Abstract:
Spectral, frequency based non-invasive procedure for determination of blood constituents utilizing in vivo NIRS (Near-Infrared Spectrum) technology, which is the measurement of the near-infrared absorption spectrum within a region of the living human body for the purpose of identifying tissue and blood components and their concentrations and more particularly to novel applications and methodology for determining the optical response, measurements and calculations relating to the concentrations of individual chromophores in the bloodstream and particularly to the level of CO chromophores in the tissues of an animal or human being.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to in vivo NIRS (Near-Infrared Spectroscopy) technology, which is the measurement of the near-infrared absorption spectrum within a region of the living human body for the purpose of identifying tissue and blood components and their concentrations and more particularly to novel applications and methodology for determining the optical response, measurements and calculations relating to the concentrations of individual chromophores in the tissues and particularly to the level of CO chromophores in the tissues of an animal or human being. 
       BACKGROUND OF THE INVENTION 
       [0002]    Transillumination is, at the most basic level, the transmission of light through tissues of the body. A common example is the transmission of light from a flashlight through fingers, producing a red glow easily observed in a darkened room. This is due to the fact that red blood cells absorbed other colors of the beam and favor transmission of the red component. Except for blood, human tissue is surprisingly translucent to near infrared light. The effect can be easily seen using a red laser “pointer” as used for slide presentations to probe one&#39;s own tissues in a dark room. The light is transmitted through thin parts (cheek, ear, fingers, etc.) and a halo of “back-scattered” light can be observed from all tissues. 
         [0003]    Transillumination is a major application of visible light in medicine. It is mainly used by pediatricians to shine light into the bodies of infants and observe the amount of scattered light. Since their skeleton is not fully calcified, light can easily penetrate tissues. Common examples of diagnosis are: Light penetrates to the inside of the skull of the infant. If there is an excess of cerebrospinal fluid (CSF), light is scattered to different parts of the skull, producing patterns characteristic to hydrocephalus. The device used in this operation is a Chun gun that uses a 150 watt projection bulb as a light source. Another example is where bright light penetrates the thin front chest wall and reflects off the back chest wall to indicate the degree of pneumothorax. To treat it, a physician will insert a needle attached to a syringe into the area of collapse to remove the air between lungs and chest wall, causing the lung to reinflate. 
         [0004]    The features and forms of transillumination have also been used for the examination and analysis of tissue. Specifically the in vivo examination of human female breast tissue, and also to the general field of optical light propagation and response technology in the examination of human female breast anatomy by use of optical response observation, response measurements and characterization, and including spectral response by way of transmissibility, reflection and scattering aspects and relationships. 
         [0005]    Blood consists of 55% plasma (water, dissolved salts and proteins), 45% red cells and a smattering of white cells. Virtually all the oxygen carried by the blood is bound to hemoglobin in the red cells (erythrocytes). There are about 280 million red-colored hemoglobin molecules within each of the 10 13  erythrocytes circulating in the blood. Each molecule of hemoglobin can carry as many as four molecules of oxygen and hemoglobin can transport over 70 times more oxygen than can be simply dissolved in the blood. Bright red hemoglobin that is 100% saturated with oxygen is called oxyhemoglobin (HbO). Hemoglobin without oxygen is called deoxyhemoglobin (or simply Hb) and is much darker and bluish. 
         [0006]    At sea level pressure (760 mm Hg) the 21% oxygen in air has a partial pressure (pO 2 ) of 160 mm of mercury. If air is bubbled through blood, the hemoglobin becomes about 100% saturated with oxygen. The saturation of hemoglobin is not linear with the partial pressure of oxygen but “s” shaped, rising very slowing at low pO 2  and then more rapidly as the oxygen tension approaches 20 mm where the slope becomes steepest. By 60 mm the saturation is already up to 90 “SAT” (percentage of full saturation). This relationship, called the “oxygen dissociation curve” is not fixed, but highly affected by other constituents such as carbon dioxide, acidity (pH) and temperature. At 30 mm oxygen tension, a 10 mm increase in CO 2  tension will lower oxygen saturation by about 4 SAT units as will a 0.1 pH decrease or 2 degree C. temperature increase. 
         [0007]    Hemoglobin takes on a maximal load of oxygen over a wide range of atmospheric oxygen partial pressures and carries it to the tissues of the body where oxygen is being consumed and the O 2  tension is low. As tissue pO 2  falls, the hemoglobin is forced to give up some of its oxygen making it available to the tissue. The greater the local metabolism, the lower the tissue oxygen tension. In rapidly metabolizing tissue, the O 2  tension falls to the steep part of the dissociation curve and much more oxygen is released by the hemoglobin. 
         [0008]    The oxygen-depleted venous blood flows back to the lungs where the hemoglobin again becomes saturated with oxygen from the alveoli of the lungs. 
         [0009]    In order to transport oxygen to cells, there needs to be at least a 20 mm (Hg) gradient between the oxygen-using part of the cell, i.e. mitochondria and the hemoglobin in the capillaries. 1  When pO 2  falls to this value little oxygen can be transferred even though the hemoglobin is still 25% saturated. 2    1 Landis E M, Pappenheimer J R, “Exchange of substances through the capillary walls”, Chapter 29, Handbook of Physiology, Circulation, Vol. II (Hamilton W F, editor), 1963, American Physiological Society, Washington, DC. 2 Anaerobic degradation of glucose to lactate (glycolysis) is an emergency way of producing chemical energy (ATP)_when the oxygen supply is cut off. In fact, red cells are always anaerobic. They do not use oxygen but depend entirely on glycolysis for their chemical energy. 
         [0010]    Much of the foregoing can be explained by the existence of two species of hemoglobin called “relaxed” hemoglobin (Hb R ) and “tense” or “tight” hemoglobin (Hb T ). 3  Although these molecules have the same chemical composition, they are dimensionally slightly different. The difference that we are mostly interested in is the distance of the iron atom in the flat “heme” groups from the effective hemoglobin surface. In Hb T , all four iron atoms are 6×10 −5  microns deeper than Hb R . The result is that oxygen can reach an iron atom in the Hb R  form allowing it to bind tightly while in Hb T  the affinity for oxygen binding is about 100 times less, only about 1% of its affinity for Hb R .  3 Perutz M F, “Hemoglobin Structure and Respiratory Transport”, 1978, Scientific American 239 #6 (Dec) pp. 92-125. (Note that Perutz and Kendrew shared the Nobel prize in chemistry in 1962 for their discovery of the structures of hemoglobin and myoglobin). 
         [0011]    Hemoglobin is either in one form or the other—there is nothing in between. The hemoglobin molecule literally “snaps” between the two forms. Deoxyhemoglobin consists of Hb T  molecules and oxyhemoglobin consists of Hb R . What happens in between explains a lot. 
         [0012]    Assume deoxyHb T  is at zero pO 2  and gradually increase the oxygen tension. At low pressures, very little oxygen will be absorbed because of the very low oxygen affinity of tight hemoglobin. This is the “toe” of the dissociation curve. As pO 2  increases, some of the Hb T  molecules will begin to add a molecule of oxygen. As pressure continues to increase, more hemoglobin molecules will bind an oxygen molecule. At some point, the pressure will be high enough for some hemoglobin molecules to bind a second oxygen. As the number of bound oxygen molecules increase so does the tendency for hemoglobin to snap from Hb T  to Hb R . Once the change takes place to the relaxed form of the hemoglobin molecule, its affinity for oxygen is so great that it quickly fills all four of its heme sites. One of the factors driving the conversion may be the tension of the bound oxygen molecule pulling on the iron atom in the heme group. Some hemoglobin molecules convert on adding the second oxygen and others ay wait until the third. Hb T  cannot exist with four oxygen molecules. Other coexisting conditions affect the conversion point. As pO 2  increases, a crescendo of clicking Hb T  to Hb R  conversions occurs giving rise to the steep part of the oxygen dissociation curve. 
         [0013]    The reverse process occurs. Arterial hemoglobin is almost entirely oxyhemoglobin (Hb R ). As it enters the capillary beds of the tissues, it encounters falling pO 2  which starts the conversion of some Hb R  to Hb T . When a hemoglobin molecule snaps to the tight form, its four heme groups retract and the remaining oxygen gets ejected making it available for tissue metabolism. The transition from Hb R  to Hb T  is strongly enhanced by H +  ions entering the red cells. A single H +  ion can trigger the conversion of a molecule of hemoglobin to the tight form and eject its remaining oxygen. This is the mechanism for the “Bohr” effect. 
         [0014]    Besides H +  ions, increasing concentrations of Cl ions, CO 2  and DPG 4  all favor Hb T . Most of the agents that drive hemoglobin to the tight form also raise  4 The release of oxygen is enhanced by 2,3-Diphosphoglycerate (DPG). There is about one molecule of DPG for every molecule of hemoglobin in red cells. More DPG both decreases the hemoglobin affinity for oxygen and steepens the slope of the “s-shaped” dissociation curve. Taken together, the effect is to force hemoglobin to release even more oxygen to tissues as pO 2  falls. It turns out that DPG plays a direct role in tightening the hemoglobin molecule and forcing its conversion to Hb T . the equilibrium constant for the first oxygen bound, which exaggerates the “s” shape of the dissociation curve and increases its maximum slop (Hill&#39;s coefficient”). 
         [0015]    Agents driving the hemoglobin the other way towards Hb R  are, besides oxygen, carbon monoxide (CO) and nitric oxide (NO). Hemoglobin has even more affinity for these gases than for oxygen. A heavy smoker will lower the O 2  concentration in the arterial blood by 20%. Increasing temperature favors Hb R  and decreases the maximum slope of the dissociation curve. 
         [0016]    Except for small pressure gradient effects between capillary blood pO 2  and mitrocondra pO 2 , and variations in the dissociation curve as discussed above, the hemoglobin oxygen saturation of the venous blood draining the capillaries is indicative of tissue oxygen partial pressure. 5  To the extent that large vessels act as “sinks” for photons, NIRS technology favors capillaries and venoles.  5 Without knowing the blood flow, it does not tell us much about oxygen consumption of the tissue (extraction). 
         [0017]    The absorption spectrum of a chromophore, a colored molecular constituent is typically shown as a graph of its attenuation as a function of light wavelength. Besides, Hb, HbO 2  and HbCO as shown in  FIG. 1 , there are many other clinically interesting chromophores found in tissue including methemoglobin, sulfhemoglobin, various cytochromes (distinguished by spectroscopy), bilirubin and glucose. 
         [0018]    Note that hemoglobins are strongly absorbing at visible wavelengths—which is why they look colored to our eyes. Water looks transparent (clear) having little absorption in the visible. On the other hand, both water and fat have complex absorption spectra in the infrared. 
         [0019]    If tissue were to contain only a mixture of HbO 2  and Hb (in blood)—and no other chromophores—then measurements at only two wavelengths would suffice to determine their relative concentrations. Two wavelengths are commonly used in commercial “pulse oximeters.” If carboxyhemoglobin (HbCO) is present, they will have errors in their readings of oxygen saturation. The latest pulse oximeters correct for carboxyhemoglobin by adding spectral measurements at additional wavelengths. 
         [0020]    Wavelength selection in these instruments is by the use of wavelength-selective filters with narrow “pass bands.” These filters are designed to be relatively transparent to a specified wavelength and reject light at all other wavelengths. In order to analyze blood and tissue for an increasing number of constitutes it becomes necessary to add more and more filters with carefully chosen transmission windows. The location of these windows must be such that they maximize sensitivity and discrimination for the desired constituents while minimizing interference from all other constituents. 
         [0021]    Therefore, for a two constituent device a minimum of two filters (wavelengths) are needed to generate two equations that can be solved for two unknowns. Three constituents require at least three filters—and so forth. But this only works if these are major constituents and spurious absorption by other chromophores does not materially interfere. The number of filters needed to analyze for many constituents and suppress interference from other chromophores becomes very large. 
       OBJECT AND SUMMARY OF THE INVENTION 
       [0022]    The present invention solves this wavelength selection issue by using a CCD, a Charge-Coupled capacitor (or other) spectrometer which looks at all wavelengths. This is equivalent to hundreds or thousands of discrete filters. And by using the nose septa as a thin “sample cell” the wavelength range is extended from the near infrared well down into the visible where there is a lot of unique wavelength absorption structure for many chromophores of interest. In all cases the final concentrations of the chromophores will be determined by deconvolving, also known as decomposition, their absorption spectra from the measured total absorption spectra. 
         [0023]    Because the body is so transparent, even a very small amount of an absorbing component in the tissue can be measured. How many photons of each wavelength are absorbed by the component depends on its concentration and characteristic absorption spectrum. 
         [0024]    For example, if the absorber is the hemoglobin in red cells, the light reduction will depend on the wavelength of the injected photons and the amount of oxygen bound to hemoglobin. The absorption spectrum of hemoglobin is strongly dependent on how much oxygen it is carrying. We are all familiar with the bright red appearance of oxygen-saturated arterial blood compared with the darker and bluer venous blood from which much of the oxygen has been removed. In the infrared, as the photon wavelength gets longer the absorption of oxygen-saturated blood gradually increases while the initially strong absorption of blood with little oxygen falls rapidly. At about 800 nm the curves cross (the “isobestic” point) and at that wavelength the absorption of photons by hemoglobin is uniquely independent of the oxygen carried. By measuring the signal at the isobestic wavelength, we can measure the total concentration of hemoglobin in the region sampled by the photons, and by comparing the absorption at another wavelength we can estimate its average oxygen saturation. This is the principle of operation of the Somanetics INVOS™ Cerebral Oximeter, the only FDA-approved medical device based on this technology (U.S. Pat. No. 4,570,638) 
         [0025]    Wherefore it is a primary object of the present invention to probe into the body using only light sources and detectors on the surface of the skin in a simple, non-invasive, cost-effective procedure. 
         [0026]    It is also an object of the present invention to overcome the above mentioned shortcomings and drawbacks associated with the prior art. 
         [0027]    Another object of the present invention is to provide a relatively simple and effective non-invasive apparatus for determining the concentrations of blood constituents and in particular the concentration of CO an animal or human subject. 
         [0028]    A further object of the present invention is to apply light to a desired tissue sample having a high concentration of blood and determine the amount of the light which is absorbed by the tissue sample. 
         [0029]    Yet another object of the present invention is to provide a light source which can impart a plurality of sequentially different light spectra or a “white” light source using a spectrometer for detection. The purpose in either case is to produce a comparison of the spectra with the light passing through the tissue sample. 
         [0030]    A still further object of the present invention is to provide a method of determining the CO level in an animal or human subject by applying the light to the nasal septum of the subject. 
         [0031]    The present invention also relates to A method for non-invasively determining the relative amounts of a plurality of chromophores in the blood of a human or animal subject, the method including the steps of imparting a wavelength range of light comprising visible and near-infrared components to a light delivery fiberoptic, passing the wavelength range of light through a selected tissue sample of the human or animal subject and receiving an unabsorbed portion of the wavelength range of light in a light collecting fiberoptic, providing a spectrometer for receiving the unabsorbed portion of the wavelength range of light from the light collecting fiberoptic, determining a total absorption spectrum of the selected tissue sample for a plurality of wavelengths selected from the wavelength range of light and comparing a first absorption of the plurality of chromophores at a first wavelength to a second absorption of the plurality of chromophores at one or more additional wavelengths to determine a relative amount of each of the plurality of chromophores in the blood of a human or animal subject. 
         [0032]    The present invention also relates to an apparatus for non-invasively determining the relative amounts of a plurality of chromophores in the blood of a human or animal subject having a light source imparting a wavelength range including visible and broadband infrared light to a light delivery fiberoptic, a light collecting fiberoptic for receiving the light after the light has passed through a selected tissue sample of the human or animal subject, and a spectrometer for measuring the intensity of substantially all wavelengths of light received by the light collecting fiberoptic. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0033]    The invention will now be described, by way of example, with reference to the accompanying drawings in which: 
           [0034]      FIG. 1  shows the absorption curves for deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), and carboxyhemoglobin (HbCO); 
           [0035]      FIG. 2  shows carboxyhemoglobin detector, according to the present invention; 
           [0036]      FIG. 3  shows the ability of a monitor to follow the growth of carboxyhemoglobin in a large (121 lb) pig; 
           [0037]      FIG. 4  is a schematic of a more sophisticated spectrometer-based technology system, according to the present invention; 
           [0038]      FIGS. 5A-5E  are spectral ratios recorded with a spectrometer as in  FIG. 4   
           [0039]      FIG. 6  is a diagram of the absorption spectra from several subjects (displaced vertically for clarity); and 
           [0040]      FIG. 7  is a diagram of absorption spectra of various species of hemoglobin in the wavelength range of 475 to 640 nm. 
       
    
    
     DETAILED DESCRIPTION 
       [0041]    At any moment, the body&#39;s total supply of oxygen is only about 1 gram (just enough to last about four minutes). The length of time that cells can survive following interruption of oxygen depends on the type of tissue. Brain neurons are unrecoverable after only a few minutes. The organs of the body get their supply of oxygen from circulating blood where it is bound to hemoglobin. Hemoglobin is a large protein carried by blood&#39;s red cells (erythrocytes) giving them their characteristic color. 
         [0042]    Blood consists of 55% plasma (water, dissolved salts and proteins), 45% red cells and a smattering of white cells. There are about 280 million hemoglobin molecules within each of the 10 13  erythrocytes circulating in the blood. Each molecule of hemoglobin can carry as many as four molecules of oxygen and can transport over 70 times more oxygen than can be simply dissolved in the blood. Virtually all the oxygen carried by the blood is bound to hemoglobin in the red cells. 
         [0043]    The lungs supply hemoglobin with oxygen. When hemoglobin is saturated with oxygen, it is called oxyhemoglobin and has the bright red characteristic of arterial (supply veins) blood. The oxygen needed for supplying energy to organs is extracted from the hemoglobin as it passes through their capillary beds. Stripped of oxygen, the hemoglobin becomes darker and takes on the “bluish” color we associate with venous (draining veins) blood. This is called deoxyhemoglobin and is returned to the lungs by the heart and absorbs a fresh supply of oxygen. 
         [0044]    Hemoglobin has even more affinity for carbon monoxide (CO) than for oxygen. A heavy smoker will bind enough CO to his hemoglobin to lower the O 2  concentration in the arterial blood by 20%. More fire victims die from a buildup of carbon monoxide in the blood, which displaces oxygen, than all other causes. The greatest threat to life from fires is not the heat or lack of oxygen in the air, but suffocation from the displacement of oxyhemoglobin (HbO 2 ) with carboxyhemoglobin (HbCO). Carbon monoxide becomes so tightly bound to hemoglobin that the half-life for CO washout after exposure is removed is five hours. 
         [0045]    Besides oxyhemoglobin, deoxyhemoglobin and carboxyhemoglobin, there are still other varieties found in special circumstances, in methemoglobin the normal ferrous iron is replaced by ferric iron and occurs in the blood of people poisoned by nitro and amide compounds. Clinically, it is seen in small amounts after the therapeutic use of sulfonamide, nitrates, methylene blue, sulfonal, KCl, etc. Sulfhemoglobin apparently arises from methemoglobin and is more frequently found postmortem. The absorption of light by hemoglobin depends both on the wavelength of the incident light and the kind of hemoglobin. This unique spectral “signature” allows the simultaneous sorting out of different forms of hemoglobin. 
         [0046]    The absorption curves for deoxyhemoglobin (Hb), oxyhemoglobin (HbO 2 ), and carboxyhemoglobin (HbCO) are shown in  FIG. 1 . HbCO is far more transparent than Hb or HbO 2  in the near infrared. In principle, measurements of absorption at least three wavelengths, between 500 to 650 nm, will be sufficient to determine relative amounts of each of these three hemoglobin varieties (Other, non-hemoglobin absorption such as water fat and variable tissue scattering make it desirable to use additional wavelengths.) 
         [0047]    While spectral measurements can be made in different extremities of a body, an important aspect of the present invention is the choice of the septum of the nose as a point of measurement. There is a high concentration of blood in this area and critically, it is not affected by the shutdown of blood flow to the extremities. An important additional feature is that the septum is thin enough to extend the wavelengths measured below 650 nm where additional spectral signatures are available. 
         [0048]    Observing  FIG. 2 , the transmission spectrum of the septa is measured by inserting two, small, very flexible fiberoptic probes  3 ,  5 , one into each nostril. They are attached to a common base  7  where they are separated by about 8 mm. It is quite easy to insert the fiberoptic probes into the human nose as they are so small as not to affect the airway for normal breathing. The combination of the base  7  with probes is cheap and disposable. They can be incorporated in a mask or presented in other ways. Broadband infrared light is directed into one nostril and the spectral distribution of the light emerging form the other nostril is measured. In the simplest embodiment, broadband near infrared light is produced by the incandescent lamp  9 . Light from the filament of the lamp is collected over a large solid angle by lens  11  and focused into the delivery fiberoptic  13 . The collecting fiberoptic  15  is held parallel to the delivery fiber by base  7 . In addition, both fiberoptics  13 ,  15  are stiffened by thin aluminum sleeves  17 . The collected light is delivered to the CCD (Ocean Optics spectrometer  19 . 
         [0049]    Jacketed 2 mm diameter plastic fibers  13 ,  15  are used giving an overall diameter of 3 mm. The outside diameter of the aluminum sleeves  17  is 4 mm and the fibers  13 ,  15  are spaced 12 mm on center giving a separation of 8 mm. By sliding the block  7  up and down, the penetration distance up the nostrils can be set. 
         [0050]    An important consideration is the treatment of the (nostril) ends of the delivery and receiving fibers  13 ,  15  defining the flexible fiberoptic probes  3 ,  5 , so that the light is directed perpendicularly through the septum of the nose. The present method is to cut the ends at 45 degrees to form an angled end surface  21  and cement on a bit of polished aluminum foil or other such reflective surface material onto the end surface  21  it is also possible to provide means for sending some of the light to a silicon photodiode to monitor lamp intensity and to the spectrometer to monitor the lamp spectral distribution. 
         [0051]    The best signal-to-noise ratios can be obtained by injecting light in short, intense pulses rather than continuous illumination so uncomfortable heating of the nose with the use of a continuous incandescent lamp is avoided. For example, an incandescent lamp may be pulsed by discharging a capacitor through its filament. Flashing also has the potential for delivering high peak power without burning. The most recent embodiment uses “spectrally tailored” light source with enriched emission in the spectral range of interest achieved with a combination of a short-arc xenon lamp and appropriate colored filters. A spinning disk acts as a shutter and makes it possible to interlace a lamp background spectrum with the transmission spectrum. 
         [0052]    Drs. Stahl and Soller, Department of Surgery at the University of Massachusetts Medical Center tested the Carboxyhemoglobin Detector in an animal model by gradually increasing the CO load of the hemoglobin and comparing the results with Co-oximeter measurements of HbCO from blood samples.  FIG. 3  shows the ability of the monitor to follow the growth of carboxyhemoglobin in a large (121 lb) pig. The data, shown as open diamonds, were recorded by the Carboxyhemoglobin Detector at intervals starting at 13:46 and continuing through 14:22. During this time carbon monoxide was momentarily added to the inhaled gas mixture three times. The larger open circles show the HbCO as measured by the University of Massachusetts&#39; co-oximeter using collected samples of arterial blood. 
         [0053]    The HbCO concentration in the arterial samples jumps immediately after CO gas is added and then falls as it equilibrates with the total circulating blood volume. Not only does the carboxyhemoglobin detector follow the equilibrium values for the arterial blood samples as measured by the co-oximeter, but also the curve appears to be a bit steeper at about 7 minutes after each inhalation of CO. This curve could be plotted upside down showing the fall in oxyhemoglobin as its oxygen is displaced by carbon monoxide with time. 
         [0054]    One particular problem addressed by the present invention is the method of determining the quantities or concentrations of chromophores such as CO in the blood sample. This problem is solved by use of the spectrometer and deconvolution, or decomposition, techniques explained below. Decomposition is best understood as a method of untangling, identifying and determining the concentrations of the various chromophores that are constituents of a blood sample from a composite absorption spectrum obtained by the use of a spectrometer. The absorption spectrum of light which has been passed through the septum of the nasal passages is a specific example. The absorption spectrum recorded by the spectrometer consists of the measured light intensity at each wavelength in the spectral range of the spectrometer. Wavelengths for which the blood sample is absorbing will produce the least light with smallest values in the spectrum while wavelength regions where the blood sample is transparent will produce the largest. 
         [0055]    The spectrometer records the composite absorption spectrum of light resulting from the sum of individual absorption spectra of chromophores in a blood sample. What is not immediately known from this composite spectrum is the relative contribution of each of the individual chromophores. 
         [0056]    In order to identify the individual chromophores and determine their concentrations we must first know their absorption spectral signatures as a function of concentration. This catalog of chromophore spectra can be assembled by measuring the spectrum of each separately, one-by-one, at a known concentration. In general, the spectral “shape” will provide a unique signature for each chromophore while the actual amount of light absorption will depend on their concentrations. 
         [0057]    In the simplest case of two chromophores, e.g. hemoglobin and oxygenated hemoglobin, absorption measurements at only two wavelengths, for which the spectral signatures are different, are required. This leads to two independent equations which can be solved for the concentrations of the two constituents. Pulse oximeters measure oxygen saturation of arterial blood using two optical filters that pass light in two defined wavelength bands. If carboxyhemoglobin is added to the list of unknowns, then three filters are required to provide three equations to solve to obtain the three chromophore concentrations. The problem in these cases is errors caused by ignoring the absorption of other constituents in the substrate—such as fat and water. 
         [0058]    The errors caused by the substrate increase as measurement of additional chromophores at increasingly smaller concentrations is attempted. Not only are more optical filters needed for the new constituents, but additional filters are required to deal with the substrate spectral absorption. It becomes increasingly difficult to devise a finite filter set that leads to a sufficiently independent set of equations. 
         [0059]    The use of a spectrometer (essentially an array of many adjacent filters) minimizes these problems by producing a large set of equations to solve for a lesser number of chromophores. A matrix can be set up and solved for the constituents with a computer. The only limits to accuracy are system noise in the composite spectrum, missing chromophores in the catalog used for setting up the equations, and lack of independence of the spectral signatures. 
         [0060]    More specifically, the constituents are solved by the following method. The intensity of light I(λ) received after passing through a sample with spectral absorption coefficient α(λ) is given by 
         [0000]        I (λ)= I   0 (λ) e   −α(λ)l(λ)    (1) 
         [0000]    where I 0 (λ) is the illumination intensity and l(λ) is the distance photons travel through the sample. In the simplest case, this distance is just the thickness of the sample, but, more generally, it might depend on wavelength if there is sufficient scattering within the sample. 
         [0061]    Define sample transmission T(λ) in the usual manner as the ratio 
         [0000]    
       
         
           
             
               
                 
                   
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         [0000]    this can be expressed in the simpler logarithmic form 
         [0000]      −ln  T (λ)=α(λ) l (λ)   (3) 
         [0062]    Total absorption is due to a collection of different absorbing species (chromophores). Assuming we know the absorption cross sections σ 1 (λ), σ 2 (λ) . . . σ N (λ) of N of these, we can write 
         [0000]    
       
         
           
             
               
                 
                   
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         [0000]    where 
         [0000]      α n (λ)= c   n σ n (λ)   (5) 
         [0063]    Any residual absorption not included in the known set is included in the term α n (λ). The coefficients c 1 ,c 2  . . . c N  appearing in (5) are the unknown concentrations of the N chromophores. The values of these are what we are interested in determining and the procedure to do so is described next. 
         [0064]    First, substitute (4) and (5) into (3) to obtain 
         [0000]    
       
         
           
             
               
                 
                   
                     
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         [0065]    Define F(λ) as 
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                               σ 
                               n 
                             
                              
                             
                               ( 
                               λ 
                               ) 
                             
                           
                         
                       
                       + 
                       
                         
                           α 
                           u 
                         
                          
                         
                           ( 
                           λ 
                           ) 
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   7 
                   ) 
                 
               
             
           
         
       
     
         [0066]    Rearrangement of this expression to isolate the unknown absorption coefficient produces 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       α 
                       u 
                     
                      
                     
                       ( 
                       λ 
                       ) 
                     
                   
                   = 
                   
                     
                       F 
                        
                       
                         ( 
                         λ 
                         ) 
                       
                     
                     - 
                     
                       
                         ∑ 
                         
                           n 
                           = 
                           1 
                         
                         N 
                       
                        
                       
                         
                           c 
                           n 
                         
                          
                         
                           
                             σ 
                             n 
                           
                            
                           
                             ( 
                             λ 
                             ) 
                           
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   8 
                   ) 
                 
               
             
           
         
       
     
         [0000]    square both sides and integrate over the measured wavelength domain to obtain 
         [0000]    
       
         
           
             
               
                 
                   
                     ∫ 
                     
                       
                         
                           α 
                           u 
                           2 
                         
                          
                         
                           ( 
                           λ 
                           ) 
                         
                       
                        
                       
                          
                         λ 
                       
                     
                   
                   = 
                   
                     ∫ 
                     
                       
                         
                           ( 
                           
                             
                               F 
                                
                               
                                 ( 
                                 λ 
                                 ) 
                               
                             
                             - 
                             
                               
                                 ∑ 
                                 
                                   n 
                                   = 
                                   1 
                                 
                                 N 
                               
                                
                               
                                 
                                   c 
                                   n 
                                 
                                  
                                 
                                   
                                     σ 
                                     n 
                                   
                                    
                                   
                                     ( 
                                     λ 
                                     ) 
                                   
                                 
                               
                             
                           
                           ) 
                         
                         2 
                       
                        
                       
                          
                         λ 
                       
                     
                   
                 
               
               
                 
                   ( 
                   9 
                   ) 
                 
               
             
           
         
       
     
         [0067]    The goal is to find the c 1 ,c 2  . . . c N  which minimize the integrated square residual absorption as defined by the LHS of the above equation. This, of course, is the same as minimizing the RHS. To keep the solution from diverging when σ 1 (λ),σ 2 (λ) . . . σ N (λ) are weakly independent, we condition the solution by adding a regularizing term that penalizes large coefficients. Hence, we seek to minimize 
         [0000]    
       
         
           
             
               
                 
                   
                     ∫ 
                     
                       
                         
                           ( 
                           
                             
                               F 
                                
                               
                                 ( 
                                 λ 
                                 ) 
                               
                             
                             - 
                             
                               
                                 ∑ 
                                 
                                   n 
                                   = 
                                   1 
                                 
                                 N 
                               
                                
                               
                                 
                                   c 
                                   n 
                                 
                                  
                                 
                                   
                                     σ 
                                     n 
                                   
                                    
                                   
                                     ( 
                                     λ 
                                     ) 
                                   
                                 
                               
                             
                           
                           ) 
                         
                         2 
                       
                        
                       
                          
                         λ 
                       
                     
                   
                   + 
                   
                     γ 
                      
                     
                       
                         ∑ 
                         
                           n 
                           = 
                           1 
                         
                         N 
                       
                        
                       
                         c 
                         n 
                         2 
                       
                     
                   
                 
               
               
                 
                   ( 
                   10 
                   ) 
                 
               
             
           
         
       
     
         [0000]    with respect to each of the c 1 ,c 2  . . . c N . The factor γ may be thought of as a Lagrange multiplier that sets the amount of penalty for large coefficients. By definition, at minimum, the derivatives of (10) with respect to each c 1 ,c 2  . . . c N  vanishes, that is 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       ∂ 
                       
                         ∂ 
                         
                           c 
                           m 
                         
                       
                     
                      
                     
                       ( 
                       
                         
                           ∫ 
                           
                             
                               
                                 ( 
                                 
                                   
                                     F 
                                      
                                     
                                       ( 
                                       λ 
                                       ) 
                                     
                                   
                                   - 
                                   
                                     
                                       ∑ 
                                       
                                         n 
                                         = 
                                         1 
                                       
                                       N 
                                     
                                      
                                     
                                       
                                         c 
                                         n 
                                       
                                        
                                       
                                         
                                           σ 
                                           n 
                                         
                                          
                                         
                                           ( 
                                           λ 
                                           ) 
                                         
                                       
                                     
                                   
                                 
                                 ) 
                               
                               2 
                             
                              
                             
                                
                               λ 
                             
                           
                         
                         + 
                         
                           γ 
                            
                           
                             
                               ∑ 
                               
                                 n 
                                 = 
                                 1 
                               
                               N 
                             
                              
                             
                               c 
                               n 
                               2 
                             
                           
                         
                       
                       ) 
                     
                   
                   = 
                   0 
                 
               
               
                 
                   ( 
                   11 
                   ) 
                 
               
             
           
         
       
     
         [0000]    for every m. This gives rise to the set of N linear equations 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       ∫ 
                       
                         
                           
                             σ 
                             m 
                           
                            
                           
                             ( 
                             λ 
                             ) 
                           
                         
                          
                         
                           ( 
                           
                             
                               F 
                                
                               
                                 ( 
                                 λ 
                                 ) 
                               
                             
                             - 
                             
                               
                                 ∑ 
                                 
                                   n 
                                   = 
                                   1 
                                 
                                 N 
                               
                                
                               
                                 
                                   c 
                                   n 
                                 
                                  
                                 
                                   
                                     σ 
                                     n 
                                   
                                    
                                   
                                     ( 
                                     λ 
                                     ) 
                                   
                                 
                               
                             
                           
                           ) 
                         
                          
                         
                            
                           λ 
                         
                       
                     
                     + 
                     
                       γ 
                        
                       
                           
                       
                        
                       
                         c 
                         m 
                       
                     
                   
                   = 
                   0 
                 
               
               
                 
                   ( 
                   12 
                   ) 
                 
               
             
           
         
       
     
         [0000]    which, after exchanging the order of integration and summation, become 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       ∑ 
                       
                         n 
                         = 
                         1 
                       
                       N 
                     
                      
                     
                       
                         c 
                         n 
                       
                        
                       
                         ( 
                         
                           
                             ∫ 
                             
                               
                                 
                                   σ 
                                   m 
                                 
                                  
                                 
                                   ( 
                                   λ 
                                   ) 
                                 
                               
                                
                               
                                 
                                   σ 
                                   n 
                                 
                                  
                                 
                                   ( 
                                   λ 
                                   ) 
                                 
                               
                                
                               
                                  
                                 λ 
                               
                             
                           
                           + 
                           
                             γ 
                              
                             
                                 
                             
                              
                             
                               δ 
                               
                                 m 
                                 , 
                                 n 
                               
                             
                           
                         
                         ) 
                       
                     
                   
                   = 
                   
                     ∫ 
                     
                       
                         
                           σ 
                           m 
                         
                          
                         
                           ( 
                           λ 
                           ) 
                         
                       
                        
                       
                         F 
                          
                         
                           ( 
                           λ 
                           ) 
                         
                       
                        
                       
                          
                         λ 
                       
                     
                   
                 
               
               
                 
                   ( 
                   13 
                   ) 
                 
               
             
           
         
       
     
         [0068]    Let 
         [0000]      α m,n =∫σ m (λ)σ n (λ) dλ   (14) 
         [0000]      and 
         [0000]        b   m =∫σ m (λ) F (λ) dλ   (15) 
         [0000]    Then (13) becomes 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       ∑ 
                       
                         n 
                         = 
                         1 
                       
                       N 
                     
                      
                     
                       
                         ( 
                         
                           
                             a 
                             
                               m 
                               , 
                               n 
                             
                           
                           + 
                           
                             γ 
                              
                             
                                 
                             
                              
                             
                               δ 
                               
                                 m 
                                 , 
                                 n 
                               
                             
                           
                         
                         ) 
                       
                        
                       
                         c 
                         n 
                       
                     
                   
                   = 
                   
                     b 
                     m 
                   
                 
               
               
                 
                   ( 
                   16 
                   ) 
                 
               
             
           
         
       
     
         [0000]    which, in matrix-vector notation, is just 
         [0000]      ( A+γI )= c=b    (17) 
         [0000]    where A is the N×N matrix with elements α m,n  given by (14), I is the N×N identity matrix, c is the N-component vector whose elements are the unknown concentrations and b is the N-component vector with elements b m  given by (15). 
         [0069]    The c which solves (17) is formally given by 
         [0000]        c= ( A+γI ) −1   b    (18) 
         [0000]    If the catalog of chromophores is complete and σ 1 (λ),σ 2 (λ) . . . σ N (λ) are linearly independent over the domain of measured wavelengths, then the solution to (18) with γ set to zero will be exact and both sides of (9) will vanish. If the catalog is not complete, there will be non-zero residual absorption exhibiting both positive and negative values. Since negative values are not physically possible the accuracy of the solution is improved by increasing γ until α u (λ) is everywhere non-negative. Furthermore, if σ 1 (λ),σ 2 (λ) . . . σ N (λ) are not completely independent, use of non-zero γ will prevent the solution from diverging when the intensity measurements are noisy. 
         [0070]    As the number of absorbers (chromophores) to be monitored increases, more spectral data is needed to sort them out. For developing this application we utilized two Ocean Optics CCD spectrometers, one with a wavelength range of 300 to 1000, and another ranging from 500 to 1200 nm. To deliver as much light as possible, a short (3 mm) arc xenon lamp S was chosen producing the very high brightness needed for getting as much of the light produced into a small fiberoptic cable. 
         [0071]      FIG. 4  is a schematic of a more sophisticated system. Light from the short arc xenon flash lamp S is collected and collimated by the f/1 aspheric Fresnel lens L. An identical lens L refocuses the light into the injector fiberoptics. The space provided between the two Fresnel lenses may be used for filters F. After transmission from the delivery fibers  13  to the receiving fibers  15  through the nose septum N, light is conducted to the CCD spectrometer  19  that records the absorption spectrum of the septum N. A separate reference fiber  27  records the lamp S spectra. The motor  25  drives a “chopper” disk C described below is seen at the center. The light delivery fiber  13  leads to the probe end  5  for being inserted in a subject nasal passage. The reference fiber  27  extends past the disk C and directly to the spectrometer  19  without passing through the nasal septum N. An electronics board  26  is used to control the motor speed from a computer  28 . Operation of the system is described in detail below. 
         [0072]    The motor-driven “chopper” disk C, with a single small hole  29 , selects the source of the light arriving at the spectrometer  23 . The disk C makes one rotation in 200 ms during which (4) four spectra S 1 , S 2 , S 3 , and S 4  are acquired at 50 ms intervals. The first spectrum S 1  is acquired from the tissue when the flash lamp is fired at the time the chopper hole is aligned with the injector fiber. The second spectrum S 2  from ambient light is acquired during the next 50 ms during which time the lamp is off. The third spectrum S 3  is acquired when the flash lamp is fired at the time the chopper hole is aligned with the reference fiber  27 . Finally, the fourth spectrum S 4  which is again ambient light is taken and is like the first ambient light spectrum. The cycle is then repeated many times gradually building up these four separately-summed spectra in the computer. 
         [0073]    Data analysis begins by subtracting the average ambient light spectra S 2 ,+S 4 /z from S 1 , the spectrum containing the tissue reflectance spectrum, and S 3 , the spectrum containing the flash lamp spectrum. This leaves: 
         [0000]    
       
      
       S 
       1 
       =S·T·F  
      
     
         [0000]      S 3 =S 
         [0000]    Where S is the flash lamp spectrum, T is the tissue transmittance, and F is the filter transmittance. Solving for T: 
         [0000]    
       
         
           
             T 
             = 
             
               
                 S 
                 1 
               
               
                 
                   S 
                   3 
                 
                 · 
                 F 
               
             
           
         
       
     
         [0074]    In an experiment, data taken from the nasal septa of a human test subject is shown is  FIGS. 5A-5E . Curve A is the reciprocal of S 1 . B is S 3  and C is the reciprocal of S 1 /S 3 . D is the filter absorptance 1/f. E is the resulting absorptance of the nasal septa 
         [0000]    
       
         
           
             A 
             = 
             
               
                 { 
                 
                   
                     S 
                     1 
                   
                   
                     
                       S 
                       3 
                     
                      
                     F 
                   
                 
                 } 
               
               
                 - 
                 1 
               
             
           
         
       
     
         [0000]    Compare  FIG. 5E  with the HbO2 curve in  FIG. 1 . The absorption spectra from five subjects is shown in  FIG. 6 . The individual spectra have been shifted vertically by 0.4 OD to avoid overlapping. 
         [0075]    This technology is an important step in the field of continuous, non-invasive, in vivo monitoring of blood gases. The carboxyhemoglobin detector described above is a subset of this technology. Because of the number of target chromophores, namely, oxy-, deoxy-, cart)oxy-, cyano-, met- and sulf-hemoglobins, non-target chromophores, can only be supplied by spectra covering the largest range of wavelengths. 
         [0076]    It is especially important to obtain data in the region of 550 nm where hemoglobins have strong absorptance and characteristic spectra. Until now, in vivo NIRS methods have been unable to collect spectral data much below about 650 nm. With the present invention it is possible to extend in vivo spectra down to 450 nm by taking transmission spectra of the thin, blood-rich septum of the nose using high-power, short-arc flash lamps and sophisticated data processing. Also, one or two, 3 mm GB-26 green filters are used to attenuate the large amount of infrared light above 600 nm in order to keep the CCD spectrometer within its dynamic operating range. 
         [0077]    Since certain changes may be made in the above described improved method and instrument for measuring carbon monoxide poisoning of humans, without departing from the spirit and scope of the invention herein involved, it is intended that all of the subject matter of the above description or shown in the accompanying drawings shall be interpreted merely as examples illustrating the inventive concept herein and shall not be construed as limiting the invention.