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Description  |
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FIELD OF THE INVENTION
This invention relates generally to apparatus and methods for determining
oxygen tension in biological systems. More particularly, the invention
concerns apparatus and methods for determining oxygen tension in
biological in vivo tissue utilizing physiologically acceptable
paramagnetic materials and electron paramagnetic resonance oximetry.
BACKGROUND OF THE INVENTION
Benefits derived from the measurement of oxygen concentrations in tissue
are known. Oxygen is the primary biological oxidant, and the measurement
of oxygen tension pO.sub.2 (positive pressure of oxygen) can improve the
evaluation and understanding of many physiological, pathological, and
therapeutic processes.
Prior art systems and methods for measuring oxygen concentrations in tissue
are also known, including: the Clark electrode, fluorescence quenching,
O.sub.2 binding to myoglobin and hemoglobin, chemiluminescence,
phosphoresence quenching, and spin label oximetry. However, these systems
and methods have certain, and often acute, limitations, especially when
used in vivo. They especially lack the qualities required for complete
experimental and clinical use, such as sensitivity, accuracy,
repeatability, and adequate spatial resolution. See J. Chapman, Radiother.
Oncol. 20, 13 (1991) and J. M. Vanderkooi et al., "Oxygen in Mammalian
Tissue: Methods of Measurement and Affinities of Various Reactions", Am.
J. Physiol. 260, C1131 (1991).
The polarographic microelectrode is one popular device for measuring oxygen
tension in tissue. However, it has obvious technical difficulties
associated with the repeated insertion of the microelectrode into the
tissue. For example, the microelectrode often damages the tissue, and
there is repeated difficulty in re-positioning the microelectrode at the
same test location. The microelectrode is also relatively insensitive to
oxygen concentrations below 10 mm Hg, which is within the required
sensitivity region for effective oximetry. Finally, the microelectrode may
itself consume oxygen, thereby altering its own environment, inducing
measurement errors, and reducing the accuracy and usefulness of the
evaluation process.
There are scattered reports which concern in vivo pO.sub.2 measurements
with such devices, especially in skeletal muscle. Whalen and Nair, Am. J.
Physiol. 218, 973 (1970), measured pO.sub.2 of cat gracilis at rest using
a recessed Au 1-51 .mu.m microelectrode, giving average pO.sub.2 values of
6.6.+-.0.4 mm Hg (n=372). Gayeski et al., Am. J. Physiol. 254, H1179
(1988), measured pO.sub.2 of dog gracilis at rest, exhibiting a partial
pressure range of 4.5-35 mm Hg (16.8 mm Hg median), and 95% VO.sub.2 max,
using a Mb saturation technique, exhibiting a partial pressure range of
0.2-2.3 mm Hg (0.9-1.8 range of mean). Nevertheless, there are effective
limitations to these pO.sub.2 measurement techniques. In the
microelectrode method, for example, it is technically difficult to monitor
or make long term evaluations of pO.sub.2. In the Mb saturation method, it
is especially difficult to measure low pO.sub.2, and the method can only
be used in muscle.
Nuclear Magnetic Resonance (NMR) techniques have been explored and
considered in the context of oxiometric measurements, especially through
the use of an oxygen dependent proton hyperfine line in myoglobin and
oxygen dependent relaxation of fluorine nuclei. NMR is a common
spectroscopic technique in which the molecular nuclei is aligned in a
magnetic field and simultaneously excited by absorption of radiofrequency
energy. The molecular relaxation from the excited state to the initial
state is an observable event that is affected by the presence of oxygen
through exchange or dipolar actions. However, the NMR techniques have not
demonstrated sufficient sensitivity and/or applicability to the measure of
pO.sub.2 in either experimental or clinical settings.
Electron Paramagnetic Resonance (EPR) oximetry is another technique for
measuring oxygen tension. Similar to NMR, EPR oximetry is a spectroscopic
technique based upon the Zeeman effect and the line-broadening effect of
molecular oxygen on the EPR spectra of paramagnetic materials. These
materials have unpaired electron spins that are aligned in a magnetic
field and excited by microwave energy. The separation between the lower,
unexcited energy state and the higher, excited energy state is
proportional to the strength of the magnetic field. The presence of oxygen
with the excited molecule measurably affects the molecular relaxation so
that the line width of the EPR spectra changes and provides an indication
of pO.sub.2.
Nitroxides exemplify one family of compounds having paramagnetic quality
that are suitable for EPR oximetry, and which have been used in a variety
of in vitro experiments. Although nitroxides have also been tested in
vivo, at least two resulting problematic areas exist in such measurements:
first, nitroxides tend to be bioreduced; and secondly, nitroxides are
relatively insensitive to low oxygen tension levels that are of the most
biological interest today, i.e., less than 10 mmHg.
Other recent discoveries of new paramagnetic materials, such as Fusinite
and lithium phthalocyanine (LiPc), have made progress as oxygen probes in
the field of in vivo EPR oximetry. These two compounds, for example, are
suitable for in vivo usage because they exhibit certain favorable
characteristics, including: accuracy; spatial resolution; sensitivity in
the physiologically important oxygen tension range; ease of use; little or
no apparent toxicity; and relative stability in tissues, permitting
prolonged measurements over periods of weeks or months after administering
the compound. Nevertheless, because these paramagnetic compounds have not
been previously tested in humans, they will have to undergo very long and
extensive toxicological evaluation before they can be used clinically.
This evaluation is likely to be prolonged because of other problems
inherent in the compounds, such as stability and inertness, which
encourage indefinite, unwanted persistence within the tissue.
There are other existing problems limiting the effectiveness of EPR
oximetry, including the inability to measure EPR spectra efficiently and
effectively, especially in vivo. Conventional EPR spectrometers, for
example, typically utilize microwave frequencies, e.g., 9 GHz, that are
strongly absorbed by tissue and water, and which reduce the useful depth
penetration and measurement sensitivities within the tissue. Prior EPR
spectrometers also cannot effectively measure EPR spectra from a
biological system such as a live animal, because movements of the animal
change the observed EPR spectra. This movement increases noise and reduces
the accuracy. Finally, conventional EPR spectrometers have the resonator
and the sample under test, e.g., tissue, within a common magnetic field.
This constrains the EPR measurement/flexibility, being subject to physical
size considerations, and potentially to the patient's dexterity.
It is accordingly an object of this invention to provide an improved EPR
spectrometer and associated methodology that are free of the
afore-mentioned difficulties.
It is another object of this invention to provide an improved apparatus and
method that enables the direct measurement of oxygen concentration in
biological systems, such as tissue.
It is a further object of the invention to provide improved methodology and
apparatus for in vivo EPR oximetry.
Other objects of the invention will be apparent from the following
description.
SUMMARY OF THE INVENTION
The invention attains these and other objects, according to one aspect, by
providing a method for evaluating oxygen tensions in a biological system,
including the steps of (1) introducing physiologically acceptable
paramagnetic material to the biological system, (2) applying a magnetic
field and an electromagnetic field to the biological system, and (3)
determining the EPR spectra of the biological system. The paramagnetic
material is of the type which has an EPR spectra responsive to the
presence of oxygen, such as India ink, constituents of India ink having
paramagnetic quality, carbon black, and other carbon-based material. The
biological system includes in vivo and in vitro biological systems,
biological tissues, cells, cell cultures, animals, and live human beings.
In another aspect, the method provides for the step of calibrating the EPR
spectra of the paramagnetic material by comparing the EPR spectra of the
biological system with the EPR spectra of the paramagnetic material in the
presence of a known concentration of oxygen. Preferably, both the measured
spectra from the biological system and the calibration spectra are
determined by the spectra's peak-to-peak line width. The peak-to-peak line
width indicates oxygen tension in the biological system, and oxygen
concentration is determined directly by comparing the measured line width
to the calibration line width.
In other aspects, the method provides for sweeping the magnitude of the
magnetic field between approximately 100 and 500 Gauss to acquire the EPR
spectra through the frequencies of the EPR resonance. The step of sweeping
preferably occurs in less than 60 seconds.
In another aspect, the magnetic field includes a first magnetic field
having lines of force in substantially one direction, and the method
provides for applying a second magnetic field to the biological system
that is substantially parallel to the first magnetic field. The second
magnetic field is thereafter slowly varied to modify, or sweep, the
magnitude of the first magnetic field between approximately 1 and 500
Gauss, to acquire the EPR spectra through the EPR resonance frequencies.
Alternatively, an electromagnet is employed to sweep the magnetic
intensities. Preferably, a third magnetic field is applied to the
biological system that is substantially perpendicular to the first
magnetic field. The third magnetic field is modulated between
approximately 1 and 500 kHz, to improve the signal-to-noise ratio for
determining the spectra. Preferably, the electromagnetic field applied to
the biological system is directed substantially perpendicular to the first
magnetic field with an oscillating frequency between approximately 100 MHz
and 5 GHz, such as in the microwave L-band.
In still another aspect, the method includes the step of determining the
EPR spectra by utilizing an EPR spectrometer that has a resonator and an
associated Q factor. The Q factor is determined and monitored for change,
such that, in another aspect, the Q factor is compensated to maintain
resonant frequency during movements by the biological system, e.g., the
tissue or animal.
The method in accordance with the invention also provides for introducing
to the biological system a paramagnetic material that has substantially
uniform particles with diameters between approximately 0.1 and 100
microns. Alternatively the paramagnetic material can include at least one
relatively large particle with a diameter between approximately 100
microns and one centimeter. This relatively large paramagnetic particle
functions as a point source to spatially determine the EPR spectra in the
biological system.
In other aspects according to the invention, the paramagnetic material is
introduced to the biological system by several appropriate methods. In
tissue, for example, the material can be injecting directly into the
biological system. If the biological system has a circulatory blood
stream, the paramagnetic material can be introduced directly into the
blood stream. Accordingly, the method can include the further steps of (1)
changing the blood flow to the biological system or tissue, and (2)
determining the change in the EPR spectra to provide a real-time
evaluation of the change in oxygen concentration in the tissue.
Additionally, the blood flow to the tissue can be reduced to reduce the
oxygen concentration in the tissue.
The paramagnetic material can also be introduced to the biological system
via lymphatics. To derive additional spatial information, the paramagnetic
material can also be selectively introduced to a localized region within
the biological system, thereby indicating oxygen tension at the localized
region. Alternatively, the paramagnetic material is introduced to a
biological system having phagocytic activity, such that the paramagnetic
material is introduced to the biological system by phagocytosis.
The invention also provides for a method to determine EPR spectra of a
biological system having a surface. When the biological system has a
surface, e.g., the skin of an animal, the EPR spectra is preferably
determined from the surface. In other aspects, an EPR resonator
constructed in accordance with the invention for use with an EPR
spectrometer directly measures EPR spectra from the surface.
In another aspect, a method is provided for evaluating oxygen tension in a
cell. Physiologically acceptable paramagnetic material--which has an EPR
spectra responsive to the presence of oxygen--is first introduced to the
cell, such as through phagocytosis. A magnetic field and an
electromagnetic field are then applied to the cell, and the peak-to-peak
line width of the EPR spectra of the cell is determined. The paramagnetic
material can include carbon black, carbon-based material, India ink, or
ingredients of India ink having physiologically acceptable paramagnetic
quality. The electromagnetic field preferably has a frequency between
approximately 100 MHz and 5 GHz.
The method additionally provides for the steps of determining the EPR
spectra peak-to-peak line width of the paramagnetic material in the
presence of a known concentration of oxygen. The spectra from the known
concentration of oxygen is then compared to the spectra of the cell to
determine the oxygen tension present in the cell.
The invention also provides a system for determining oxygen concentrations
in biological systems, including (1) physiologically acceptable
paramagnetic material in the biological system, and (2) an EPR
spectrometer to determine the EPR spectra of the biological system. The
paramagnetic material can include India ink, an ingredient of India ink
having physiologically acceptable paramagnetic quality, carbon-based
material, and carbon black. The biological system can be in vitro and in
vivo biological tissue, biological tissue having phagocytic activity, one
or more phagocytic cells, living animals and humans. The paramagnetic
material is introduced to the biological system via an appropriate manner,
including: direct injection into the biological system; direct injection
into the blood stream; via lymphatics; and through ingestion.
Preferably, in another aspect, the system includes means for determining
the peak-to-peak line width of the EPR spectra. This line width is then
compared with the peak-to-peak line width of the EPR spectra of the
paramagnetic material in the presence of a known concentration of oxygen.
A system according to the invention also preferably includes a magnet, for
applying a magnetic field to the biological system, and means for sweeping
the magnitude of the magnetic field between approximately 100 and 500
Gauss. The magnitude is typically varied in a period less than sixty
seconds.
In another aspect, a system according to the invention includes means,
e.g., a magnet or an electromagnet, for applying a first magnetic field to
the biological system that has lines of force in substantially one
direction. The system further has means, e.g., a magnet or an
electromagnet, for generating a second magnetic field with lines of force
substantially parallel to the first magnetic field to modify and sweep the
magnitude of the first magnetic field between approximately 1 and 500
Gauss. Preferably, the system has means for generating a third magnetic
field, with lines of force substantially perpendicular to the first
magnetic field, wherein the third magnetic field is modulated between
approximately 1 and 500 kHz to improve the signal-to-noise ratio of the
measured spectra.
In still another aspect, the system has an oscillating electromagnetic
source for applying electromagnetic radiation to the biological system.
The electromagnetic radiation, preferably within the range 100 MHz to 5
GHz, such as the L-band microwave frequencies, is directed to the
biological system and is substantially perpendicular to the magnetic
field.
In still another aspect according to the invention, the EPR spectrometer
has a resonator and means for determining the resonator Q. Preferably, the
resonator Q is compensated in response to movements of the biological
system to maintain the resonant frequency.
In other aspects, the paramagnetic material of the system is substantially
uniform, with particle diameters between approximately 0.1 micron and 100
microns. The paramagnetic material can also be one or more relatively
large particles with diameters between approximately 100 microns and one
centimeter. These relatively large particles function much like a point
source for the spectra in the biological system. In one aspect, for
example, the paramagnetic material is localized within the biological
system, thereby providing a selectable spatial indication of the oxygen
tension in the biological system.
In other aspects, the system provides means to determine the EPR spectra
directly from the surface of the biological system, e.g., the skin of a
human. If the biological system is biological tissue having a circulatory
blood flow, the system can include means for changing the blood flow to
the tissue and means for determining the change in the EPR spectra,
thereby providing a real-time evaluation of the change in oxygen
concentration in the tissue. Accordingly, the system can also include
means, e.g., a tourniquet, for reducing the blood flow to the tissue to
reduce the oxygen concentration at the tissue.
The invention also provides, in another aspect, a spectrometer for the in
vivo measurement of oxygen concentration in tissue. The spectrometer
includes (1) magnets for selectively applying a magnetic field of
selectable strength to the tissue, (2) electromagnetic oscillator for
selectively applying electromagnetic radiation having a frequency between
approximately 100 MHz and 5 GHz to the tissue, (3) detector for detecting
the electron paramagnetic spectra of the tissue, (4) resonator arranged to
maintain a substantially constant resonant frequency, (5) console in
communication with the detector for displaying the EPR spectra, and (6)
computer connected to the console for controlling the spectrometer, and
for analyzing the EPR spectra.
Preferably, the resonator includes an automatic frequency control circuit
to tune the resonator to the frequency of the oscillator. The detector is
preferably arranged with a preamplifier for combined, high-dynamic range
detection of EPR spectra.
In other aspects, the spectrometer includes an electromagnetic bridge with
automatic frequency control, a fixed frequency oscillator, and a varactor
diode tuned resonator. The electromagnetic bridge, especially in the
microwave region, is arranged to tune the resonator to the resonant
frequency, thereby compensating for movements of the tissue. In another
aspect, the resonator has a high Q LC circuit coupled with an external
planar loop via a .lambda./2 symmetrical line. Further, the computer can
be arranged for (1) determining the peak-to-peak line width of the EPR
spectra, (2) storing calibration EPR spectra of paramagnetic material in
the presence of known concentrations of oxygen, and (3) comparing
calibration spectra with EPR spectra of the tissue.
In a preferred aspect, the spectrometer system comprises India ink, a
constituent of India ink having physiologically acceptable paramagnetic
quality, or other physiologically acceptable paramagnetic materials, in
the tissue to be measured.
The methods of the invention preferably utilize an EPR spectrometer
constructed in accordance with the invention, such that the EPR spectra is
determined without significant interference from the configuration or
movement of the biological system; and further such that the measurement
is compatible with EPR spectra from physiologically acceptable
paramagnetic materials, e.g., India ink, in in vivo tissue.
These and other aspects and advantages of the invention are evident in the
description which follows and in the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 graphically shows calibration EPR line width spectra of India ink
and Fusinite over a wide range of oxygen tensions;
FIG. 1A graphically shows EPR line width spectra from India ink in the
presence of other materials, such as water, serum and oleic acid;
FIG. 1B graphically shows the EPR spectra of India ink in nitrogen and air
using a X-band EPR spectrometer;
FIG. 2 graphically shows microwave power and saturation data on line height
in nitrogen and in air;
FIG. 3 is an EPR spectrometer constructed in accordance with the invention;
FIG. 4 is a microwave resonator for use in the EPR spectrometer of FIG. 3;
FIG. 5 shows the signal response of EPR India ink spectra before and after
restricting the blood flow to the gastrocnemius muscles of an adult mouse
injected with India ink;
FIG. 6 graphically shows the de-oxygenation in in vivo mouse muscle
injected with India ink, subsequent to the tightening of a tourniquet;
FIG. 7 graphically shows the de-oxygenation characteristics of mouse muscle
injected with India ink over a period of thirty-nine days;
FIG. 8 shows a histological slide of mouse leg muscle forty days after
implantation by India ink;
FIG. 9 illustrates the tattoo of a human volunteer; and
FIG. 10 graphically shows EPR spectra from a human tattoo based on India
ink with and without blood flow restriction.
DETAILED DESCRIPTION OF THE INVENTION
The invention concerns apparatus, systems, and methods for determining the
partial pressure of oxygen, pO.sub.2, in biological systems, including in
vivo or ex vivo tissue. The invention provides improvements to EPR
oximetry by improving the sensitivity, accuracy, and repeatability of EPR
techniques. The invention further provides an EPR spectrometer and a
paramagnetic material that are physiologically compatible with in vivo
measurements. This paramagnetic material is already approved for use with
humans; and the material exhibits a measurable correlation between EPR
spectra and oxygen tension over a clinically effective pressure,
sensitivity, and resolution range. These methods, systems, and apparatus
have immediate and important application to clinical and experimental
problems which exist today.
The invention utilizes physiologically acceptable paramagnetic materials,
and in particular carbon black, especially in the form of India ink, as
new paramagnetic probes for EPR oximetry. India ink is an injectable
compound that is widely used in clinical settings, with no apparent
toxicity. India ink has extensive prior use in humans as the basis for
black tattoos, used for medical purposes as well as for personal
decoration. It has also been widely used in surgery to trace pathways in
tissues. India ink additionally exhibits the desired physical and chemical
properties required for effective clinical EPR oximetry, having EPR
spectra that is very sensitive to the presence of oxygen. In accordance
with the invention, physiologically acceptable paramagnetic
materials--such as India ink, constituents of India ink, carbon black, or
carbon-based material--are used to directly determine the pO.sub.2 in
biological systems, such as tissue. Previously, no known paramagnetic
material has exhibited the requisite properties to enable direct, in vivo
evaluation of humans.
The description below discusses the relevant properties of India ink, and
the methodology and apparatus for determining pO.sub.2 in vivo via EPR
oximetry. Experimental results are given from tests conducted with live
animals, and from tests demonstrating that oxygen dependent changes in
India ink EPR spectra can be detected in humans. The latter experimental
results are based upon the presence of India ink within an ornamental
human tattoo, and the response of India ink EPR spectra to differing
oxygen concentrations present at the tattoo.
India ink is a stable paramagnetic material. It has a single EPR signal
spectra with a peak-to-peak line width that is calibrated to directly
determine oxygen tension pO.sub.2 in vivo. FIG. 1 illustrates one set of
calibration data in a graph of the EPR spectra line width of India ink 20
and Fusinite 22 against pO.sub.2. With reference to FIG. 1, the India ink
line width 20 is approximately 600 mGauss in the absence of oxygen and
approximately 4500 mGauss in the presence of air. When India ink is within
biological tissues, the shape of the EPR spectra is between these values,
which is correlated to determine the in vivo oxygen tension. On the other
hand, over the same partial pressures, the Fusinite line width only
changed from 500 mGauss at 0 mm Hg to 1200 mGauss at 35 mm Hg.
At least two other noteworthy characteristics are apparent with reference
to FIG. 1: first, the India Ink line width spectra is sensitive to oxygen
tension levels below 1 mm Hg; and secondly, the slope of the India Ink
calibration data 20 shows that the EPR spectra line width is particularly
sensitive to changes in oxygen tensions of less than 30 mm Hg, which is a
critical realm for effective oxiometric measurements. As compared to
fusinite 22, for example, the line-broadening effects of the India ink EPR
spectra per unit pO.sub.2 are greater, improving sensitivity.
India ink is additionally less sensitive to the external conditions, and to
the compounds present in the biological system under investigation, which
might otherwise affect or reduce measurement accuracy. Over the broad
range of conditions that can occur in vivo, for example, the response of
India Ink EPR spectra to pO.sub.2 is essentially independent of pH,
oxidants, reductants, and the nature or lipophilicity of the biological
medium. FIG. 1A graphically shows the line width of India ink EPR spectra
24 in the presence of various media, including oleic acid 25, serum 26,
and water 27. The data 24 is the same as the calibration data 20 of FIG.
1, to within the accuracy of the measurement.
The experimental India ink data illustrated in FIGS. 1, 1A and 1B, and in
the principal experimental data presented in FIGS. 5-8, derive from India
ink purchased at SHIKAYA, JAPAN, having a concentration of 80 mg/ml. The
India ink particles were homogenous in size, and were approximately 1
.mu.m in diameter. Other chemicals for the principal experiments discussed
herein were purchased from Sigma, in St. Louis, Mo.
The calibration of India ink and other in vitro experimental studies of
India ink were performed on a Varian E-109 EPR spectrometer, which has an
X-band, 9.6 GHz microwave oscillator. Typical control settings for the
Varian spectrometer were: (1) 3210 Gauss of magnetic field strength; (2)
10 mW of microwave power; and (3) a modulation amplitude less than one
third of the line width. Experimental temperatures were controlled with a
Varian gas flow variable temperature control unit. And EPR spectra were
collected using EW software, from Scientific Software Inc., in Normal,
Ill., which was installed on an IBM-compatible personal computer. DPPH was
used as a secondary standard for spin density measurements.
More particularly, the calibration of India ink was as follows. Ten
micro-liters of India ink in PBS was drawn into a gas permeable teflon
tube from Zeus Industrial Products, Inc., in Raritan, N.J. This teflon
tube had a 0.623 mm inner diameter and a 0.138 wall thickness, and was
folded twice and inserted into a quartz EPR tube open at both ends. The
sample was then equilibrated with different O.sub.2 : N.sub.2 gas
mixtures. pO.sub.2 in the perfusing gas was monitored and measured by a
modified Clark electrode oxygen analyzer from Sensor Medics Co., Model
OM-11, in Anaheim, Calif., which was calibrated with pure air and
nitrogen. FIG. 1B shows that the response of the India ink EPR line width
spectra 30 in air, as compared to the spectra 32 in nitrogen, is severe,
indicating the ink's usefulness for oximetry.
The quantitative dependence of the EPR spectra on pO.sub.2 was obtained by
measuring the line width as a function of pO.sub.2 in the perfusing gas.
EPR line widths are usually reported as the difference in magnetic field
between the maximum and minimum of the first derivative recording of the
signal. In other words, the EPR line width is the peak-to-peak separation
of the first derivative, with respect to frequency, of the
Lorentzian-shaped absorption spectra.
The experiments presented herein also considered the microwave saturation
effects of the environment. FIG. 2 summarizes microwave power data on the
line height within nitrogen 34 and air 35. Because power saturation
occurred only at high microwave powers, the in vitro experimental testing
utilized 10 mW of unsaturated X-band microwave radiation.
With further reference to FIGS. 1 and 1A, the g-value, spin density, and
line width of the EPR India ink spectra were measured at room temperature.
The g-value (2.0027.+-.0.0008) and spin density (2.5.times.10.sup.19
spin/g) of India ink were not affected by oxygen. While the g-value of
India ink was approximately equal to Fusinite, the number of spins for
India ink spectra was more than twice the number of spins for Fusinite
(1.0.times.10.sup.19 spin/g). As illustrated in FIG. 1, the India ink EPR
probe is very sensitive, as compared to Fusinite, at low pO.sub.2,
especially less than 30 mm Hg of oxygen tension. Conveniently, the
principal pO.sub.2 dependencies for clinical and biomedical applications
occur in the range of 0-30 mm Hg pO.sub.2, making India ink EPR oximetry a
valuable measurement tool.
India ink EPR spectra exhibited no self-broadening due to changes in the
concentration of India ink particles. No effect, for example, was observed
in the EPR spectra of India ink in the presence of a paramagnetic agent,
K.sub.3 Fe(CN).sub.6, an oxidant, H.sub.2 O.sub.2, or a reductant,
ascorbic acid. The line width of India ink was also not affected by
variation in temperatures between 25.degree. C. and 50.degree. C., nor by
variations in the pH between 4 to 14. FIG. 1A illustrates that the
response of EPR India ink spectra in the presence of oxygen is essentially
independent of the media, including oleic acid 25, serum 26, and water 27.
For in vivo EPR measurements, discussed below, an EPR spectrometer
constructed in accordance with the further features of the invention was
utilized, having a L-band, low-frequency microwave oscillator
(approximately 1.2 GHz) with an extended planar loop antennae connected to
a resonator.
FIGS. 3 and 4 illustrate an EPR spectrometer apparatus 40 constructed in
accordance with the invention, and which has significant structural
differences as compared to conventional EPR spectrometers. Most
significantly, the spectrometer 40 permits the accurate measurement of EPR
spectra from in vivo biological systems, such as live animals, by retuning
its resonator 42 to maintain resonant frequency during movements of the
animal.
A spectrometer 40 constructed according to the invention solves certain
technology problems which make existing EPR spectrometers incompatible
with oxiometric measurements using physiologically acceptable paramagnetic
materials. Existing EPR spectrometers are especially incompatible with in
vivo measurements of live beings using paramagnetic probes either
implanted in tissue or administered through another route, such as orally,
intravenously, or by injection.
The spectrometer system 40 is a low frequency EPR spectrometer that
measures the EPR spectra of India ink or other physiologically acceptable
materials in animals, including humans, and other biological systems. The
spectrometer 40 has a resonator 42 and an associated microwave bridge 44.
The spectrometer 40 further has a magnet 46, powered by a power supply 48,
and modulation coils 50. The power supply 48, the coils 50, and the
microwave bridge 44 connect to a standard spectrometer console 52. A
computer 54 connects to the console to control elements in the
spectrometer 40.
In a conventional microwave bridge for an EPR spectrometer, an Automatic
Frequency Control (AFC) circuit locks the microwave oscillator to the
resonant frequency of the resonator. This is problematic for the purpose
of measuring animals, or a patient, with EPR oximetry. Movements in the
subject being studied cause a retuning of the oscillating bridge frequency
by .+-.5 MHz, which is equivalent to a shift in the position of the EPR
line width by 2000 mGauss. In the spectrometer 40 of FIGS. 3 and 4, the
AFC circuit has been constructed so that the resonator is tuned to the
microwave source, using a varactor diode with a range of approximately
.+-.8 MHz. Consequently, the microwave frequency is stable and independent
of movement of the experimental subject, tissue, or being under
investigation.
In operation, and with reference to FIG. 3, the magnet 46 applies a
magnetic field to the subject under investigation, which is adjacent to
the resonator 42. This magnetic field aligns and separates spins of
unpaired electrons of the subject within the field so that microwave
energy is absorbed by the subject's molecules. The microwave bridge
oscillator 44 and resonator 42 jointly apply a microwave electromagnetic
field to the subject while maintaining a single resonant microwave
frequency in the high Q resonator circuitry, illustrated in FIG. 4. The
microwave energy is absorbed by the molecules according to a functional
dependence with the magnetic field strength. At one magnetic field
strength, the photon energy of the microwave field is matched to the
excited molecular state of the electron spins, and peak absorption is
attained. Other frequencies of the EPR resonance are attained by gradually
changing, or "sweeping", the strength of the magnetic field generated by
the magnet 46. At the other frequencies, the microwave absorption is less.
A full sweep by the magnet 46 generates an absorption spectra having a
Lorentzian line-shape, or, more typically, spectra presented as the first
derivative of that line shape.
The presence of oxygen in a subject or tissue having a physiologically
acceptable paramagnetic material, e.g., India ink, affects the relaxation
rate of the excited paramagnetic molecule, thus causing an increased
time-integrated intensity, or line-broadening effect within the spectra,
as discussed above.
FIG. 4 illustrates the external loop resonator 42 constructed in accordance
with the invention and which improves oscillator stability and sensitivity
for possible resonant mismatching caused by movements of the biological
tissue. The resonator 42 includes an input 60 for Automatic Frequency
Control (AFC) circuitry, a high frequency input 62 for a 50 .OMEGA.
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