CA2168872A1

CA2168872A1 - Apparatus and methodology for determining oxygen in biological systems

Info

Publication number: CA2168872A1
Application number: CA002168872A
Authority: CA
Inventors: Harold M. Swartz; Fuminori Goda; Tadeusz Walczak; Ke Jian Liu
Original assignee: Individual
Current assignee: Dartmouth College
Priority date: 1993-08-12
Filing date: 1994-07-15
Publication date: 1995-02-23
Also published as: EP0752111A1; US5833601A; US5494030A; JPH09507763A; WO1995005611A1

Abstract

The invention provides apparatus and methods for measuring oxygen tensions in biological systems utilizing physiologically acceptable paramagnetic material, such as India ink or carbon black, and electron paramagnetic resonance (EPR) oximetry. India ink is introduced to the biological system and exposed to a magnetic field and an electromagnetic field in the 1-2 GHz range. The EPR spectra is then measured at the biological system to determine oxygen concentration. The EPR spectra is determined by an EPR spectrometer that adjusts the resonator to a single resonator frequency to compensate for movements of the biological system, such as a human or animal. The biological system can also include other in vivo tissues, cells, and cell cultures to directly measure pO2 non-destructively.

Description

wo 95/05611 2 ~ 6 8 ~ 7 2 PCT/USg4/07719 APPARATUS AND METHODOLOGY FOR DETERMINING
OXYGEN IN BIOLOGICAL SYSTEMS

Field of the Invention This invention relates generally to a~dLus and methods for del~ ini"g oxygen 10 tension in biological systems. More particularly, the invention concerns a~p~dlus and methods for ~ e.lllil,;"g oxygen cn,~rP~ ion, or PO2, in biological in vivo tissue ntili7in~
physiologically acceptable par~m~nt~tiC m~teri~le and electron p~r~m~gn~tic resonance oximetry.

15 Back~round ofthe Invention Benefits derived from the mea~ul~ ent of oxygen concentrations in tissue are known.
Oxygen is the primary biological oxi~l~nt, and the mea~ulellle,l~ of PO2 can hll~lov~ the evaluation and lm~lerst~ntling of many physiological, pathological, and th~;ld~ulic processes.
Prior art systems and methods for me~e-lring oxygen concentrations in tissue are also known, incll1rling the Clark electrode, fluolesce.lce ~ rln~g~ 2 binding to myoglobin and hemoglobin, chemil--minescen~e, pliosphoresence qneJlching, and spin label oximetry.
However, these systems and methods have certain, and often acute, limitations, especially 25 when used in vivo. They especially lack the qualities required for complete e~ ;lllental and clinical use, such as sensitivity, accuracy, repeatability, and adequate spatial resolution. See J.
Ch~pm~n, Radiother. Oncol. 20, 13 (1991) and J.M. V~n~erkt-oi et al., "Oxygen inM~mm~ n Tissue: Methods of Measurement and .Affinities of Various Reactions", Am. J.
Physiol. 260, Cl 131 (1991).
The polarographic microelectrode is one popular device for me~llrin~ 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 35 location. The microelectrode is also relatively insensitive to oxygen collc~;llL,~Lions below 10 mm Hg, which is within the required St;llsiLiviLy region for effective oximetry. Finally, the microelectrode may itself consul~le oxygen, thereby altering its own e"vi,o,~,lent, in~ ring measurement errors, and re~lnr,ing the accuracy and llsefillness of the evaluation process.

WO 95/05611 PCTtUS94/07719 7 ~

There are scattered reports which concern in vivo PO2 mea~ lents with such devices, especially in skeletal muscle. Whalen and Nair, Am. J. Physiol. 218, 973 (1970), measured PO2 of cat gracilis at rest using a recessed Au 1-5~1m microelectrode, giving average PO2 values of 6.6+0.4 mm Hg (n=372). Gayeski et al., Am. ~ Physiol. 254, H1179 (1988), measured PO2 Of dog gracilis at rest, exhibiting a partial ples~ule range of 4.5-35 mm Hg (16.8 mm Hg median), and 95% V02 max, using a Mb saturation technique, exhibiting a partial plCS:iUlC range of 0.2-2.3mm Hg (0.9-1.8 range of mean). Nevertheless, there are effective limit~tiQns to these PO2 measurement techniques. In the microelectrode method, for example, it is technically difficult to monitor or make long term evaluations of PO2. In the Mb saturation method, it is especially difficult to measure low PO2, and the method can only be used in muscle.

Nuclear Magnetic Resonance (NMR) techniques have been explored and considered in the context of oxiometric mea~ulclllents, ~speci~lly through the use of an oxygen dependent proton hy~lrllle line in myoglobin and oxygen dependent relaxation of fll10rin~
nuclei. NMR is a common s~e.iL.oscopic technique in which the molecular nuclei is aligned in a m~nt-tic field and .~imnlt~nPously excited by absorption of radiofrequency energy. The molecular re~ tic-n from the excited state to the initial state is an observable event that is ~ffecte(l by the l~lcsellce of oxygen through ryl'~ pe or dipolar actions. However, the NMR
techniques have not demonstrated sllfficient sensitivity and/or applicability to the measure of PO2 in either experimtont~l or clinical settings.

Electron p~r~m~gn~tic Resonance (EPR) o~hllclly is another technique for m~
oxygen concentrations. Similar to NMR, EPR oximetry is a spectroscopic technique based upon the Zeeman e~ect and the line-bro~ ning effect of molecular oxygen on the EPR
spectra of p~ lic m~t~ri~l~. These m~teri~l~ have unpaired electron spins that are aligned in a m~gn~tic field and excited by micluw~ve energy. The sep~r~ti-n bclweell the lower, lm~Y-~it~-l energy state and the higher, excited energy state is pl~"..,llional to the strength of the m~gn~tic fiéld. The presence of oxygen with the excited molecule mP~cl-r~hly 30 affects the molecular relaxation so that the line width of the EPR spectra changes and provides an in-lic~tiQn of PO2-Nitroxides exemplify one family of compounds having p~r~m~gn-otic quality that are suitable for EPR oximetrv, and which have been used in a variety of in vitro c~
35 Although nitroxides have also been tested in vivo, at least two res-lltin~ problematic areas exist in such measulc;lllents: first, nitroxides tend to be bioreduced; and secondly, nitroxides are not very sellsilive to the low conccl~ lions of oxygen that are of the most biological interest today, i.e., less than 10 Torr.

wo 95/05611 2 ~ 6 ~ 8 7 2 PCT/USg4/07719 Other recent discoveries of new par~m~gnPtiC m~tPri~lc, such as Fusinite and lithium phthalocyanine (LiPc), have made progress as oxygen probes in the field of in vivo EPR
oximetry. These two c~.ll~o~,ds, for example, are suitable for in vivo usage because they exhibit certain favorable char~ctPri~tics, inrln~ling: accuracy; spatial resolution; scll~i~ivily in S the physiologically important conrpntr~tion range Of P02; ease of use, liKle or no a~p~c"~
toxicity, and relative stability in tissues, pc.,ll;~ lg prolonged measurements over periods of weeks or months after ~tlmini~tPring the compound. Nevertheless, because these p~ranl~gnptic compounds have not been previously tested in hnm~n~, they will have to undergo very long and extensive toxicological evaluation before they can be used clinically.
10 This evaluation is likely to be prolonged because of other problems inherent in the compounds, such as stability and inertness, which encourage inrl~finite, ullw~lled per~ictenre within the tissue.

There are other exi~ting problems limiting the effectiveness of EPR oximetry, 15 inrln-ling the inability to measure EPR spectra efficiently and effectively, especially in vivo.
Conventional EPRspectromPtPrs, for example, typically utilize microwave fic~luencies, e.g., 9 GHz, that are strongly absorbed by tissue and water, and which reduce the useful depth penetration and mea~ulcl"ent sensitivities within the tissue. Prior EPR ~e~;Ll.".~tPr.~ also cannot effectively measure EPR spectra from a biological system such as a live animal, 20 because movements of the animal change the observed EPR spectra. This movc,lltlll illc~cases noise and reduces the accuracy. Finally, co,lv~ lEPR sl,e~;Llo",eters have the e~o,.;1lr~ and the sample under test, e.g., tissue, within a common magnetic field. This collsLldills the EPR mea~ llent/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
spe~;Ll~ll"eter and ~oci~tecl methodology that are free of the afore-mentioned difficulties.

It is another object of this invention to provide an hll~lovcd a~p~d~us and method 30 that enables the direct mea~ulcment of oxygen conc~ntr~tion in biological systems, such as tissue.

It is a further object of the invention to provide improved methodology and a~l,~dLus for in vivo EPR oximetry.
Other objects of the invention will be a~cllL from the following description.

WO 95/05611 ~ 1 6 8 ~ 7 2 PCT/US94/07719 Sl~mm~ry 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, inrhl-linp the steps of (1) S introducing physiologically acceptable ~ .n~ tic m~tPri~l to the biological system, (2) applying a m~gnPtic field and an electrom~gnP-tic field to the biological system, and (3) de~ ...;..;..g the EPR spectra of the biological system. The p~r~m~gnetic m~teri~l is of the type which has an EPR spectra lc~oll~ive to the presence of oxygen, such as India ink, c~ l;L-~ of India ink having p~r~m~gnptic quality, carbon black, and other carbon-based 10 m~tPri~l The biological system inr.hldes in vivo and in vitro biological systems, biological tissues, cells, cell cultures, ~nim~l~, and live human beings.

In another aspect, the method provides for the step of calibrating the EPR spectra of the p~r~m~gnPtic m~tPri~l by co."~ g the EPR spectra of the biological system with the 15 EPR spectra of the p~r~m~gnPtic m~tPri~l in the presence of a known conrP-ntration of oxygen. Preferably, both the mca~u~cd spectra from the biological system and the c~lihr~tion spectra are detPrminPd by the s~c~,L~a's peak-to-peak line width. The peak-to-peak line width in-lir,~t~s oxygen tension in the biological system, and PO2 is ~let~rmined directly by c~....p~ the measured line width to the c~libr~tion line width.
In other aspects, the method provides for ~wct;~ g the m~gnit~ e of the m~gnPticfield between a~l~,xi..l~ely 100 and 500 Gauss to acquire the EPR spectra through the frequencies of the EPR reson~nre The step of SWC;C~ g preferably o~curs in less than 60 seconds.
In another aspect, the m~nPtic field incl~ldP~ a first magnetic field having lines of force in snhst~nti~lly one direction, and the method provides for applying a second m~EnPtic field to the biological system that is subst~nti~lly parallel to the first magnetic field. The second m~gn~tic field is thereafter slowly varied to modify, or sweep, the m~Eni~lde of the 30 first m~EnPtic field bclwccn a~pl.)x;...~lPly 1 and 500 Gauss, to acquire the EPR spectra through the EPR resonance frequencies. ~ livcly, an electromagnet is employed tosweep the m~nPtic i~ ilies. Preferably, a third m~gnPtic field is applied to the biological system that is ~u1,~ ly perpendicular to the first m~EnPtiC field. The third m~gnPtic field is mocllll~te~l between approximately 1 and 500 kHZ? to improve the signal-to-noise ratio for 35 cl~lr.llli.,il-g the spectra. Preferably, the electrom~gnPtic field applied to the biological system is directed ~--h~ 11y perpendicular to the first m~nptic field with an oscill~ting frequency between ~f~xhllately 100 MHz and S GHz, such as in the miclow~vc L-band.

~ ~8~72 S
In still another aspect, the method includes the step of (1~1~ ",i"i"g the EPR spectra by ntili7ing an EPR spe~ilrolneter that has a resonator and an associated Q factor. The Q factor is ~letPrmin~d and monitored for change, such that, in another aspect, the Q factor is co...pe.ls~led to m~int~in resonant frequency during movements by the biological system, S e.g., the tissue or animal.

The method in accordance to the invention also provides for introducing to the biological system a par~m~gnPtic m~tPri~l that has subst~nti~lly ulliroll-l particles with diameters between 2lppru~h--ately .1 and 100 microns. Alternatively the par~m~gnPtic 10 m~teri~l can include at least one relatively large particle with a ~ mPter 1~a~loxi",~tely 100 microns and one centimpter. This relatively large p~r~m~gnPtic particle functions as a point source to spatially ~etPrmine the EPR spectra in the biological system.

In other aspects according to the invention, the paramagnetic m~tPri~l is introduced to 15 the biological system by several a~lopl;ate methods. In tissue, for example, the m~tPri~l can be injecting directly into the biological system. If the biological system has a circulatory blood stream, the p~r~m~gn~tic material can be introduced directly into the blood stream.
Accordingly, the method can include the further steps of (1) ~h~nging the blood flow to the biological system or tissue, and (2) .lt;l~.lllil,i"~ the change in the EPR spectra to provide a 20 real-time ev~ tion of the change in oxygen con~entr~tion in the tissue. Additionally, the blood flow to the tissue can be reduced to reduce the oxygen concPntr~tion in the tissue.

The p~r~m~gnPtic m~tPri~l can also be introduced to the biological system via lymph~tics. To derive additional spatial inforrn~tion, the par~m~gnPtic m~tPri~l can also be 25 selectively introduced to a localized region within the biological system, thereby intlic~ting oxygen tension at the localized region. ~ ively, the p~r~m~gnPtic m~tPri~l is introduced to a biological system having phagocytic activity, such that the par~m~gnPtic m~t~ri~l is introduced to the biological system by phagocytosis.

The invention also provides for a method to clet~rminP 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 ~ from the surface. In other aspects, an EPR
resonator constructed in accordance with the invention for use with an EPR spe~ ul,~
directly measures EPR spectra from the surface.
In another aspect, a method is provided for evaluating oxygen tension in a cell.Physiologically acceptable p~r~m~pnPtiC m~teri~l - which has an EPR spectra responsive to presence of oxygen - is first introduced to the cell. such as through phagocytosis. A m~gnPtic field and an electrom~gnP,tic field are then applied to the cell, and the peak-to-peak line width g ~ 2 of the EPR spectra of the cell is ~let~Prmin~ The p~r~m~gnPtic m~teri~l can include carbon black, carbon-based material, India ink, or ingredients of India ink having physiologically acceptable p~r~m~gnPtic quality. The electromagnetic field preferably has a frequency between ~lux;...~l~ly 100 MHz and 5 GHz.
The method additionally provides for the steps of d~ .g the EPR spectra peak-to-peak line width of the par~m~gnP-tic m~t~Pri~l in the presence of a known conrPntr~tion of oxygen. The spectra from the known c~ .l . nl ;-)n of oxygen is then cul-lp~ed to the spectra of the cell to d~ P the oxygen tension present in the cell.
The invention also provides a system for ~ r~ g oxygen cu~c~ n~ in biological systems, inr~ ing (1) physiologically acceptable p5.,~...z~g..Ptic m~riz~l in the biological system, and (2) an EPR spectrometer to detPrminP the EPR spectra of the biological system. The p~r~m~gnPtic m~teri~l can include India ink, an ingredient of India ink 15 having physiologically acceptable p~r~m~gnptic quality, carbon-based m~teri~l, 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 hllm~n~ The p~rZlm~gnt~tic m~tPri~l is introduced to the biological system via an a~lu~fi~ m~nnPr, inrlllrlinF direct injection into the biological system; direct injection into the blood stream, 20 via ly.l.l.hnl;r,s; and through ingestion.

Preferably, in another aspect, the system inrlllfl~Ps means for cl(,t~ g the peak-to-peak line width of the EPR spectra. This line width is then col~dled with the peak-to-peak line width of the EPR spectra of the paramagnetic m~tPri~l in the presence of a known 25 concellLIdlion of oxygen. A system according to the invention also preferably inr.lllrl~s a m~gnPt for applying a m~gnPtic field to the biological system, and means for :iWt;epUl~, the m~nihlrle of the m~gnPtic field between apprc~xil~ ly 100 and 500 Gauss. The m~iblrlP
is typically varied in a period less than sixty secon~

In another aspect, a system according to the invention includes means, e.g., a magnet or an ele~ u. . .~ l, for applying a first m~gnPtic field to the biological system that has lines of force in su~ y one direction. The system further has means, e.g., a magnet ûr an electrom~gnPt7 for gellelc.~ g a second m~gnptic field with lines of force s~lbst~nti~lly parallel to the first m~nPtic field to modify and sweep the m~gnitllde of the first m~gnP,tic field b~lw~t;ll approximately 1 and 500 Gauss. Preferably, the system has means for genc;ldlillg a third m~gnetic field, with lines of force sllbst~nti~lly perpendicular to the first magnetic field, wherein the third m~gnPtic field is m~ ts(l between ~ hllately 1 and 500 kHz to improve the signal-to-noise ratio of the measured spectra.

WO 95/05611 ~ 2 PCT/US94/07719 .

In still another aspect, the system as an osçill~ting electrom~PnPtic source forapplying electrom~gnPtic radiation to the biological system. The ele~iLlu,.-~gnPtic radiation, preferably within the range 100 MHz to S GHz, such as the L-band microwave freqll~nçiP,s, is directed to the biological system and is ~ub~ 1y perpendicular to the m~gnPtic field.
In still another aspect according to the invention, the EPR spc~,Llullleter has a resonator and means for rle~ g the resonator Q. Preferably, the lçso.-~lur Q is compensated in response to mo~ , of the biological system to m~int~in the resonant frequency.
In other aspects, the par~m~gnPtic m~t~ri~l of the system is substantially ulirOllll, with particle ~ mP~ters between a~plo~ tP,ly .1 micron and 100 microns. The p~r~m~gnptic m~teri~l can also be one or more relatively large particles with diameters b~:Lweell a~ploxilllately 100 microns and one cPntimPtPr. These relatively large particles function 15 much like a point source for the spectra in the biological system. In one aspect, for example, the p~r~m~gnetiC m~tPri~l 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 cletPnninP the EPR spectra directly 20 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 ch~npin~
the blood flow to the tissue and means for d~ ,..;,.;"g the change in the EPR spectra, thereby providing a real-time evaluation of the change in oxygen conrPntr~tion in the tissue.
Accordingly, the system can also include means, e.g., a tourniquet, for retl~1cing the blood 25 flow to the tissue to reduce the oxygen concentration at the tissue.

The invention also provides, in another aspect, a ~e~;Llolllcter for the in vivomea~ule.ll~;llL of oxygen collcr~ ;on in tissue. The .,~e~llullleter includes (1) magnets for selectively applying a m~gn~tic field of selectable strength to the tissue, (2) electromagnetic 30 oscillator for selectively applying electrnm~nPtic radiation having a ~le4uell-;y b~w~ell ap~roxilllately 100 MHz and S GHz to the tissue, (3) ~lPtPctor for detecting the electron par~m~gnetic spectra of the tissue, (4) l~SO~ 1ul~ ~rr~nFecl to m~int~in a s~lbst~nti~lly coll~L7~
resonant frequency, (5) console in c~ mml-nic~tion with the detector for displaying the EPR
spectra, and (6) co~ uL~l connPcted to the console for controlling the spectrometer, and for 35 analyzing the EPR spectra.

Preferably, the resonator includes an auLulnalic frequency control circuit to tune the resonator to the frequency of the oscillator. The detector is preferably arranged with a prç~mplifier for comhinPc1 high-dynamic range detection of EPR spectra.

~8~2 ~

In other aspects, the ~e-;llullleter inr~ çs an ele-;LIulllagnetic bridge with ~llts)m~tic frequency control, a fixed frequency oscillator, and a V~d~;~Ol diode tuned lcso~ ol. The electrl m~ n~tic bridge, especially in the microwave region, is arranged to tune the l`eSOl~

5 to the resonant frequency, thereby comp~ g for movements of the tissue. In another aspect~ the l-,SO~ Ol has a high Q LC circuit coupled with an rxtPrn~l planar loop via a ~/2 symmPtric~l line. Further, the culll~uh. can be arranged for (1) ~lr~ the peak-to-peak line width of the EPR spectra, (2) storing calibration EPR spectra of p~r~m~gnrtic m~t~ri~l in the plcscllce of known co~-rr~ ;ons of oxygen, and (3) co..~ g calibration spectra with 10 EPR spectra of the tissue.

In a plercllcd aspect, the ~c~ llleter system comprises India ink, a co..~ .l ofIndia ink having physiologically ~ccept~hle par~m~gnrtic quality, or other physiologically acceptable p~r~m~gn.otic m~t~n~l~, in the tissue to be measured.
The methods of the invention preferably utilize an EPR ~e~iL,~,llleter con~L-u;led in accorda,lce with the invention, such that the EPR spectra is dete minrcl without .~ignifir~nt hllclrclcnce from the confi~lration or movement of the biological system, and further such that the measurement is colll~la~ible with EPR spectra from physiologically accc~t~ble 20 p~r~m~gnPtic m~trri~l~, 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 accon~allyillg drawings.

Brief Description of the nrawir~s FIGURE 1 gr~phir~lly shows s~lihr~tion EPR line width spectra of India ink and 30 Fusinite over a wide range of oxygen ten~ion~;

FIGURE lA graphically shows EPR line width spectra from India ink in the ~re~ lce of other m~ttori~l~, such as water, serum and oleic acid;

FIGURE lB gr~phir,~lly shows the EPR spectra of India ink in nitrogen and air using a X-band EPR ~e~;llullleter;

FIGURE 2 gr~phir,~lly shows miclowavc power and saturation data on line height in nitrogen and in air;

WO 95/OS611 ~ 1 6 8 8 ~ 2 PCT/USg4/07719 .

FIGURE 3 is an EPR ~I,e~ l-eter constructed in accordance with the invention;

FIGURE 4 is a microwave resonator for use in the EPR spectrometer of FIGURE 3;

FIGURE 5 shows the signal response of EPR India ink spectra before and after restricting the blood flow to the gastro~ muscles of an adult mouse injected with India ink;

FIGURE 6 grarhi~ally shows the de-oxygenation in in vivo mouse muscle injected with India ink subsequent to the tight~ning of a tourniquet;

FIGURE 7 grarhic~lly shows the de-o~ygell~lion characteristics of mouse muscle injected with India ink over a period of thirty-nine days;
FIGURE 8 shows a histological slide of mouse leg muscle forty days after jmplantation by India ink;

FIGURE 9 ill l l~ s the tattoo of a human volunteer; and FIGURE 10 graphically shows EPR spectra from a human tattoo based on India ink with and without blood flow restriction.

I:)etailed Description of the Invention The invention concern~ appa~lus, systems, and methods for ~ the c- nc~ntration or partial ~ e of oxygen, PO2, in biological systems, inch~rling in vivo or ex vivo tissue. The invention provides il~ruv~ments to EPR oximeky by il"provi"g the se~ ivily, accuracy, and repeatability of EPR techniques. The invention further provides an 30 EPR spe~ "c;lt;l and a paramagnetic mat~rial that are physiologically colllr~;1l;hle with in vivo mea~ulG",c"~. This paramagn~tic m~terial is already appr~,ved for use with hllllla~, and the mat~rial exhibits a measurable correlation between EPR specka and oxygen tension over a clinically effective ~x,ule. sensitivity, and resolution range. These methods, systems, and apl)d,dllls have imm.o(1iate and important application to clinical and t;~ ntal problems 35 which exist today.

The invention utilizes physiologically acceptable paramagnetic m~t~rial~, and inparticular carbon black, espec-ially in the form of India ink, as new paramagn.otic probes for EPR oximetry. India ink is an injectable compound that is widely used in clinical setting~

WO 95/05611 ~ :~L 6 8 8 7 2 PCT/US94/07719 with no al,pa~Glll toxicity. India ink has GAIGllsive prior use in hllm~ne as the basis for black tattoos, used for mçrlir~l purposes as well as for personal decoration. It has also been widely used in surgery to trace pdLhwdy~ in tissues. India ink ~d~lition~lly exhibits the desired physical and rhrmjr~l plope,lies required for effective clinical EPR ~hllGLly, having EPR
5 spectra that is very sGn~ ivily to the p,~,sGnce of oxygen. In accoldi~lce with the invention, physiologically acceptable p~r~m~EnPtic m~teri~le - such as India ink, c~ of India ink, carbon black, or carbon-based m~teri~l - are used to directly detrrminto the PO2 in biological systems, such as tissue. Previously, no known p~r~nl~Enptic rn~t~ri~l has e~rhihi~d the requisite ~ Gl Lies to enable direct, in vivo evaluation of h-lm~ne The description below ~iiecllcees the relevant plOp~ ~ Lies of India ink, and the methodology and a~udlus for ~ g PO2 in vivo via EPR oximetry. F~ .;...rnt~lresults are given from tests con~ cted with live ~nim~le, and from tests d~mo,~l.d~ g that oxygen dependent changes in India ink EPR spectra can be ~tecte(l in hllrn~ne The latter 15 G~. ;lllr~ l results are based upon the p,~,s~lce of India ink within an orn~ment~l hum~n tattoo, and the rG~ollse of India ink EPR spectra to ~lifferinE oxygen co,~ "~ ne present at the tattoo.

India ink is a stable ~ g.~lic m~teri~l It has a single EPR signal spectra with a 20 peak-to-peak line widt_ that is c~lihr~ted with known oxygen c~ e~ l;ons to directly e PO2 in vivo. FIGURE 1 ill~.~;l"1les one set of calibration data in a graph of the EPR
spectra line width of India ink 20 and Fusinite 22 against PO2. With lcrGlGllce to FIGURE 1, the India ink line width 20 is a~pr~,x;...i1lely 600 mGauss in the absence of oxygen and a~proxi."ately 4500 mGauss in the plesGllce of air. When India ink is within biological 25 tissues~ the shape of the EPR spectra is bG~wGell these values, which is correlated to determine the in vivo collcGl~ Lion of oxygen. On the other hand, over the same partial plGS~ulGs, the Fusinite line width only rh~nged from 500 mGauss at 0 mm Hg to 1200 mGauss at 35 mm Hg.

At least two other n~lGw-~,Lhy ~`.h~ 1 ;r~e are a~c"l with reference to FIGURE 1:
first, the India Ink line width spectra is SG"silivG to oxygen conr~ntr~tions 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 ~Lr~-;livt; oxi- metric mea~ .,~"l~. As coll,~ ed to fusinite 22, for çx~mple the line-bro~(ito-ning effects of the India ink EPR spectra per unit PO2 are greater, improving s~ iLivily.

India ink is additionally less sensitive to the ~xt~rn~l conditions, and to the colllpuu,lds present in the biological system under investig~tinn, which might otherwise affect or reduce 21f~J83~l2 mea~u~ ent accuracy. Over the broad range of conditions that can occur in vivo for example, the response of India Ink EPR spectra to PO2 is essPnti~lly independent of pH, oxi~l~nt~ re~ ct~nt~, and the nature or lipophilicity of the biological medium. FIGURE lA
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 c~libt~tion data 20 of FIGURE 1, to within the accuracy of the mea~u elllent.

The c~t-illlental India ink data illustrated in FIGURES 1, lA and lB, and in theprincipal e~Ly~ Pnt~l data p.escll~ed in FIGURES 5-8, derive from India ink purchased at SHIKAYA, JAPAN, having a co~ Qn of 80 mg/ml. The India ink particles were homogenous in size, and were approxim~tely lllm in diameter. Other chPmic~l~ for the principal ;;~. . ;...Pnt~ discussed herein were ~LIlcllased from Sigma, in St. Louis, Missouri.

The calibration of India ink and other in vitro c~pt? ;l~nt~l studies of India ink were pelr~lllled on a Varian E-109 EPR ~ecLl~,llleter, which has an X-band, 9.6 GHz micl~ w~vt;
oscillator. Typical control settings for the Varian ~e-;l,.,llleter were: (1) 3210 Gauss of magnetic field strength; (2) 10 mW of micl~,w~vc power; and (3) a mo~ tion ~mplitllde less than one third of the line width. E~F ;...~..t;~l lclll~ ulcs were controlled with a Varian gas flow variable ~elllpc.dLulc control unit. And EPR spectra were collected using EW software, 20 from Scientific Software Inc., in Normal, Illinois, which was in~t~lle(l on an IBM -c~mp~tible personal COlll~U~.,.. DPPH was used as a sec~n~l~ry standard for spin density mea~u cl,lents.

More particularly, the calibration of India ink was as follows. Ten micro-liters of India 25 ink in PBS was drawn into a gas permeable teflon tube from Zeus ~n-illetri~l Products, Inc., in Raritan, New Jersey. This teflon tube had a .623 mm inner ~ m~tPr and a .l38 wall thicl~n~5~, and was folded twice and inserted into a quartz EPR tube open at both ends. The sample was then equilibrated with diLr~cllt 2: N2 gas nli~Lules. P02 in the ~lru~hlg gas was morlitored and measured by a mo-lifiecl Clark electrode oxygen analyzer from Sensor Medics Co., 30 Model OM-11, in ~n~h~im, C~liforni~ which was c~lihr~tecl with pure air and nitrogen.
FIGURE lB shows that the response of the India ink EPR line width spectra 30 in air, æ
Colll~ to the spectra 32 in nitrogen, is severe, indicating the ink's usefulness for o~illlclly.

The 4..~.-l;l;1liv-e depen-l~nl~e of the EPR spectra on P02 was obtained by mP~llrinF the 35 line width as a function of P02 in the pclrusillg gas. EPR line widths are usually reported as the diLr~.ence in m~gn~tic field between the m~ximl-m and lll;l~ ll of the first dc.;v~Livc 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.

WO 95/05611 ~ i 6 8 8 7 2 PCTrUS94/07719 The c~ plcs~ cd herein also conei~lrred the microwave .e~tllr~tiol~ effects of the el.vh-------rnt FIGURE 2 ~ s microwdvc power data on the line height within nitrogen 34 and air 35. Because power saturation occurred only at high microwave powers, the in vitro ~xl l~ .; " ,t-nt~l testing utilized 10 mW of lm~ e-l X-band microwave radiation.
With further reference to FIGURES 1 and lA, the g-value, spin density, and line width of the EPR India ink spectra were measured at room tr~ . The g-value (2.0027 +
0.0008) and spin density (2.5xlOl9 spin/g) of India ink were not ~f~ctrcl by oxygen. While the g-value of India ink was al~proxilllalcly equal to Fusinite, the number of spins for India ink spectra was more than twice the number of spins for Fusinite (1.0x10I9 spin/g). As illustrated in FIGURE 1, the India ink EPR probe is very sc -~ilivc, as col.-p~cd to Fusinite, at low PO2, especially less than 30 mm Hg of oxygen tension. Conveniently, the lJ~ ;llr';P~l PO2 dependencies for clinical and biomedical applications occur in the range of 0-30 mm Hg PO2, making India ink EPR o~i..-cLl~y a valuable measu c...ent tool.

India ink EPR spectra exhibited no self-bro~lPning due to cha..ges in the conc~ntr~tinn of India ink particles. No effect, for ry~mrle~ was observed in the EPR spectra of India ink in the ~ scllce of a p~r~m~gnPtic agent, K3Fe(CN)6, an oxidant, H22 or a recl~lct~nt ascorbic acid. The line width of India ink was also not ~ffected by variation in tr~ cs bclwcen 25 C and 50 C, nor by variations in the pH bcLwccll 4 to 14. FIGURE lA illu~;l"1l~s that the response of EPR India ink spectra in the p.~,;,c..ce of oxygen is rcqrnti~lly independent of the media, inrhl~ling oleic acid 25, serum 26, and water 27.

For in vivo EPR me~u c---ents, liecllceed below, an EPR s~e~;L-oll.eter consllu-;led in 25 accordance with the further rcdlult;s of the invention was ntili7r-l, having a L-band, low-frequency microwave oscillator (d~l`Ox;lll~ y 1.2 GHz) with an r-~trn~e(l planar loop ~ntt~nn~e connrctecl to a ~eson~lu-.

FIGURES 3 and 4 illustrate an EPR spectrometer a~a dl~lS 40 con~LIu~;lcd in 30 accordallce with the invention, and which has ei~nific~nt ~ diL~.~i..ces as compared to co..vc..Lional EPR spectrnm~t.ors. Most eignifir~ntly, the spectrometer 40 permits the accurate mea~ ;...ent of EPR spectra from in vivo biological systems, such as live ~nim~le, by retuning its resonator 42 to l~ resonant frequency during movements of the animal.

A ~e~ omelt; 40 constructed acco.dillg to the invention solves certain technology problems which make e~cietinp EPR spe.;l...lllt~ inco..-pdlible with oxiomPtnc measurements using physiologically acceptable p~r~m~gnPtic m~tPri~le F~cieting EPR
~e~.ul..eters are especially incompatible with in vivo mea~u-e nents of live beings using wo 95/05611 ~ ~ ~ 8 ~ ~ 2 PcT~us94lo77l9 p~r~m~gn~otic probes either impl~nte~i in tissue or ~lmini~tPred through another route, such as orally, hlLI~vt;llously, or by injection.

The ~e~llumeter system 40 is a low frequency EPR spectrometer that measures the S EPR spectra of India ink or other physiologically acceptable m~t~ri~l~ in ~nim~l~, in~ ing hllm~n~, and other biological systems. The spectrometer 40 has a resonator 42 and an associated microwave bridge 44. The a~e~ vllleter 40 further has a magnet 46, powered by a power supply 48, and mo~lnl~fi~-n coils 50. The power supply 48, the coils 50, and the microwave bridge 44 connect to a standard spectrometer console 52. A coll~ulel 54 c~ nn~c 10 to the console to control elements in the specllulll~L~. 40.

In a conv~ ion~l microwave bridge for an EPR spe-;~lul.-e~el, an Automatic Frequency Control (AFC) circuit locks the microwave oscill~tor to the resonant frequency of the resonator. This is problematic for the purpose of measuring ~nim~l~, or a patient, with 15 EPR oximetry. Movelllell~ in the subject being studied cause a retuning of the oscill~ting bridge frequency by +/- S MHz, which is equivalent to a shift in the position of the EPR line width by 2000 mGauss. In the spe~ ulllt;~l 40 of FIGURES 3 and 4, the AFC circuit has been constructed so ~at the lc;solla~ol is tuned to the micluw~ve source, using a V~ ;tOl diode with a range of ap~lu~hllately +/- 8 MHz. Consequently, the llli-;luw~ve frequency is 20 stable and independent of movement of the rxl c ;l,.ent~l subject, tissue, or being under investip~tion In operation, and with l~Ç~ ce to FIGURE 3, the magnet 46 applies a m~gn.~tic field to the subject under investigation, which is ~ r~nt to the resonator 42. This m~n~tic field 25 aligns and s~ s spins of ullpail~d electrons of the subject within the field so that microwave energy is absorbed by the subject's molecules. The micluw~v~ bridge osc~ tor 44 and resonator 42 jointly apply a microwave electrom~ ntotic field to the subject while m~;"l;~ ;"~ a single leso~ micluw~ve r~ uelll;y in the high Q reson~tor ci~
illuskated in FIGURE 4. The mil;ruw~v~ energy is absorbed by the molecules accoldil1g to a 30 functional depen~lPn~e with the m~n~-tic field strength. At one m~gnptic field strength, the photon energy of the microwave field is m~trhPd to the excited molecular state of the electron spins, and peak absorption is att~inp~l Other frequencies of the EPR r~son~nce are attained by gradually ch~ngin~, or "~wce~ g", the strength of the m~gnPtic field gelle.~Led by the magnet 46. At the other freq~lP-nries, the microwave absorption is less. A full sweep by the 35 magnet 46 genPr~tPs an absorption spectra having a Lorentzian line-shape, or, more typically, spectra ~lest;llLt;d as the first derivative of that line shape.

The plcse~lce of oxygen in a subject or tissue having a physiologically acceptable par~m~gnetic m~tPri~l, e.g., India ink, affects the relaxation rate of the excited par~m~gnPtic ~8872 molecule, thus causing an increased time-hlL~.d~ed hllellsiLy, or line-bro~ ning effect within the spectra, as discussed above.

FIGURE 4 ill~ s the e~rt~rn~l loop resonator 42 con~L,u~iL~d in accordance with the S invention and which h~ ves osçill~tor stability and sellsiLivily for possible lcsoll~
mi~m~trllin~ caused by movements of the biological tissue. The resonator 42 inrh~ s an input 60 for Automatic Frequency Control (AFC) ~;h~;uiLly, a high frequency input 62 for a 50 n coaxial line, and a variable inductive coupling 64. The ,~son~Lol 42 further has a high Q
LC resolla"L circuit 66, a varactor diode 68, a two-wire ~/2 ~y ..., . ,~ . ;c~l line 70, and a planar 10 loop 72.

The ~sollalo, 42 avoids the physical access problems faced by conv~ ;on~l EPR
spectrometers in co-locating the l~50ll~lo, and subject within a common m~gn~tic field. The resonator 42 m~t~hes and Ill~ the l~sOlldlll frequency of the ~so,~ cavity by use of a high Q LC circuit 66 coupled with an ~.~tf~.rn~l planar loop 72 via a ~/2 symmetric~l ~nt~nn~-like line. The LC circuit 66 is m~t~ ed to a 50 Q coaxial line at the input 62 via a v~iable inductive coupling 64. The coupling 64 con~i~t~ of a coupling loop, a ;~J4 flexible i",~ed~ce tr~n~former, and a mecl~ ", that f ll~n~s the position of the loop relative to the LC circuit 66. The application of the ;~ ,e(l~ e ~ r~. .,.1 . makes it possible to t;rfe~;livt;ly match the 20 lcso~ l to the 50 Q line. The loop portion 72 is the ~nt~nn~e like elem~nt which is placed in pl02sil~lily to the region to be studied. The loop 72 can be confi~lred to optimally fit the subject, e.g., by going around a protruding tumor, because the ,~so~Lo, need not be in the m~gn~tic field. This is not, however, how a co,lv~ ;on~ sol~ ol operates, where the subject and the resonator are within a common m~gnPtic field, thereby co,,~ ill;llg 25 mea~ ",ent flexibility.

Movt;",~"L of the subject also ;~ es the ,esol~LuPs match to the 50 Q coaxial line, which hlc,c;ases the high r~4uell~;y voltage level at the output. This could potentially produce an overload of the y~i.."l)lirier and rlet~ctor, and, therefore, the ~e~,Llu",eter 40 of 30 FIGURE 3 prcrt;ldbly utilizes a wide-dynamic preamplifier and ~etector to measure the EPR
absorption spectra.

The ~l,e~;llo",eter 40 described in FIGURES 3 and 4 also o~.,ldLes at a lower frequency than conv~ntiQn~l EPR spectrorn~ot~r~ Typically, conv~ntion~l systems have 35 oscill~ting frequencies of ap,u,u2~ ,ately 9 GHz, which are strongly absorbed by high t1ielectric m~tt~ri~l~ such as water or tissue. MiCluwdvt; absorption at 9 GHz op~,r~Les much like a micluwdv~ oven, creating u-~w~l~d heating in clinical applic~ti~n~ Thus, the specL,u~eter 40 of FIGURE 3 upeldl~s with a lower frequency mi~;luwdve oscill~tor. One acceptable frequency range used is within L-band freq len~ies~ i.e., 1100-1200 MHz, which WO 95/05611 2 :L ~ 8 8 ~ 2 PCTtUS94107719 provide an acceptable co.l.~ --lise between depth penetration and sensitivity. L-band microwave frequencies are suitable for p~r~m~gnetic probes, such as India ink, located at depths of up to ten millimf tere S However, as those skilled in the art can appreciate, the specllon~eter 40 is easily constructed according to the invention at lower freqllf~nriPc~ such as within the radiofrequency range of 100 to lOOOMHz, to increase ~cll~LLdlion depth while de~;.casi..g s~ ~iLivily, which may be desirable in some applic~til~ne Those skilled in the art also nnflPr~st~n~ the prinrir~l operation of the other components of the spectrometer 40, FIGURE 3, and of other PeePnti~l co..~ollents not illustrated, as they are filnction~lly similar to cl-."p~ ble, co-lvt;lllional EPR spectrometer components.

The advantages provided by the spectrometer 40 in the context of EPR oximetry using p~r~m~gnPtic probes are several. First, the s~e~ ~LOlll~ 40 attains m;1x;...l.~.. possible depth within the target tissue while ret~inin~ sufficient s~l~iLivi~y for ~ccllr~tp~ and rapid clinical and biological appli~tione The s~c~ el 40 further is undrrt;.;~;d by the particular ~limPneionS of the target tissue, or body, to be studied because the resonator 42 is not limited 20 by the configuration of the ~eson~ LIul;LuLc; employed as the ~lptf ctor. Finally, the inevitable motions of living ~nim~l~, e.g., heart beats, f ~;~ ;on, and small physical movements, are c.~ e..~ d by adj~lctn~P~nte to the l~ ~O..hl~l r~ ut;llcy to m~int~in a b~l~n~e(l bridge.

Thus, the spectrometer 40 of FIC~URE 3 is especially well-suited for EPR
25 measurements of ~nim~le or patients when combined with the p.op~llies of physiologically acceptable par~m~gnptic m~tf ri~le, such as India ink. This combination in accordance with the invention is suitable for many clinical and t;xl.. .;...ent~l uses for the direct measure of PO2 in in vivo tissues.

In vivo measurements were first conrluGtP~I in the gastroçnPmiue mlle~les of adult mice.
A 10~11 slurry of India ink was injected into these mue~lee, whc.c;~l~l the ~nim~le were measured for EPR spectra by an EPR spectrometer, such as the spectrometer 40 of FIGURE
3. The coupled planar loop ~ntPnn~P 72, FIGURE 4, was positioned over the area of the leg co~ ;--g the India ink. When required, blood flow was restricted by a ligature around the upper leg. The ~nim~le were conscious throughout the ~ Pnt The stability of the l~sponse of India ink EPR spectra to oxygen con~Pntr~tion in the ~nice was studied by mç~enring the EPR spectra before and after restricting the blood flow.
FIGURE 5 shows the EPR signal spectra 80 of India ink-injected gastrocnemius muscle of WO 95/05611 . PCT/US94/07719 the mouse with unrestricted blood flow one day after imrl~nt~tion. When blood flow to the leg was restricted by a ligation around the upper leg, the EPR spectra response to a re-iuçtit)n of PO2 is in-lis~tçcl by the n~lu~vhlg line width and increased line height, as shown by the signal spectra 82. The corresponding PO2 before and after the constriction of the blood flow 5 were 11.4 mm Hg and 0.7 mm Hg, ~ e(;Livt;ly.

The kin~tics of de-oxygenation in in vivo mouse muscle, subsequent to the tig~ g of the tourniquet, was also monit-~red. FIGURE 6 graphically shows that the response of India ink is sufficif ntly rapid to follow the de-oxygenation, typically within 20 secon~ic This 10 lc~ol1se lasted for at least thirty-nine days, as shown by the periodic t;A~ IPnt~l data of FIGURE 7, with little reslllt~nt toxicity, as shown in FIGURE 8. The upper data points of FIGURES 6 and 7 lC~lcSt;ll~ unrestricted oxygen flow to the muscle, while the lower data points represent restricted oxygen flow. The multiple, co-located data points lc~lcstllL the several mice tested.
FIGURES 6-8 illn~tr~tç the very favorable biological properties of India ink, in~ ing stability, FIGURE 7, low toxicity, FIGURE 8, and the rapid response of the spectra to ~h~ngPs in pO2, FIGURE 6. Once India ink is injected into the tissue of interest, PO2 is measured conveniently, rapidly, and le~,lilivcly in a non-hlv~ive manner, i.e., through EPR
20 oximetry. The enormous sen~ilivi~y of carbon-based m~t~ori~l~, such as India ink, to oxygen, combined with its inert physical and ch~mic~l prop~ .~ies, make carbon-based physiological p~r~m~gnPtic m~tPri~l~ ideal probes for oxygen mea,ulc.-lents in tissues, in~ lAing that of ~nim~l~ and hnm~n~.

India ink, being clinic~lly approved m~tPri~l, can immçrli~tPIy be used within hnm~n~
to measure oxygen co,~ ".~lions in clinical settingC. The EPR ~e-;Llu.lleter col~llu~ d accol.lh~g to the invention, e.g., the ~ecllullleter 40 of FIGURE 3, with the ç~t~rn~l loop Icsol~Lul and microwave bridge, provides clinically effective EPR spectra mea~ul~.llent capability from p~r~m~gn~tic m~t~ri~lc in living ~ ..;",~nt~l ~nim~l~ and human subjects.
30 The whole process of mea~u.clllent in accordance with the invention takes less than 30 seconds.

The invention offers the ~ lition~l advantage of providing spatially resolved information of PO2 directly, because the measured EPR spectra is ~letectecl at the specific 35 point where the India ink is inserted. This technology is .oxp~n~1~hle, in accol~lce with the invention, for the ~imlllt~n~ous measu,..--ent of PO2 at two or more test sites. A single particle of India ink can also be inserted at a sçlect~hle spatial location within the biological system or tissue to provide a selectable and spatial test probe within the system. The particle is selected according to the test biological system and can be cellular in size, e.g., .1 ,um, or ' WO 95/05611 ~ 8 ~ ~ PCTtUS94/07719 relatively large in size, e.g., one centimeter. By inserting such a particle to the system, the EPR spectra is measured from a selectable and localized region in the biological system, such as within a cell or within the liver.

EPR oximetry in vivo mea~ulclllents of a human subject injected with physiologically acceptable p~r~m~gnptic m~teri~l~ were p~rùlllled through use of an extensive tattoo, illustrated in FIGURE 9, compti~inE India ink. The human subject was a volunteer who had the tattoo on his role~lll. Accordingly, the EPR spectra of the tattoo intlic~ted the oxygenation of the skin. Similar to the ~;~.. . ;.,.~nt~ cnn~lluted on the mice, EPR spectra 10 measurements were made of the tattooed skin before and after constricting the blood flow to the folc:~lll. FIGURE 10 graphically shows the India ink EPR spectra line width v~ri~tinn due to the constriction of the blood flow, providing a direct mea~u,clllent of PO2.

The particular details of the mea~ulelllents in FIGURE 10 are as follows. The ru15 with the tattoo was placed between the poles of a magnet of an L-band mic,ow~ve ~e~;Llollleter cons~ ed in accordance with the invention, such as described in FIGURE 3.
A promin~ntly black area of the tattoo was positil n~c~ on the detector and spectra were obtained before and during constriction of the blood flow by means of a rubber tourniquet around the arm and above the tattoo. When the blood flow was restricterl the EPR spectra 20 line width narrowed while its line height increased. The line width rh~nEecl from 4050 mGauss, unrestricted, to 3400 mGauss, restricted.

Methods and ~d~dLu~ for ~æL~...;..;..E oxygen conc~ ;on in tissue having one or more of the foregoing features accoldi.,g to the invention have several advantages. These 25 include the ability to directly ~ ..nin~ oxygen cn~ e~ lion in in vivo tissues in order assess their state and response to therapy. This capability is especially desirable for p1~nninE, and for ev~hl~tinE tumor therapy and vascular insufficiency. FulLL~lllore, the sensitive, accurate, and repeated mea~ulci~llents of PO2 in tissues provided for by the invention has clinical significance, especially for the Ol~Lill~i dLion and utilization of cancer therapy, and for 30 the diagnosis and tr~tm~nt of vascular disease. A number of other potential clinical applications, including the evaluation of other ~lie~ces which concern oxygen ~les~ within tissues can also benefit from the invention by providing clinically useful information. The modern hospital may eventually utilize the te~ching~ of the invention in an integral clinical role, especially in the oncology and cardiovascular sections of the hospital.
The invention further provides for a wide range of ~ studies that may be undertaken in small and large ~nim~lc These studies include the clinical areas described above, and may further include a wide range of studies in basic biology and physiology, because of the importance of oxygen conc~.l.,.lions in most physiological and -72 ~

pathophysiological processes. The results ~lcst;llLed herein, particularly from the EPR studies of India ink in mice and hllm~n~, additionally in~iC~te that methods and d~l~LldLUS in accordance with the invention achieve good signal-to-noise ratios and repeatable in vivo EPR
mea~ulclllcnL~, often without ~ The availability and safety of the p~r~m~nptic India 5 ink m~teri~l provide for the imme~ tç and in vivo usage of these methods in ~nim~l.c and hllm~n~.

India ink has been extensively used in patients as a marker for surgical procedures and radiation therapy, in addition to its t;A~ lsive non-mP~lic~l use for decoration. In general 10 surgery, India ink has been used to mark surgical resection lll~h~S. For Px~mrle tattooing with India ink has been described as a precise and practical method for identifying a biopsy site when there is cignific~nt delay bGlwt;en biopsy and d~rllliLiVc surgery. E. Fpst~Pin, J.
Dermatol, Surg Oncol. 15, 272 (1989). India ink has also been used to intlin~tP the location of lymph nodes and lylllph~lic ~h~nnPle For eY~mrle, M~.ly~na et al., Nippon Geka Gakkai 15 Zasshi 901,318 (1989), injected India ink in the pPri~tric lymph nodes of 3,785 patients who had st~ m~rh cancer at the operation in order to find mPt~ct~tic lymph nodes and reported that this technique made it easier to find lymph nodes, thereby hll~ ving prognoses. I~
radiation therapy, India ink is routinely used to mark fields for i~ tic)n. For ~ lc S. J.
Walker, Radiography Today 54, 617 (1988), made a survey of mPth~ for m~rking fields in 20 twelve radioLhcl~y centers in Britain, and ~c~lLed that tattooing with India ink was a dald procedure in most departments. There was no ~ugge~Lion of any serious problems in tattooing. In the endoscopic field, India ink is used as a long-term colonic mllco~l marker.
Fennerty et al., The American Journal of Gastroenterology 87, 79 (1992), imrl~ntell India ink tattoos to colorectal polygas of patients who were followed for at least six month~, and 25 reported no side effects or compli~tion~

The basis for the app~cllL lack of toxicity of India Ink is fairly straight-rul ~ l. India ink consists of a suspending vehicle, an em~ ifier, and the "active ingredient", which is carbon black. From analyses of its physical properties, and from experience in ~nim~l~ and 30 p~fiPnt~, the carbon black appears to be both non-reactive and non-allergenic. The particles of India ink are also very small, homogenous, and in-lPpçn-l~nt from each other. When the ink is injected hlL"lv~llously, the particles are trapped by the reticuloendothelial system, i.e., the liver and spleen, and not in the c~pill~ries of the lung. In vitro c~ Pnt.C have shown that India ink is easily taken into cells via phagocytosis, without showing any toxicity, as 35 measured by the colony-forming ability and exclusion of trypan blue. Th~lcîolc, in accordance with the invention, India ink is also useful for the selective mea~ulcll-cllL of intr~-~Pll~ r PO2.

WO 95/05611 2 ~ 7 ~ PCT/US94/07719 The invention thus attains the objects set forth above, among those a~ cnl from prece~lin~ description. Since certain changes may be made in the above a~ lus and methods without departing from the scope of the invention, it is intPn-led that all matter contained in the above description or shown in the accompanying drawing be h~ lcd as S illustrative and not in a limiting sense.

It is also to be nn~prstood that the following claims are to cover all generic and specific fc~lu cs of the invention ~es~-ribecl herein, and all st~tPmPn~.c of the scope of the invention which, as a matter of language, might be said to fall there bclwccll.

Claims

1. Apparatus for measuring oxygen tension in a biological system containing a paramagnetic material, comprising (a) magnetic means for selectively applying a magnetic field of selectable strength to the biological system, (b) electromagnetic means for selectively applying electromagnetic radiation having a selected substantially constant frequency to the biological system, (c) detection means for determining the electron paramagnetic spectra of the biological system, said spectra having a selected peak-to-peak line width that is indicative of said oxygen tension in the biological system, (d) resonator means coupled to the electromagnetic means for forming a resonator, said resonator means including means for tuning said resonator means to the constant frequency of said electromagnetic means to maintain a substantially constant resonant frequency in response to movements in the biological system.

(e) console means in communication with said detection means for displaying saidspectra, and (f) computer means connected to said console means for controlling said apparatus, and for analyzing said spectra.

2. Apparatus according to claim 1 wherein said detection means includes preamplifier means and a detector for combined, high-dynamic range detection of said electronparamagnetic spectra.

3. Apparatus according to claim 1 wherein said tuning means comprises microwave bridge means having an automatic frequency control means with a fixed frequency oscillator and a varactor diode tuned resonator, said bridge means being arranged for tuning said resonator to said fixed frequency oscillator, thereby compensating for movements in the biological system.

4. Apparatus according to claim 1 wherein said resonator means comprises a high QLC
circuit coupled with an external planar loop via .lambda./2 symmetrical line.

5. A system for determining oxygen tension within a biological system, comprising means for introducing india ink or constituents of india ink into the biological system, magnetic means for applying a first magnetic field to the biological system, radiation means for applying electromagnetic radiation having a frequency between about 100 MHz and about 5 GHz to the biological system to excite the india ink to a higher energy state, said excited india ink or constituents of india ink then relaxing at a rate dependent upon the presence of oxygen in the biological system, and means for determining the electron paramagnetic resonance spectra of the biological system, said spectra being indicative of the oxygen tension within the biological system.

6. The system of claim 5 further comprising means for sweeping the magnitude of said first magnetic field between about 1 Gauss and about 500 Gauss.

7. The system of claim 5 further comprising modulation means for generating an alternating magnetic field generally orthogonal to said first magnetic field, and means for modulating said first magnetic field between about 1 KHz and about 500 KHz.

8. The system of claim 5 wherein said means for determining comprises a resonator having an operating resonant frequency.

9. The system of claim 8 wherein said radiation means applies radiation having asubstantially constant frequency, said system further comprising resonant frequency control means for adjusting said resonant frequency of said resonator in response to movements of the biological system to match said constant frequency of said radiation means.

10. The system of claim 5 further including a resonator operable at a selected resonant frequency.

11. The system of claim 10 wherein said radiation means applies radiation having a substantially constant frequency, said system further including tuning means for tuning said resonant frequency of said resonator to said constant frequency of said radiation means to maintain a substantially constant resonant frequency.

12. The system of claim 11 wherein said tuning means includes an automatic frequency control circuit.

13. The system of claim 10 wherein said resonator includes an LC resonant circuit, and an external planar inductive loop in communication with said resonant circuit.

14. The system of claim 13 wherein said resonator further includes a variable inductive coupling and a varactor diode in electrical communication with said resonant circuit.

15. A system for determining oxygen tension within a biological system containing a paramagnetic material, comprising magnetic means for applying a magnetic field to the biological system, radiation means for applying electromagnetic radiation having a selected substantially constant frequency to the biological system to excite the paramagnetic material, resonator means operable at a selected resonant frequency, tuning means for tuning said resonant frequency of said resonator to match said constant frequency radiation means to maintain a constant resonant frequency in response to movements in the biological system, and means for determining the electron paramagnetic resonance spectra of the biological system, said spectra being indicative of the oxygen tension within the biological system.

16. The system of claim 15 wherein said tuning means comprises automatic frequency control means having a fixed frequency oscillator and a varactor diode tuned resonator.

17. The system of claim 15 wherein said electron paramagnetic material comprises carbon black.

18. A method for determining the oxygen tension in a biological system, comprising the steps of introducing into the biological system carbon black, india ink or selected constituents of india ink having a physiologically acceptable paramagnetic quality, and determining the electron paramagnetic resonance spectra of the biological system, the spectra being indicative of oxygen tension within the biological system.

19. The method of claim 18 wherein the step of determining the electron paramagnetic resonance spectra comprises the step of determining the peak-to-peak line width of said spectra.

20. The method of claim 18 wherein said carbon black, india ink, or constituents of india ink comprise substantially uniform particles having diameters between approximately 0.1 and 100 microns.

21. The method of claim 20 wherein said paramagnetic material comprises at least one particle having a diameter between approximately 100 microns and one centimeter, said particle functioning as a point source for said spectra in the biological system.

22. The method of claim 18 comprising the steps of applying an electromagnetic field having a frequency between approximately 100 MHz and 5 GHz to the system, and determining the electron paramagnetic resonance spectra of the system, said spectra being indicative of the oxygen tension of the biological system.

23. A method for measuring oxygen tension in a biological system, said method comprising the steps of applying a magnetic field of selectable strength to the biological system, applying electromagnetic radiation having a substantially constant resonant frequency to the biological system, detecting the electron paramagnetic spectra of the biological system, and spectra having a peak-to-peak line width that is indicative of said oxygen tension in the biological system, providing a resonator having an operable resonant frequency, tuning said resonator to the constant frequency of the electromagnetic radiation to maintain a substantially constant resonant frequency in response to movements in the biological system, and displaying and analyzing said spectra.

24. The system of claim 23 further comprising the step of introducing a paramagnetic material to the biological system.