Three-dimensional electron spin resonance imaging

JOURNAL

OF MAGNETIC

RESONANCE

84,241-254 ( 1989)

Three-Dimensional Electron Spin Resonance Imaging RONALD K. WOODS, *GORANG.BACIC,*PAULC.LAUTERBUR,-~ ANDHAROLD M . SWARTZ* * ESR Center, SO6S. Mathews St., and t Biomedical Magnetic Resonance Laboratory, 1307 W. Park St., University of Illinois at Urbana-Champaign, College of Medicine, Urbana, Illinois 61801 Received September 20, 1988; revised December 28, 1988 Three-dimensional electron spin resonance images have been obtained by reconstruction from projections, permitting unambiguous determinations of the distribution of unpaired electrons in several complex objects. Examples of applications to several objects are presented. 0 1989 Academic Press, Inc.

Electron spin resonance imaging (ESRI) is an emerging new technique with unique capabilities to m a p the distribution of paramagnetic speciesin macroscopic systems. Simple two-dimensional imaging has been done with solid-state and biological samples, ranging from free radicals in mesophasepitch ( 1) to a tumor in a living mouse (2). In addition, various ESRI techniques have been reported such as spinecho-detected imaging and spectral-spatial ESR imaging (3, 4). Although most biological work has focused on in vitro systems, some in vivo investigations have been reported (2, 5). In earlier work, we used 2D ESRI to carry out studies of biological function in vitro, using, for example, the effects of oxygen on nitroxide metabolism, line broadening, and m icrowave power saturation to describe qualitatively its local concentration (6-8). ESRI does not have the general applicability of NMRI because of the infrequent occurrence of endogenous unpaired electron species in useful concentrations. The stable nitroxide free radicals, however, are useful for ESRI becausetheir distribution and kinetics and the shapesof their ESR spectra can provide information about important biological parameters such as oxygen concentration and redox metabolism. Satisfactory imaging of most objects, however, requires the use of three-dimensional techniques. In order to avoid this requirement, most prior ESRI studies have used sampleswith at least one axis of symmetry in order to facilitate the interpretation of image results. That is, a twodimensional image of a three-dimensional object necessarily superimposes information from regions above and below the imaging plane (assuming the data are in the form of plane integrals). W e show here that threedimensional ESRI can be implemented in a straightforward manner, using a slightly m o d ified apparatus to acquire plane integral data, and standard projection reconstruction techniques. Three examples are presented: a phantom containing six small samples of 2,2-diphenyl- 1-picrylhydrazyl (DPPH), a piece of y-irradiated quartz, and a segment of a rat femur soaked in “N-substituted perdeuterated Tempone (2,2,6,6-tetramethylpiperidine-d,6-N-oxyl-4-one, “N-PDT). 241

0022-2364189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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a

mm

Well Depths:

.7 mm

e-6.0

mm+

1-5.5

mm*

b .5 mm

x

C

2

/JY

FIG. 1. Schematic diagrams of (a) DPPH phantom, (b) irradiated quartz cylinder, and (c) rat femur showing the orientations in the laboratory frame.

MATERIALS

AND METHODS

ESR spectra. Data were obtained at X band (9.3 GHz) with a Varian E- 112 spectrometer, using a Varian TE- 102 cavity. First-derivative-mode projections were recorded with 100 kHz modulation. Incident microwave powers varied from 2 to 5 mW; modulation amplitudes varied from 0.5 to 4 G; scan ranges and scan times varied from 40 to 80 G and 6 to 20 s, respectively; and gradients varied from 25 to 70 G/cm. The specific parameters were selected on the basis of the requirements of the particular experiment.

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FIG. 2. Surface plots of planes selected from the 3D image of the DPPH phantom: (a) plane z = 24, (b) z = 34, (c) z = 44. Data acquisition parameters included microwave power = 5 mW, modulation amplitude = 0.5 G, scan range = 80 G, scan time = 10 s, and field gradient = 50 G/cm. The view is from the positive y direction.

Field gradient coils. A more complete description of the gradient coil assembly has been published (8). Briefly, it consists of circular z gradient coils mounted in an antiHelmholtz fashion upon the main magnet pole faces, upon which are also mounted paired rectangular transverse (x/y) gradient coils which can be manually rotated about the z axis. Gradients at any angle to the z axis are achieved by computercontrolled changes of the current in the z and x/y coils. Gradient angles in the x/y plane can be selected by rotating the x/y coils. The combined result is the ability to orient the projection axis at any angle. Unlike more familiar “slice-select” techniques, the method of volume excitation produces projections in the form of plane integrals, thus providing a higher degree of sensitivity. The current in the coils was kept constant by a homebuilt current stabilizer. In order to avoid effects of the gradient fields on the Hall probe, it was moved to the periphery of a main magnet pole face.

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WOODS ET AL. TABLE 1 DPPH Phantom Image Fidelity Measurements Intervala

Actual (mm)

Observed (arbitrary units)

Calculated calibration factor

Distance between cylinder centers A(Y)B WD C(xP NYK

3.6 5.2 3.0 1.8

18.0 22.5 14.5 8.5

0.20 0.23 0.21 0.21

Cylinder

Actual (mm)

Observed (mm) b (uncorrected)

Observed (mm)‘ (corrected)

Full cylinder width at half-height A B C D

1.0 1.0 2.0 1.0

1.03 1.10 1.97 1.05

0.91 0.98 1.90 0.93

u For example, A(y)B is the distance between they coordinates of the centers of cylinders A and B (see Fig. la). ’ Calculated using the experimental calibration factor. ‘Correction factors were obtained by convolving a Lorentzian function (width of 2 G) with the appropriate density distribution functions (cylinder shape).

Data collection and imagingprocedure. Data were collected and stored as arrays of 1024 points per spectrum using a Zenith Z- 100 dedicated computer with I / 0 Technologies analog and digital I/O boards. For each experiment, 19 projections were obtained for each of the 19 x/y coil settings at angles equally spaced between 0” and 17 lo, inclusive. Using 15-s scans, the total time for data collection was approximately 110 minutes. Spectra were integrated (in the case of rat femur, only the low-field line of the “N-PDT hyperfine spectrum was used) and then filtered using a three-point (second-difference) filter ( 9, IO). Two-stage back-projection reconstruction was then performed. Stage one produced 19 2D projections, and stage two created the final 3D image array from the 2D projections, as previously described for 3D NMR zeugmatography ( II ) . A description of 2D techniques can be found elsewhere (8). Materials. “N-PDT was purchased from MSD Isotopes, Los Angeles, California, and DPPH was purchased from Molecular Probes, Eugene, Oregon. Figure la is a schematic drawing of the DPPH phantom. The cube was constructed by assembling four polymethylmethacrylate plates (6.0 X 6.0 X 1.5 mm), three of which contained cylindrical cavities (radii = 0.5 or 2.0 mm and depths of 0.7 mm) containing DPPH crystals. Figure 1b is a schematic drawing of the quartz cylinder. The sample was yirradiated ( r3’Cs) at room temperature for approximately 40 hours (total dose = 100 kGy). Figure lc depicts the excised and thoroughly cleaned segment of rat femur. The bone segment was soaked in 1.O mM “N-PDT for 96 hours and blotted dry.

3D ELECTRON

a

SPIN IMAGING

b

d FIG. 3. Surface plots of planes, viewed from the negative y direction, selected from the 3D image of the DPPH phantom as compared to a 2D image viewed from the negative x direction: (a) plane x = 18, (b) s = 30, (c)x = 38, and (d) 2D image of the DPPH phantom. RESULTS

The results are presented as surface plots or gray scale images of planes selected from the 3D image arrays (65 3), corresponding to x/z, x/y, or y/z planes in Fig. 1. Figures 2a-2c show x/y image planes ( 13.6 X 13.6 mm) of the DPPH phantom at various values of z. Irregularities in the shape and intensity of the peaks are due to nonuniformity of the distribution of DPPH. In addition, 192 is a theoretically insufficient number of projections for reconstructing a 65 3 image. Table 1 lists image fidelity data for the x/y plane at z = 34. The average value of 0.2 1 m m /grid agrees quite well with the expected value of 0.20 m m /grid determined by the gradient, scan range, and effective range of the data used for the reconstruction. The values of cylinder diameter given by the image demonstrate the effect of convolving a density distribution with a broad spectral function (linewidth of the DPPH was approximately two gauss, peak-to-peak). Unless spectral deconvolution is performed, the spatial boundary of the density will necessarily be extended, as shown by the positive error in the observed uncorrected cylinder diameters. The appropriate comparison of the corrected diameters with the actual values leads to differences that are within experimental error. Figures 3a-3c depict y/z planes ( 13.6 X 13.6 mm) at various points along the x axis. It can be noted that the image reconstructed from 2D data (Fig. 3d) is a superimposition of information along the x axis. Figures 4a-4d are y/z image planes ( I 1 X 11 mm) of irradiated quartz at successive points along the x axis, while 4e-4h are x/z image planes ( 11 X 11 mm) at successive

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FIG. 4. Surface plots of planes selected from the 3D quartz image: (a) plane x = 16, (b) x = 22, (c) x = 28, (d) x = 34, (e) y = 20, (f) y = 32, (g) y = 44, (h) y = 48. Data acquisition parameters included microwave power = 2 mW, modulation amplitude = 4 G, scan range = 80 G, scan time = 6 s, and field gradient = 70 G/cm. The view is from the positive y direction (planes e-h have been rotated to show the internal surface ofthe cylinder).

points along the y axis. Image fidelity data were not obtained; however, the image qualitatively provides a good description of the internal and external geometries of the cylinder. The y/z image plane at x = 28 (Fig. 5a) and the image reconstructed from 2D data (Fig. 5b) provide another example of the superiority of 3D techniques. Figure 6 shows the x/z image plane ( 16.2 X 16.2 mm) of a rat femur at y = 28. The image provides an acceptable qualitative description of the femur and demonstrates the capability of the technique applied to systems with hyperfine lines in their ESR spectra.

3D ELECTRON

SPIN IMAGING

a

253

b

FIG. 5. Gray scale plots of the plane x = 28 selected from the 3D quartz image compared to a 2D image (projected on the yz plane): (a) plane x = 28, (b) 2D quartz image. The view is from the positive x direction. DISCUSSION

This paper describes a practical 3D ESR imaging technique. The data listed in Table 1 indicate that an accuracy of about kO.1 m m , sufficient for macroscopic samples, can be achieved with the apparatus and methods used. Several modifications should be made to achieve m icroscopic resolution. An electronically controlled threecoil gradient system would eliminate the error introduced by mechanically rotating the coil assembly as well as reduce the time required to collect the data. Sensitivity would be increased by replacing the rectangular cavity (TE 102) with a loop-gap resonator. In addition, larger gradients and more projections will be required to improve resolution. Deconvolution of broad spectral features would also increase resolution. An ESR m icroscope would be a valuable tool for materials research as well as for studying certain biological model systems. In particular, we plan to use

FIG. 6. Surface plot of the plane y = 28 of the 3D rat femur image. Data acquisition parameters included microwave power = 5 mW, modulation amplitude = 0.5 G, scan range = 40 G, scan time = 20 s, and field gradient = 25 G/cm. The view is from the negative zdirection.

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this method to perform microscopy on spheroids, an in vitro living model of a solid tumor ( 22). ACKNOWLEDGMENTS The authors thank Professor H. S. Ducoff for his help in irradiating the quartz sample and Mark Sandrock, operator of the VMS 1 1 / 780 DEC 4.1 system, for providing technical assistance as well as accommodating the computing demands of this work. Also, special thanks are owed to Professor R. Linn Belford and Dr. Robert Clarkson for the use of their computer facilities as well as accepting R.K.W. as a Summer Craig I Fellow. This research was partially supported by NIH Grant RR0 18 11 and by the Servants United Foundation. R.K.W. received support from the University of Illinois College of Medicine Research Fellowship Fund. REFERENCES 1. K. OHNO AND T. YOKONO, Carbon 24,s 17 ( 1986).

2. 3. 4. 5. 6. 7. 8. 9. IO. J 1. J2.

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