Spectrophotometric method for quantitatively determining the concentration of a dilute component in a light- or other radiation-scatteri

What is claimed is:

1. A spectrophotometric method of quantitatively determining the concentration of a dilute component in an environment containing the dilute component of known identity but of unknown concentration in combination with a reference component of known concentration, by a series of successive, substantially contemporaneous measurements of transmitted and/or reflected radiation at selected wavelengths, comprising:

(a) determining the apparent effective pathlength in said environment;

(b) directing at said environment incident electromagnetic radiation of a first wavelength in a selected spectral region at which the dilute and/or reference component(s) exhibit absorption for the electromagnetic radiation;

(c) measuring the first wavelength radiation transmitted and/or reflected by the environment;

(d) directing at the environment incident electromagnetic radiation of at least one other wavelength in the selected spectral region at which the dilute and/or reference component(s) exhibit absorption of different relative intensities than for the first wavelength incident radiation;

(e) measuring the other wavelength radiation transmitted and/or reflected by the environment;

(f) determining extinction coefficient values for the dilute component at said first and other wavelengths in said environment; and

(g) based on the apparent effective pathlength determined for the environment, the extinction coefficient values for the dilute component at said first, and other wavelengths, and the measured absorbed and/or reflected radiation at said wavelengths, determining the relative amount of the dilute component to the amount of the reference component, as the concentration of the dilute component in the environment.

2. A method according to claim 1, wherein said apparent effective pathlength of said environment is determined by the steps comprising:

(i) measuring the absorbance of said reference component at different selected wavelengths in said selected spectral region;

(ii) calculating the differential absorbance from the measured absorbance values;

(iii) determining the extinction coefficient values for said reference component at the different selected wavelengths of step (i);

(iv) calculating the differential extinction coefficient from the determined extinction coefficient values for said reference component; and

(v) dividing the differential absorbance by the differential extinction coefficient to yield the apparent effective pathlength of said environment.

3. A method according to claim 1, wherein said determination of step (f) is effected by establishing simultaneous modified Beer-Lambert equations for each of said absorption/scattering measurement steps, and solving said equations for the concentrations of said dilute component and said reference component is said environment.

4. A method according to claim 1, wherein a second dilute component of unknown concentration is contained in said environment, and wherein electromagnetic radiation of a third wavelength in said selected spectral region is directed at said environment at which said second dilute component exhibits an absorption for the electromagnetic radiation, and the third wavelength radiation transmitted and/or reflected by the environment is measured, and employed to determine the relative amount of the second dilute component to the amount of the reference component.

5. A method according to claim 4, wherein three simultaneous modified Beer-Lambert equations are established for the concentration of the dilute components and reference component in said environment.

6. A method according to claim 1, wherein said environment effects scattering of said electromagnetic radiation which is independent of wavelength of said radiation, and wherein the transmitted and/or reflected radiation is measured at a single additional wavelength.

7. A method according to claim 1, wherein said environment effects scattering of said electromagnetic radiation to an extent which is of sloped linear relationship to wavelength, and wherein the transmitted and/or reflected radiation is measured at an additional two wavelengths.

8. A method according to claim 1, wherein said environment effects wavelength scattering of said electromagnetic radiation which is a non-linear function of wavelength, and wherein the transmitted and/or reflected radiation is measured at an additional at least three wavelengths.

9. A method according to claim 1, wherein said environment is a corporeal environment.

10. A method according to claim 9, wherein said environment is selected from the group consisting of corporeal tissue and corporeal organs.

11. A method according to claim 9, wherein the dilute component is selected from the group consisting of tissue components and blood-borne species.

12. A method according to claim 9, wherein the dilute component is selected from the group consisting of enzymes, metabolites, substrates, waste products and poisons.

13. A method according to claim 9, wherein the dilute component is selected form the group consisting of glucose, hemoglobin, oxyhemoglobin, and cytochrome a,a.sub.3.

14. A method according to claim 9, wherein said corporeal environment comprises a body portion selected from the group consisting of heads, fingers, hands, toes, feet, and earlobes.

15. A method according to claim 9, wherein said reference component is water.

16. A method according to claim 1, wherein said environment comprises an externally added indicator solution as a reference component.

17. A method according to claim 14, wherein said environment is a corporeal corpse body moiety, and said indicator is an aqueous solution of indocyanine green dye.

18. A method according to claim 1, wherein a first dilute component and reference component exhibit absorption for the electromagnetic radiation in said selected spectral region and a second dilute component does not exhibit absorption for the electromagnetic radiation in said spectral region, and wherein the first and second dilute components exhibit absorption for electromagnetic radiation in a second spectral region closely proximate to said selected spectral region and in which the reference component does not exhibit absorption for the electromagnetic radiation, comprising determining the relative amount of the first dilute component to the amount of the reference component in said selected spectral region as the concentration of the first dilute component in the environment, and utilizing the first dilute component whose concentration is thus determined, as a reference component for the second dilute component in said second spectral region.

19. A method according to claim 1, wherein said electromagnetic radiation is infrared radiation having a wavelength in the range of from about 700 to about 1400 nanometers.

20. A method according to claim 1, wherein subsequent to determination of the concentration of the dilute component in the environment, the environment is monitored for changes in said concentration.

21. Apparatus for spectrophotometrically quantitatively determining the concentration of a dilute component in an environment containing the dilute component of known identity but of unknown concentration in combination with a reference component of known concentration, comprising:

(a) means for producing electromagnetic radiation of known wavelengths and directing said radiation into the environment to be characterized for the dilute component;

(b) means for detecting electromagnetic radiation emanating from and/or reflected from the environment and producing therefrom an electrical signal corresponding thereto;

(c) means for receiving said electrical signal and producing therefrom electrical signals corresponding to said different wavelengths;

(d) means receiving and operatively responsive to said electrical signals corresponding to said different wavelengths, to establish absorbance equations responsive to said electrical signals corresponding to said different wavelengths, wherein absorbance at each of the wavelengths is expressed as a function of the relative intensities of the absorption contributions of the dilute and reference components and the concentrations of the dilute and reference components, and for calculating the amounts of the dilute and reference components by solution of said absorbance equations; and

(e) means for displaying the calculated concentrations of said dilute and reference components.

22. A spectrophotometric method of quantitatively determining the concentration of a dilute component in an environment containing the dilute component of known identity but of unknown concentration in combination with a reference component of known concentration, comprising:

(a) directing at the environment incident electromagnetic radiation at a number of wavelengths in a selected spectral region at which the dilute and/or reference components exhibit absorption for the electromagnetic radiation, the number of said wavelengths being determined by the number of dilute and reference components in the environment, and the scattering characteristics of the environment;

(b) determining the absorbance by the environment of the electromagnetic radiation at the various wavelengths and the relative intensities of the absorption contributions of the dilute and reference componets and scattering losses from the environment at each of said wavelengths;

(c) at each of said wavelengths, establishing absorption equations of the form: ##EQU6## wherein: Abs.sub.w is the absorbance by the environment, containing the dilute and reference components, of the incident electromagnetic radiation of wavelength w; x.sub.i is the relative intensity of the absorption contribution of the associated dilute component A.sub.i, and wherein terms of the form x.sub.i A.sub.i are set forth for each of the dilute components; n is the number of dilute components; z is the relative intensity of the absorption contribution of the reference component; R is the concentration of the reference component; and S is the normalized scattering of the environment at wavelength w; thereby establishing for each of said wavelengths an absorbance equation, to yield a set of simultaneous equations whose number equals the number of dilute and reference components and the number of wavelengths required to characterize the scattering of the environment;

(d) deriving algorithms by matrix solution of said simultaneous equations, said algorithms being of the form: ##EQU7## wherein: [c] is the concentration of the specific dilute or reference component; a.sub.i is a determined numerical constant; m is the number of said wavelength determinations; Abs.sub.w.sbsb.i is the absorbance at wavelength w; thereby establishing the concentration of each of the dilute and reference components in the environment.

23. A method according to claim 22, wherein subsequent to determination of the concentration of each of the dilute and reference components in the environment, the environment is monitored to determine changes in said concentrations.

Description Submit all comments and votes

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to a spectrophotometric method of quantitatively determining the concentration of a dilute component in a light- or other radiation-scattering environment containing the dilute component in combination with a reference component of known concentration.

2. Description of the Related Art

In many fields of technology there is a need for quantitative determination of dilute component concentrations in environments where the dilute component is in combination with a reference component of known concentration. Examples of illustrative environments of such type include enzymes, proteins, and metabolites in corporeal fluids; acidic fumes or gaseous components (e.g., hydrogen sulfide and sulfuric acid, nitric acid, carbon monoxide, etc.) in the atmosphere; salt concentrations in sea water undergoing desalination; ozone in ozone-enriched air utilized in waste water ozonation systems, etc.

In particular, there has been a specific need in the medical and health care fields for a non-invasive, continuous, atraumatic, in vivo, in situ determination of amounts of critical metabolic indicators in body fluids or tissues of human patients. Examples of such body fluids include the blood and fluids associated with the lymphatic and neurological systems of the body. Further specific examples involving the human circulatory system include the monitoring of glucose and of oxygenated/de-oxygenated, arterial/venous colored hemoglobin in the blood stream. In addition, monitoring in localized tissue, such as brain and muscle, of certain enzyme species such as the cytochrome c oxidase enzyme (unofficially better known as cytochrome a, a.sub.3) or metabolic substrates (such as glucose) or products (such as carbon dioxide) is becoming an increasingly urgent practical application of spectrophotometric technology.

Spectrophotometric methods have been proposed in the art to monitor metabolites in corporeal fluids. Such methods involve the impingement of radiation, typically in the visible or near-infrared region, onto the exterior body portion of the subject for transdermal and interior tissue penetration of the radiation, which is monitored as to its reflectance or transmission, at a wavelength condition at which the metabolite or other monitored component is selectively absorptive for the radiation. This technique is mainly limited to yielding a qualitative determination from the measured output radiation (reflected or transmitted) of the qualitative character of the metabolism. At best a semi-quantitative result can be obtained in a so-called trend monitoring mode where concentration changes can be monitored in terms of an original baseline condition of unknown concentration.

Solute concentrations in dilute fluid media can be theoretically quantified by the Beer-Lambert law, i.e.,

log (I.sub.o /I)=d.times.E.times.c

wherein:

I.sub.0 =intensity of source radiation impinged on the sample;

I=intensity of radiation transmitted through the sample;

E=absorption (extinction) coefficient of the solute species at the wavelength of the source radiation impinged on the sample;

d=optical distance (travel pathlength of the radiation transmitted through the sample); and

c=concentration of the solute (dilute component) in the solution sample.

Although the foregoing Beer-Lambert Law equation permits a ready determination of solute concentration to be made in in vitro or other non-corporeal discrete sample systems utilized for conventional spectrophotometric assays, such direct, quantitative measurement is not possible in the intact body, even though the influent radiation is penetrative of the body elements of the corporeal system, e.g., bones, musculature, organs, and the like, since the scattering of radiation during its passage through the corporeal system is extensive and highly variable in character. Such scattering not only adds an unknown loss of radiation to the required information regarding specific absorption but by multiple scattering it also lengthens to an unknown degree the path length of those photons eventually emerging from the body element. As a result, it has not been possible to determine in an in vivo situation what the effective path length, d, of the impinged radiation actually is, prior to measurement of the transmitted or reflected radiation derived therefrom. In consequence, the absolute quantitation of solute concentrations in corporeal systems has been severely adversely limited.

Faced with the alternatives of invasive and traumatic sampling of the corporeal fluids of interest, or spectrophotometric methods which realize only qualitative or at best semi-quantitative measurement of changes in tissue or body fluid solute concentrations, there is a substantial perceived need in the art for a non-invasive, in vivo method of quantitatively determining the concentration in corporeal solution of a solute in a body fluid solvent.

A similar need exists in numerous other fields in which absolute concentrations of dilute component species in fluid media would materially assist the characterization of the fluid system. An example is atmospheric monitoring of "acid rain", i.e., airborne acidic contaminants which have in recent years proliferated and been determined to cause widespread biospheric damage, including the defoliation of forest stocks and spoliation of natural bodies of water and other aqueous environments. It is anticipated that in coming years with the increasing severity of the acid rain problem, correspondingly greater scientific and legislative efforts will be focused on the monitoring of acid rain with a view to controlling and minimizing its adverse impacts. Determinations in the naturally existing medium such as turbid water and hazy or cloudy atmosphere will be a great boon for direct, effective and rapid monitoring of these environments.

In numerous other industrial and natural systems there is a need to quantitatively monitor solute species in an indirect manner not involving the time, effort, and cost of discrete sample collection, purification and analysis.

U.S. Pat. No. 4,281,645 to F. F. Jobsis describes a spectrophotometric system for monitoring cellular oxidative metabolism by non-invasively measuring in vivo changes in the steady state oxidation-reduction of cellular cytochromes together with changes in blood volume, the oxidation state of hemoglobin and the rate of blood flow in the brain, heart, kidneys, other organs, limbs, or other parts of a human or animal body.

The methodology described in the Jobsis patent involves transmitting near-infrared radiation in at least two different and periodically recurring wavelengths through the corporeal environment, and detecting and measuring the radiation intensity which emerges at another, distant point or on the opposite side of the body, for the monitoring of biochemical reactions, utilizing an approximation of the Beer-Lambert law. One of such wavelengths selected for the measurement is in a range for which oxidized cytochrome a, a.sub.3, is selectively highly absorptive. One or more reference signals are provided at corresponding wavelengths outside the peak of the cytochrome absorption band but preferably in close proximity to the measuring wavelength. The difference or ratio between the measuring and reference signals is determined and non-specific changes in the intensity of transmitted radiation not attributable to absorption by the cytochrome species are eliminated. Thus, the system of this patent produces an output signal representing the difference in or ratio of absorption of the measuring and reference wavelengths by the organ or other corporeal portion of the body as a function of the state of the metabolic activity in vivo, which may be converted to a signal providing a substantially continuous measure of such activity. A related spectrophotometric reflectance technique is disclosed in U.S. Pat. No. 4,223,680 to F. F. Jobsis.

U.S. Pat. No. 4,655,225 to C. Dahne et al discloses a spectrophotometric system for non-invasive determination of glucose concentration in body tissue. The system involves irradiation of the exterior body portion with an optical light whose transmittance or reflectance is collected at selected band wavelength values for the glucose absorption spectrum and at a selected band wavelength value for the absorption spectrum of background tissue containing no or insignificant amounts of glucose. The measuring and reference radiation collected is then converted into electrical signals and utilized to determine glucose concentrations.

It is an object of the present invention to provide an improved method and apparatus for indirectly quantitatively, spectrophotometrically determining the amounts of a dilute component by using a reference component in the environment of interest.

It is another object of the invention to provide such a method for non-invasive, in vivo quantitative determination of the concentration of a dilute solute component in a corporeal solvent environment.

Other objects and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims.

SUMMARY OF THE INVENTION

The present invention relates to a method of determining the true concentration (e.g., in terms of grams or moles of a dilute component per volume of a reference component) in environmental media in which the optical pathlength is ill-defined due to the extensive occurence of scattering of incident radiation, such as in very long distance atmospheric monitoring as well as in more intensely light scattering media during transillumination as well as diffuse reflectance modes of spectrophotometry.

As used herein, the term "environment" refers to a selected spatial region in which the directed and measured radiation is transmitted and/or reflected along substantially the same path.

The crux of the invention is to measure the transmitted and/or reflected radiation for both the dilute component of unknown concentration and the reference component of known concentration with which it is associated. Multiple scattering spoils the optical pathlength parameter in the Beer-Lambert equation and does so to different degrees depending on the wavelength. It is, therefore, necessary to measure the dilute and reference components in closely the same spectral region. Measuring the intensity of the light absorption and/or reflectance by the two types of molecules, dilute component and reference component, in the environment and applying the extinction coefficients of each provides the opportunity to relate them to one another in terms of relative amounts, which is recognized as being the essential character of concentration. Thus, by absorption (and/or reflectance) measurements and with knowledge of the extinction coefficients, the amount of dilute component in the light path and the amount of reference component it is associated with, determined similarly at other near-by wavelength(s), may be employed to calculate the concentration of the dilute component relative to the reference component.

In a system in which the reference component is present in known concentration, the apparent pathlength may be determined by absorption measurements taken in the environment of unknown pathlength in the same electromagnetic spectral region. The difference between the resulting absorption values is calculated as the differential absorbance in the environment whose pathlength is to be determined. The tabulated or previously determined extinction coefficient values of the pure reference component at such wavelengths are then employed to calculate the differential extinction coefficient, as the difference between the respective extinction coefficient values. When the differential absorbance is then divided by the differential extinction coefficient, the result is the apparent effective pathlength of the environment. When the electromagnetic radiation emitter and detector spacing distance is measured, the pathlengthening factor for the system is determined as the ratio of the apparent effective pathlength to the actual emitter-to-detector spacing distance.

In another selected, specific aspect, the invention relates to a spectrophotometric method of quantitatively determining the concentration of a dilute component in an environment containing the dilute component of known identity but of unknown concentration in combination with a reference component of known concentration, by a series of successive, substantially contemporaneous measurements of transmitted and/or reflected radiation at selected wavelengths, comprising:

(a) determining the apparent effective pathlength in said environment;

(b) directing at the environment incident electromagnetic radiation of a first wavelength in a selected spectral region at which the dilute and/or reference component(s) exhibit absorption for the electromagnetic radiation;

(c) measuring the first wavelength radiation transmitted and/or reflected by the environment;

(d) directing at the environment incident electromagnetic radiation of at least one other wavelength in the selected spectral region at which the dilute and/or reference component(s) exhibit absorption of different relative intensities than for the first wavelength incident radiation, whereby absorption is exhibited in said selected spectral region comprising said first and at least one other wavelength by both the dilute and the reference component(s);

(e) measuring the other wavelength radiation transmitted or reflected by the envinroment; and

(f) determining extinction coefficient values for the dilute component at said first and other wavelengths in said environment; and

(g) based on the apparent effective pathlength determined for the environment, extinction coefficient values for the dilute component at said first and other wavelengths, and the measured absorbed and/or reflected radiation at said wavelengths, determining the relative amount of the dilute component to the amount of the reference component, as the concentration of the dilute component in the environment.

In another aspect of the method broadly described above, the determination of step (f) is effected by establishing simultaneous modified Beer-Lambert equations for each of the radiation absorption and/or reflectance measurement steps, and solving the equations for the concentrations of the dilute component and the reference component in the environment.

A further aspect of the invention relates to a spectrophotometric method of quantitatively determining the concentration of a dilute component in an environment containing the dilute component of known identity but of unknown concentration in combination with a reference component of known concentration, comprising:

(a) directing at the environment incident electromagnetic radiation at a number of wavelengths in a selected spectral region at which the dilute and/or reference components exhibit absorption for the electromagnetic radiation, the number of such wavelengths being determined by the number of dilute and reference components in the environment, and the scattering characteristics of the environment;

(b) determining the absorbance of the environment of the electromagnetic radiation at the various wavelengths and the relative intensities of the absorption contributions of the dilute and reference components and scattering losses from the environment at each of such wavelengths;

(c) at each of such wavelengths, establishing absorption equations of the form: ##EQU1## wherein: Abs.sub.w is the absorbance by the environment, containing the dilute and reference components, of the incident electromagnetic radiation of wavelength w; x.sub.i is the relative intensity of the absorption contribution of the associated dilute component A.sub.i, and wherein terms of the form x.sub.i A.sub.i are set forth for each of the dilute components; n is the number of dilute components; z is the relative intensity of the absorption contribution of the reference component; R is the concentration of the reference component; and S is the normalized scattering of the environment at wavelength w, thereby establishing for each of such wavelengths an absorbance equation, to yield a set of simultaneous equations whose number equals the number of dilute and reference components and the number of wavelengths required to characterize the scattering of the environment;

(d) deriving algorithms by matrix solution of the aforementioned simultaneous equations, said algorithms being of the form: ##EQU2## wherein: [c] is the concentration of the specific dilute or reference component, a.sub.i is a determined numerical constant; m is the number of said wavelength determinations;