Abstract:
An improved method for non-invasively measuring a concentration of a target analyte dissolved in a fluid flowing through a sample is presented. It includes directing a probe beam of electromagnetic radiation, consisting of time multiplexed components of different wavelengths, through the sample and measuring the difference of the absorption of the radiation at at least one wavelength pair at different sample states. During sample state changes, the amount of fluid containing the target analyte within the sample is changing, which varies the total amount of target analyte in the sample, as well as the absorption properties of the sample. The sample states are produced, for instance, by compressing and uncompressing the tissue sample. The accuracy of the presented method is enhanced by including continuous estimation of the amount of the fluid containing the target analyte within the sample, and measurement of the variations of the absorption at a wavelength at which the target analyte absorbs significantly. The method is particularly useful in measuring the concentration of a target analyte, such as glucose, in tissue containing blood. An apparatus for performing this method also is disclosed.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    This invention relates to the non-invasive measurement of the concentration of substances that absorb electromagnetic radiation, such as light or infrared radiation, in absorbing and turbid matrices, such as human or animal body tissue, using a probe beam of electromagnetic radiation. The invention is particularly applicable to glucose measurement in human tissue using near-infrared radiation. It is, however, generally applicable to measurements of the concentration of any species that absorbs electromagnetic radiation, especially in strongly absorbing and turbid matrices.  
           [0002]    The infrared measurement methods known in the art are not well adapted to the problem of quantifying an analyte, such as glucose, dissolved in a strongly absorbing solvent, such as blood. The known methods include separate or directly alternating measurements at a “glucose” wavelength and at a “reference” wavelength, where glucose does not absorb, as well as differential wavelength modulation about a glucose absorption band (C. Dähne, D. Gross, European patent 0 160 768 and references therein). In the known methods, the signal is easily masked with the variations of the strong background presented by water and other constituents in the tissue and in the capillary blood flow.  
           [0003]    The present invention is an improvement over U.S. Pat. No. 5,099,123, issued to Harjunmaa (hereafter, the “&#39;123 patent”), which is incorporated herein in its entirety by reference. The balanced differential (or “optical bridge”) method disclosed in the &#39; 123  patent utilizes two wavelengths for target analyte concentration measurements. The first, or principal wavelength is chosen such to be highly absorbed in the target analyte. The second, or reference wavelength is chosen using a balancing process so that both wavelengths have substantially identical extinction coefficients in the background matrix. A radiation beam is generated that contains these two wavelengths in alternate succession at a suitable frequency. When the beam is properly balanced for the measurement, a sample detector, placed to measure radiation transmitted or reflected by the sample matrix that contains only a residual amount of the target analyte, will detect only a very small alternating component in the radiation, regardless of the thickness of the sample. When there is a relatively substantial amount of the target analyte in the sample matrix, however, the detector will detect a significant alternating signal synchronous with the wavelength alternation. This alternating signal is amplified and is then detected using a phase-sensitive detector (or lock-in amplifier). The optical bridge balancing process entails nulling out the alternating signal from the sample detector by systematically varying the relative intensities and/or wavelengths of the repetitive radiation periods. An auxiliary detector is also used to sample the combined beam before it enters the tissue in order to enhance the measurement stability. Although suitable for the purposes intended, the realization of the precautions taken to deal with the unavoidable differences in the spectral response between the auxiliary detector and the sample detector make the system somewhat complicated.  
           [0004]    Subsequently in U.S. Pat. No. 5,178,142, (hereafter, the “&#39;142 patent”), which is also incorporated by reference herein in its entirety, Harjunmaa et al. disclosed an improved method and apparatus in which the concentration measurement is performed using a two-wavelength alternating radiation probe beam which interacts with the tissue. One of the wavelengths is used as a reference wavelength, and the other is the principal wavelength. The reference wavelength is tunable to account for the expected variability of the background spectrum. After passing through the matrix that contains a given reference concentration of analyte, detected signals from the probe beam are balanced or nulled by tuning the variable wavelength beam over a range of frequencies. Next, the blood content of the sample is changed. The alternating component of the interacted probe beam is then detected. The amplitude of the alternating component of the signal given by the sample detector is proportional to the concentration of analyte or the difference from a preset reference analyte concentration.  
           [0005]    Other related patents include U.S. Pat. Nos. 5,112,124; 5,137,023; 5,183,042; 5,277,181 and 5,372,135, each of which is incorporated by reference herein in its entirety.  
         SUMMARY OF THE INVENTION  
         [0006]    This invention relates to systems and methods for non-invasively estimating the concentration of a target analyte in a sample. For the purpose of simplicity, and to aid in the understanding of the principles of this invention, the sample is defined as consisting of three components: non-fluid, bound fluid, and unbound fluid. The non-fluid and bound fluid components are generally fixed, and together are referred to as the background matrix. In the case of human tissue, for example, the background matrix comprises the cellular matrix and intra-cellular fluid. The third main component, the unbound fluid, is generally not fixed in the sample, and can freely circulate through the fixed background matrix of the sample. In human tissue, for instance, the unbound fluid consists of blood and other substances dissolved in or otherwise contained within the blood. The unbound fluid can be displaced by, for instance, compressing (squeezing) the sample. Also, in human tissue, the interstitial fluid can be considered to be bound fluid if the sample compression lasts for less than 20-30 seconds.  
           [0007]    If not specified otherwise, the term “fluid” as used herein refers to the unbound fluid only. Generally, according to the present invention, the concentration of the target analyte in the unbound fluid is different from the concentration of the target analyte in the background matrix.  
           [0008]    The present invention relates to a series of improvements to the known balanced differential, or “optical bridge,” systems for measuring the concentration of a target analyte in a sample. As used herein, “optical bridge” refers to an apparatus and/or method for quasi-simultaneous differential optical measurement of very small absorbance differences of a sample, performed at one or more wavelength pairs. According to one aspect, the improved optical bridge method and system of present invention includes: 1) time-series measurements during and after a sample thickness variation; 2) synchronization of the measurements with the unbound fluid (e.g. blood) inrush into the sample; and, 3) the use of parameters extracted from the time-series measurements to compensate for daily and long-term variations in the absorption of the sample background matrix. An advantage of the present invention is the ability to record signals from a sample whose composition varies with time, while maintaining the sample at a substantially constant thickness, thus removing the thickness change as a major contributor to the signal. Accordingly, a simpler measurement system is provided which is capable of improved accuracy of target analyte concentration estimation.  
           [0009]    An apparatus according to this invention includes a source for producing a beam of electromagnetic radiation. This beam consists of time multiplexed components (principal and reference) of desired narrow line-width wavelengths, and is produced, for instance, using a tunable filter. In alternative embodiments, two or more separate substantially monochromatic sources, whose outputs are combined into a single beam, can also be employed.  
           [0010]    During a measurement, the alternating-wavelength probe beam passes through (or is reflected from) a sample mounted in a compression device. The compression device controllably varies the thickness of the sample (and consequently its unbound fluid content) during the measurement. A sample detector is positioned to receive the probe beam after it passes through the sample. The sample detector then feeds a signal to an analog signal pre-processing module that includes the hardware implementation of the optical bridge. The output optical bridge signal is then fed to a processor, which is programmed to calculate the target analyte concentration in the unbound fluid, based on parameters extracted from the sample detector signal and other auxiliary variables and time-varying signals.  
           [0011]    One of the auxiliary signals used in the calculation of the target analyte concentration is preferably a time-varying estimate of the unbound fluid (e.g. blood) content within the sample. This estimate can be obtained, for example, by a separate, auxiliary blood signal detector measuring the sample transmission (or reflection) of light from a separate light source that provides radiation distinct from the wavelengths used for the target analyte measurement, preferably at a wavelength where hemoglobin absorbs, and even more particularly at a wavelength where hemoglobin absorption is independent of its oxidation state (i.e., isosbestic point). In other embodiments, a laser Doppler flow meter may be used to obtain a measurement of sample blood content.  
           [0012]    Preferably, the movement of the compression device is synchronized with the unbound fluid inrush into the sample. In case of glucose measurement in the blood, the synchronization can be achieved using a separate, auxiliary synchronization detector measuring the sample transmission (or reflection) of light from a separate light source that provides radiation distinct from the wavelengths used for the target analyte measurement, and preferably at a wavelength where hemoglobin absorbs. The synchronization can also be achieved using a pulse oximeter.  
           [0013]    Additional enhancements to the target analyte measurement can be achieved by measuring the temperature of the sample and/or the temperature of the detectors, and incorporating these variables into the processing of the detector output.  
           [0014]    A method, according to this invention, for non-invasively measuring a concentration of a target substance (e.g., glucose) in a matrix (e.g., tissue) includes the following steps. First, the sample is compressed by the compression device to force out the unbound fluid that contains the majority of the target analyte. The sample is then illuminated with the probe beam of electromagnetic radiation. Preferably, the beam includes a principal period and a reference period, wherein during the principal period the effective wavelength of the radiation is more strongly absorbed by a target analyte, such as glucose, than is the effective wavelength of the radiation during the reference period. By way of illustration, the wavelength that is strongly absorbed by glucose can be between approximately 1550 and 1700 nm, and the wavelength lightly absorbed by glucose can be between approximately 1350 and 1430 nm.  
           [0015]    In one embodiment, both the principal and the reference wavelengths are universally pre-set, or pre-set individually for each patient. In another embodiment, the reference and/or principal wavelengths are selected during a balancing process. This balancing process can be performed prior to measurement. The balancing process comprises, for example, tuning the wavelength and/or intensity of at least one of the alternating radiation periods to obtain a substantially-zero alternating component of the sample detector signal (i.e. the optical bridge signal) at chosen sample thicknesses/pressures exerted by the sample compression device. The optical bridge is “balanced” when there is substantially no alternating component in the signal generated by the sample detector. A properly balanced optical bridge means that the principal and reference wavelengths are equally absorbed by the sample matrix, which contains only residual amounts of the target analyte.  
           [0016]    A measurement sequence comprises a series of individual measurements of intensities of the transmitted/reflected probe beam wavelength components obtained by the sample and auxiliary detector(s). This series of measurements is obtained during an alteration of sample thickness, and also over the subsequent sample content equilibration process that follows the alteration of sample thickness. The measurements are preferably obtained while the unbound fluid content of the sample is changing.  
           [0017]    In a preferred embodiment of the invention, the sample thickness change is synchronized with the heartbeat. One advantage of this is that since the influx speed of blood depends on the blood pressure, performing the uncompression at a constant phase of the cardiac cycle produces blood refill time profiles that are substantially constant in shape. The cardiac phase can be chosen so as to also provide the fastest possible blood content change.  
           [0018]    Measurements of auxiliary parameters (including, for example, unbound fluid content, temperature of sample and detector, sample thickness, and/or electronic control system operational parameters) accompany the measurements of the probe beam intensities. The recorded data is further combined with corresponding estimates of the time-varying unbound fluid content over the same time. An algorithm, based on modeling, is used to extract characteristic parameters from the time-series profiles, and combines these parameters with other measured parameters to achieve improved specificity and sensitivity in the estimation of the target substance concentration.  
           [0019]    Using the method of the present invention, the accuracy of the target analyte measurement is improved by isolating and quantifying the component of the optical bridge signal that results from the presence of the analyte rather than other “parasitic” factors. More specifically, where the targeted analyte is located primarily within the unbound fluid rather than the fixed structure of the matrix, the magnitude of the optical bridge signal depends directly on the amount of fluid within the sample. Thus, if the varying unbound fluid content of the sample is estimated and plotted against the magnitude of the optical bridge signal over time, the result is a substantially straight line whose slope is directly related to the concentration of analyte in the sample, assuming that the other factors contributing to the “parasitic” signal, including shifts in the effective wavelength due to changes in sample thickness, remain relatively constant during the measurement process.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0020]    The foregoing and other objects, features and advantages of the invention will be apparent from the following, more particular description of preferred embodiments of the invention, as illustrated in the accompanying figures. The drawings are not necessarily to scale, emphasis instead is placed on illustrating the principles of the invention.  
         [0021]    [0021]FIG. 1 is a schematic block diagram of an apparatus of the invention; and  
         [0022]    [0022]FIG. 2 is a block diagram schematically illustrating an embodiment of the components and steps for processing the time-varying detector signals and other measured parameters to obtain an estimate of analyte concentration, in accordance with one aspect of the invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0023]    The features and other details of the method of the invention will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.  
         [0024]    One embodiment of an apparatus for performing a method of the invention to measure glucose concentration in blood based on transmitted light through the sample will now be explained in detail in connection with FIG. 1. A similar apparatus may be designed which uses reflected light instead of transmitted light.  
         [0025]    The light source  10  is preferably a quartz-halogen lamp powered by power supply  20 . Using optical elements  21 , the lamp light is directed to an optical tunable filter  30 . The characteristics (wavelengths, intensities and duration of each wavelength component) of the probe beam (output  202  from the optical tunable filter  30 ) are controlled by filter driver  111 . In performing the methods of this invention, an acousto-optical tunable filter has been used.  
         [0026]    In an alternate embodiment, a pair of tunable monochromatic light sources, such as tunable laser diodes, may be used to produce the probe beam  202 .  
         [0027]    Clock generator  110  produces a timing signal at the desired chopping frequency f ch  needed for time multiplexing of the principal and reference components of the probe beam. The CPU  104  generates signals for controlling the principal intensity I P , both wavelengths λ P  and λ R , and the chopping frequency f ch  of the probe beam  202 .  
         [0028]    The probe beam  202  exiting from optical filter  30  is directed, using optical elements  40 , to beam splitter  50 , from which a fraction  208  of probe beam  202  is directed to auxiliary detector  60 . The auxiliary detector  60  is connected to a PID (proportional—integral—derivative) controller  102  that extracts the intensity difference between the two components (principal and reference) of the detected beam, compares that difference voltage with a pre-set voltage, and by means of controlling the intensity of the reference wavelength component I R    314 , maintains the difference of the two wavelength intensities constant. An alternative embodiment maintains the ratio of the two wavelength intensities constant. These features make the system stable against changes in light source output caused by, for instance, aging of the lamp or other components, or from light source power variations.  
         [0029]    The majority of the probe beam is directed onto diffuser plate  70 . Placing a diffuser plate in the beam path before the sample provides the advantage of minimizing the effects of the variation in the scattering properties of the sample. The sample specimen  80 , such as an earlobe, lip, cheek, nasal septum, tongue, or the skin between the fingers or toes of the subject, is placed between diffuser plate  70  and sample detector lens  92 , and is compressed by moving the measurement head  90 , mounted on compression mechanism  400 . The probe beam  203  transmitted through sample  80  is focused by sample detector lens  92 , and directed to dichroic mirror  93 . The major portion of the probe beam is transmitted by dichroic mirror  93  to sample detector  91 . The sample detector  91  detects the intensity at each of the wavelength periods of the probe beam  205  transmitted through sample  80 , and sends an electrical signal  302  to preamplifier  26  and phase sensitive detector (or lock-in amplifier)  24 . The output signal  308  from the phase sensitive detector  24  is proportional to the difference (or ratio) of the principal and reference intensities detected by sample detector  91 . This signal  308  is referred to as the optical bridge signal.  
         [0030]    In this embodiment, also shown in FIG. 1, a separate auxiliary radiation source such as an infrared or visible-light LED  44 , is used to provide an estimate of the sample blood content. This auxiliary radiation source  44  produces a blood detection beam  204  that is directed onto the diffuser plate  70  and into the sample. An LED operating at a wavelength of, for instance 525 nm (an isosbestic wavelength for hemoglobin), provides a good sensitivity to blood. The sample detector  91  can be used to detect the transmitted portion of the blood detection beam  204 . However, since there is a significant ambient light in this wavelength range, it is advantageous to use a separate blood signal detector  94  to detect the blood detection beam  204 . To achieve this, the transmitted blood detection beam is reflected by the dichroic mirror  93  to the blood signal detector  94 , producing a blood signal  300 . The blood signal  300  is then sent to the blood signal processing preamplifier  22 . The dichroic mirror  93  in this embodiment also eliminates ambient light from the sample detector  91  by transmitting only infrared wavelengths.  
         [0031]    In accordance with another embodiment, the blood content is estimated using a laser Doppler flowmeter integrated into the system, with a needle probe mounted on the optical axis. The laser Doppler flowmeter measures the number of moving red blood cells in its field of view, which extends to about 1 mm into the tissue. While the needle probe may block some light from the central portion of the optical bridge beam, the loss of light is tolerable. While the optical bridge measurements are performed, the laser Doppler instrument simultaneously takes its own readings of blood circulation under the skin. Accordingly, an estimate of the amount of blood in the measurement field at the time of measurement is provided.  
         [0032]    Other possible techniques for obtaining an estimate of the blood content include ultrasound and electrical impedance plethysmography.  
         [0033]    In the embodiment shown in FIG. 1, the pulse detection for synchronizing the measurements with the unbound fluid (e.g. blood) inrush into the sample is accomplished using an additional radiation source, similar to auxiliary radiation source  44 . This, radiation source  46 , can also be a LED operating at a wavelength of, for instance 525 nm, an oxyhemoglobin isobestic point. This radiation source  46  should be directed at a portion of the sample that at all times maintains good circulation, such as a section of the sample that is not compressed by the measurement head. The radiation source  46  generates a pulse detection beam  206  that is aimed at the sample  80 . This beam is scattered by the tissue, and a fraction of the original beam  206  is collected by sample detector lens  92 , is reflected by dichroic mirror  93 , and is detected by blood signal detector  94 .  
         [0034]    Preferably, the two auxiliary radiation sources  44  and  46  are not operated at the same time. The pulse synchronization source  46  should be operated prior to the measurement step in order to synchronize the start of the measurement process with a variation of the unbound fluid (e.g. blood) pressure. The blood detection source  44  should be operated during the measurement process to provide a time-varying estimate of the unbound fluid content within the sample. The intensities of the two auxiliary radiation beams  204  and  206  are pre-set or can be controlled by the CPU  104 .  
         [0035]    To perform a measurement, the sample  80  is introduced between diffuser plate  70  and sample detector lens  92 . The measurement head  90  is moved by compression mechanism  400  to gently compress sample  80  until a predetermined pressure is exerted on sample  80 . The preferred embodiment of compression mechanism  400  includes a miniature linear actuator. Its step size, speed and travel distance are controlled by the CPU  104 . Although this embodiment uses an electrical actuator, a hydraulic or a pneumatic actuator could also be used, with the ensuing advantages of compactness of the compression mechanism. A position sensor  402  is used to monitor the effects of the motor movement.  
         [0036]    In this description, three different types of probe beam attenuations are distinguished. First is the background matrix, the second is the target analyte, while the third is the unbound fluid attenuation.  
         [0037]    The background matrix attenuation results from the absorption of probe beam  202  by sample constituents whose concentrations are substantially constant throughout fixed sample compartments. The target analyte attenuation is caused by absorption of probe beam  202  by the target analyte (e.g. glucose), which is mostly concentrated in the unbound fluid (e.g. blood). When the tissue is sufficiently compressed, the unbound fluid, along with the target analyte (e.g. glucose), is substantially displaced from the sample  80 . Since the concentration of the target analyte in the unbound fluid is different than its concentration in the background matrix (e.g. intracellular concentration), its average concentration in the beam path changes as a result of the compression. This concentration change allows the target analyte to be detected by this method.  
         [0038]    The principal wavelength λ P  of probe beam  202  is selected in such a way to have high attenuation by the target analyte. The principal wavelength intensity I P  is set to achieve an optimal transmitted signal intensity. The reference wavelength λ R  of the probe beam is either pre-set or selected during the optical bridge balancing process. Its intensity I R  should be adjusted before each measurement as explained below in the description of the measurement process.  
         [0039]    In the following text, a simple to understand example of a bridge balancing process is presented. It will be readily understood by those skilled in the art that different, more complex, bridge balancing procedures can also be used, with corresponding variations of the signal processing algorithm.  
         [0040]    In the first step of bridge balancing, sample  80  is sufficiently compressed to remove the major amount of unbound fluid from the sample tissue. The principal wavelength parameters λ P  and I P  are set, and the reference wavelength λ R  is initialized. The probe beam  202  is directed at the sample, and the optical bridge is balanced or nulled by adjusting the intensity of the probe beam reference wavelength intensity I R  to obtain a substantially-zero optical bridge signal  308 . In other embodiments, the reference wavelength intensity I R  is set, while the principal wavelength intensity I P  is adjusted to balance the bridge. Next, the sample compression pressure is released by a predetermined amount, called “step 1 incremental thickness” (typically 0.1 mm) and the probe beam reference wavelength λ R  is adjusted by a signal from CPU  104  so as to again achieve a substantially-zero optical bridge signal  308 . The initial compression pressure is chosen such that, even after releasing sample  80  by the step 1 incremental thickness, there is nearly no unbound fluid reflow into the sample. Changes in the optical bridge signal  308 , due to this thickness increase result merely from increased background matrix thickness and not from any influx of fluid. Sample  80  is then compressed again back to its original compressed thickness, and the intensity at the reference (or principal) wavelength is again adjusted by the CPU  104  to achieve minimum optical bridge signal.  
         [0041]    This two-thickness procedure may be repeated until a substantially-zero optical bridge signal is obtained at both thicknesses. At this point, the absorption coefficient of sample  80  in its compressed state is substantially equal at the two wavelengths λ P  and λ R . Although the reference wavelength can be balanced to completely zero the optical bridge signal, a non-zero signal must generally be contended with in practice. During the measurement process, this non-zero optical bridge signal can be subtracted from detector outputs to improve the accuracy of the measurement.  
         [0042]    In one embodiment, the balancing is limited to only one cycle in order to speed up the measurement and reduce the compression stress on the sample.  
         [0043]    Due to the monochromatic components of the probe beam, and a completely diffused light field, the sample constituents that have substantially constant concentrations throughout fixed sample compartments do not give rise to any optical bridge signal, irrespective of their absorbance spectra. This holds true even for constituent substances that have differential absorbance across the wavelength pair. Accordingly, these constituents do not interfere with an optical bridge measurement, regardless of the sample thickness.  
         [0044]    This completes the optical bridge balancing phase; at this point both wavelengths and their intensities have been established. The instrument is ready to perform a measurement. A typical sequence for measurement of glucose in blood will be described in the following text, with reference to the measurement apparatus of FIG. 1, and the processing steps shown in FIG. 2. It will be understood that, in accordance with at least one embodiment of the invention, all blocks, signals, and paths shown in FIG. 2 reside within CPU  104 .  
         [0045]    With the probe beam still directed through the fully compressed sample, pulse-detection LED  46  is turned on, the measurement of blood signal  304  from blood signal detector  94  starts, and a pulse detection subroutine  501  (FIG. 2) is performed using a real-time analysis of the digitized blood signal  304 . Pulse detection subroutine  501  recognizes systolic and diastolic phases of blood signal  304 . After the subroutine  501  locks onto the pulse of the sample, the CPU  104  turns off pulse LED  46  and turns on blood detection LED  44 , The CPU  104  then waits for a period determined by subroutine  501 , and generates a trigger signal to start the measurements synchronized with the heart beat phase. First, a set of system parameter measurements is performed, as instructed by subroutine  502  (FIG. 2). As shown in FIG. 1, a plurality of system parameter signals are generated, which can be sent to data acquisition unit  106  that is in communication with CPU  104 . Examples of these system parameter measurement signals include residual optical bridge value  308 , principal and reference wavelength intensities measured by the sample and auxiliary detectors ( 306  and  310 , respectively), and PID and position sensor values ( 314  and  312 , respectively). The sample and detector temperatures can also be measured and recorded.  
         [0046]    Generally, the sample  80  is maintained in the compressed state to displace the unbound fluid content for a time period of approximately 1 to 100 seconds.  
         [0047]    Next, continuous measurements of the time-varying signals begin, including time-varying measurements of the optical bridge output  308 , blood signal  304 , and position sensor output  312 .  
         [0048]    Once these measurements begin, the compression mechanism  400  then starts opening the measurement head  90  by an amount and rate set by the CPU  104 . According to one aspect, the head opening may have an initial fast phase, followed by a secondary slow phase. The amount of head opening may be fixed (e.g. 0.5 mm for a human ear), or may be thickness dependent (e.g. 30% of the compressed sample thickness). It is directly controlled from the subroutine for compression control  505 , via connection  365 . The purpose for the fast opening phase is to allow the unbound fluid that contains the target analyte to return into the sample. The optional slow phase head opening is designed to compensate for the background matrix thickness displacement resulting from the fluid influx and is also controlled by compression control subroutine  505 .  
         [0049]    The opening of the compression mechanism causes a change in the sample composition, which makes the sample absorb differently at the two wavelengths. This change in absorption of the two wavelengths results in a non-zero optical bridge signal  308 . The measurements continue until stopped by CPU  104 . Typically, the time-varying signal series should contain several hundred data sets, which are recorded over a measurement time period of approximately 0.1 to 10 seconds after the sample uncompression begins.  
         [0050]    This concludes the measurement process, which is then followed by signal processing. An example of the steps for processing the time-varying detector signals and other measured parameters and calculating an estimate of the concentration of the target analyte is illustrated schematically in FIG. 2.  
         [0051]    The optical bridge signal can be represented with the following simplified equation:  
           OBS=CCS·UFA·TAC+CCI   (Eq. 1)  
         [0052]    where:  
         [0053]    OBS=optical bridge signal,  
         [0054]    CCS=calibration constant slope,  
         [0055]    UFA=unbound fluid amount,  
         [0056]    TAC=target analyte concentration,  
         [0057]    CCI=calibration constant intercept, and  
         [0058]    TAA=target analyte amount=UFA·TAC.  
         [0059]    In the case where the optical bridge has been ideally balanced for a measurement, the magnitude of the measured optical bridge signal, OBS (see Eq. 1), represents the difference in the absorbed light intensity at the two wavelengths resulting from the absorption of the target analyte within sample  80 . This difference is proportional to the difference of the absorption by the target analyte at the two wavelengths, as well as to the amount of the target analyte in the sample. The amount of target analyte in the unbound fluid of the sample can be calculated as the product of the unbound fluid amount, UFA, and the target analyte concentration, TAC, in the unbound fluid. The difference of the absorption by the target analyte at the two wavelengths is known (CCS and CCI in Eq. 1), and is determined during a calibration process described in greater detail below. In order to obtain the concentration of the target analyte TAC (e.g. glucose) in the unbound fluid (e.g. blood), the optical bridge signal, OBS  308 , is normalized with the amount of the unbound fluid, UFA.  
         [0060]    As shown in FIG. 2, the unbound fluid amount, UFA, is calculated using subroutine  507 . This calculation is based upon the fact that the time-series recording of the transmitted principal wavelength intensity signal  306  is dependent on the variation of the total amount of fluid in the optical path. This dependence is non-linear and relative. Similarly, the time-series recording of the blood signal  304  is dependent on the variation of the amount of unbound fluid in the optical path, and this dependence is also non-linear and relative. Subroutine  507  can thus perform mathematical modeling and self-normalization using time-series recordings  304  and  306  to calculate an estimate the time-varying amount of the unbound fluid, UFA.  
         [0061]    Theoretically, once the system is calibrated, and the unbound fluid amount is calculated, the target analyte concentration can be calculated directly from Equation 1 using the calibration constants, CCS and CCI, and the instantaneous values of the optical bridge signal, OBS, and the unbound fluid amount, UFA. Due to physiological noise (variations of sample&#39;s physiological properties within the measurement interval), however, measurement enhancements should generally be performed to increase the accuracy of the target analyte concentration estimation. For this reason, multiple (preferably several hundred) measurements of the time-varying signals are obtained during and after the sample thickness alteration. These measurements are then processed using calculation subroutine  509 .  
         [0062]    In accordance with at least one embodiment of the invention, calculation subroutine  509  performs a linear regression of the optical bridge time-series measurements (OBS) vs. the calculated unbound fluid amount (UFA) time-series, over a time window beginning after the end of the fast phase of the opening of measurement head  90 . The slope of the regression line is the parameter that is, in principle, directly correlated to the target analyte concentration. In principle, this slope is also independent of the amount of unbound fluid entering the sampling area, and also independent of the speed at which the unbound fluid enters the sampling area.  
         [0063]    As the unbound fluid enters the sample volume, it displaces some of the non-fluid and bound fluid. This displacement can affect the accuracy of the optical bridge signal in a predictable manner. Subroutine  511  is designed to cancel this effect by using a fast phase correction that is calculated from the change to the signal during the opening (i.e. fast phase) movement of the measurement head.  
         [0064]    Subroutine  517  then calculates an estimate of the target analyte concentration (TAC) in the unbound fluid using the measured system parameters and the previously-described parameters calculated by subroutines  507  (UFA) and  509  (regression), in combination with the calibration coefficients, CCS and CCI, determined by calibration algorithm  521 . The target analyte concentration (TAC) value is calculated based upon the relationship described in Equation 1, and this value may be displayed, digitally or otherwise, at step  522 .  
         [0065]    The measurement system is calibrated prior to performing predictive estimations of the target analyte concentration. The calibration constants, CCS and CCI, are determined by calibration algorithm  521 . The calibration algorithm  521  performs the previously-described measurement process, except that during the calibration process, the concentration of the target analyte is a known quantity. Preferably, at least two and typically between 4 and 10 measurement sequences are performed on samples with varying and known concentrations of the target analyte. The measurement sequence for calibration is identical to the one used in predictive estimation, except that at the end of the procedure, the calibration constant(s), rather than the analyte concentration, are calculated and stored for future reference. Using the relationship of Equation 1, the calibration algorithm  521  calculates the calibration constant(s) by performing best fit regression between the known concentrations of the target analyte and the above-described calculated parameters determined by subroutine  517 , and determines the calibration constants from the regression. Typically, there is one multiplication calibration constant, CCS, and another additive constant, CCI. These calibration constants are later used by subroutine  517  to calculate the target analyte concentration (TAC) in a predictive measurement where the analyte concentration is unknown.  
         [0066]    Having thus described a few particular embodiments of the invention, various alterations, modifications and improvements will readily occur to those skilled in the art. Such alterations, modifications and improvements as are made obvious by this disclosure are intended to be part of this description though not expressly stated herein, and are intended to be within the spirit and scope of the invention.  
         [0067]    For example, while the method is here described as applied to an optical bridge employing an acousto-optical tunable filter, it can also be applied to different implementations of the optical bridge, such as one equipped with tunable diode lasers or other means to generate a beam containing the required wavelength pairs. Moreover, although the method is here described with a focus toward measuring the concentration of glucose in blood, the method and apparatus of this invention may also be employed to detect the concentration of other analytes such as cholesterol, urea, heavy metals, alcohol, nicotine or drugs in blood or other fluids. Further, sinusoidal, rather than square, modulation waveforms that are set 180° out of phase and result in a substantially constant total intensity, can alternatively be used to form the combined radiation beam. Also, measurements of radiation reflected by the tissue, rather than transmitted radiation, can be performed to obtain the desired data.  
         [0068]    Accordingly, the foregoing description is by way of example only, and not limiting. The invention is limited only as defined in the following claims and equivalents thereto.