myocardial oxygen consumption during ischemia by spin label oximetryin the intact heart. Free Rad. Biol. Med. 9. Sl. 93.

Berliner L. J. and Wan Xl (1989) In viuo pharmacokinetics by electron magnetic resonance spectroscopy. Magn. Reson. Med. 9, 430-434.

Berliner L. .I., Fujii H., Wan X. and Lukiewicz S. J. (1987) Feasibility study of imaging a living murine tumor by electron paramagnetic resonance. Magn. Reson. Med. 4, 380-384.

Chen K., Morse P. D. and Swartz H. M. (1988) Kinetics of enzyme-mediated reduction of lipid soluble nitroxide spin labels by living cell. Biochim. biophys. Acta 943, 477484.

Chen K., Glockner J. F., Morse P. D. and Swartz H. M. (1989) Effects of oxygen on the metabolism of nitroxide spin labels in cells. Biochemistry UI, 24962501. Clarkson R. B., Boyer S. J., Wang W., Nilges M. J., Swartz H. M. and Gast P. (1990) Two new chemical probes for in vivo E.P.R. oxymetry. Society of Magnetic Resonance in Medicine, Ninth Annual Scienttfic Meeting, New York, p. 1324. Feldman A., Wildman E., Bartolinini G. and Piette L. H. (1975) fn viva electron spin resonance in rats. Phys. Med. Biol. 20, 602-612. Ferrari M., Colacicchi S., Gualtieri G., Santini M. T. and Sotgiu A. (1990) Whole mouse nitroxide free radical pharmacokinetics by low frequency electron paramagnetic resonance. Biochem. biopkys. Res. Cammun. 166, 1688173. 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.

Gadian D. G. (1979) Radiofrequency losses in NMR experiments on electrically conducting samples. J. Magn. Res. 34, 449455.

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