Abstract:
Instrumentation for measuring luminescence phase lag to quantitate an analyte concentration is corrected to eliminate or reduce extraneous phase lag noise. A calibration factor is determined in steps that are interspersed between quantitative measurements. An optical pathway is provided to accomplish the calibration by the provision of a second optical source that emits in the luminescence emission band of a luminescent material. The calibration factor may be subtracted from measurement of the quantification phase lag to correct for extraneous phase lag.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims benefit of priority to provisional application Ser. No. 60/506,813 filed Sep. 29, 2003 which is incorporated by reference herein. 
     
    
     BACKGROUND 
       [0002]    1. Field of the Invention 
         [0003]    This disclosure pertains to methods and instruments that use luminescence phenomena to achieve quantitation measurements, for example, in optical sensing measurements to assess concentration of an analyte. More particularly, the methods and instruments may achieve automatic compensation for extraneous phase response when measuring a luminescent lifetime by use of an auxiliary light source. 
         [0004]    2. Description of the Related Art 
         [0005]    Luminescence pertains to the emission of light by materials. Fluorescence and phosphorescence are luminescence phenomena that occur following stimulation or excitation of a material by photons or electrons. These phenomena are of particular interest when used in sensors. Fluorescence and phosphorescence have been used to determine temperature and strain, as reported by A. Arnaud, D I Forsyth, T Sun, Z Y Zhang and K T V Grattan, “Strain and temperature effects on Erbium-doped fiber for decay-time based sensing,” Rev. Sci. Instrum., 71, pp. 104-8 (2000); oxygen as reported in U.S. Pat. No. 4,845,368 issued to Demas and U.S. Pat. No. 5,043,286 issued to Khalil; pH as reported by Hai-Jui Lin, Henryk Szmacinski, and Joseph R. Lakowicz, “Lifetime-Based pH Sensors: Indicators for Acidic Environments,”  Analytical Biochemistry  269, 162-167 (1999); CO 2  as reported by Q. Chang, L. Randers-Eichhorn, J. R. Lakowicz, G. Rao.,  Biotechnology Progress  1998, 14, 326-331), and ions as reported by Sheila Smith et. al. “Fluorescence energy transfer sensor for metal ions,” Proc. SPIE Vol. 2388, p. 171-181, Advances in Fluorescence Sensing Technology II; Joseph R. Lakowicz; Ed. May 1995. 
         [0006]    The effect of a particular analyte on a luminescent material generally results from a change in the quantum efficiency of the luminescence process. This change manifests itself as a difference in the observed decay rate or lifetime of luminescent material. While the observed steady state luminescence intensity also changes and may be measured to quantify the analyte, the measurement of lifetime is more accurate because is it less affected by changes of intensity due to optical alignment, ambient light or changes in concentration of the luminescent sensor material, as discussed in  Topics in Fluorescence Spectroscopy , ed J. Lakowicz, Vol. 4, Chap 10. 
         [0007]    For a given optical sensor the lifetime, τ, is generally related to analyte concentration, [A], according to the following relationship from  Topics in Fluorescence Spectroscopy , vol. 1, Chap 8, 2 nd  ed, ed. J. R. Lakowicz, 1999: 
         [0000]    
       
         
           
             
               
                 
                   τ 
                   = 
                   
                     1 
                     
                       1 
                       + 
                       
                         K 
                          
                         
                           [ 
                           A 
                           ] 
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   1 
                   ) 
                 
               
             
           
         
       
     
         [0000]    In Equation (1), K is a quenching constant that is determined for each luminescence sensor formulation. Once K is determined for a particular sensor, one needs only to measure the lifetime to calculate the analyte concentration. The above Equation (1) represents a sensor with ideal quenching kinetics. Luminescent sensors may depart significantly from this model, in which case an empirically derived equation relating lifetime to analyte concentration may be used. If the sensor is in an environment where temperature fluctuates significantly, it may be necessary to include the measured temperature as part of the empirically derived relationship. Equation (2) below represents generally the manner of empirical relationship, which may be provided as a least squares fit or other type of correlation: 
         [0000]        [A]=f (τ, T ),  (2) 
         [0000]    Here τ is luminescent lifetime and T is temperature of the sensor. 
         [0008]    Numerous methods exist for of measuring the lifetime of a luminescent oxygen sensor. For example, U.S. Pat. No. 5,043,286 issued to Khalil describes the use of a time domain technique which calculates the ratio of a two box car integrations following rapid turn off of luminescence excitation provided by an LED. Other methods use a frequency domain where luminescence excitation incident on the oxygen sensitive coating is modulated in a periodic fashion, either sinusoidally, as a square wave, or other periodic waveform.  FIG. 1  illustrates the emission of the luminescent material as a dashed line that lags in phase with respect to the excitation, which is shown as a solid line. The phase lag, generally measured in degrees, is denoted Δφ. From the phase lag the lifetime may be calculated using the following relationship: 
         [0000]    
       
         
           
             
               
                 
                   τ 
                   = 
                   
                     
                       1 
                       
                         2 
                          
                         
                             
                         
                          
                         π 
                          
                         
                             
                         
                          
                         f 
                       
                     
                      
                     
                       tan 
                        
                       
                         ( 
                         
                           Δ 
                            
                           
                               
                           
                            
                           φ 
                         
                         ) 
                       
                     
                   
                 
               
               
                 
                   ( 
                   3 
                   ) 
                 
               
             
           
         
       
     
         [0000]    For sensing applications using frequency domain, also known as phase domain, the phase can be substituted in Equation 2 to give the relationship 
         [0000]      [ A]=f (Δφ, T )  (4) 
         [0000]    where Δφ is the phase lag induced by the luminescent material, T is temperature, and [A] is analyte concentration. 
         [0009]    Other calculation techniques exist, such as are described by Venkatesh Vadde and Vivek Srinivas in “A closed loop scheme for phase-sensitive fluorometry”, American Institute of Physics, Rev. Sci. Instrum., Vol. 66, No. 7, July 1995, p. 3750, where a phase-locked loop is used to determine the phase shift and luminescence lifetime. U.S. Pat. No. 4,716,363 issued to Dukes describes a method of monitoring the frequency that is required to achieve a constant phase delay through a luminescent material. Frequency may be related to lifetime by rearranging Equation (3) above U.S. Pat. No. 6,157,037 issued to Danielson describes the simultaneous variation of frequency and phase using a digital signal processor (DSP) to achieve a luminescence lifetime measurement. An analog oscillator circuit may be used to measure the lifetime of a fluorescent material, for example, as described in U.S. Pat. No. 6,673,626 B1 issued to Rabinovich et al. 
         [0010]    Pulse laser measurements described by Lakowicz et. al. in “2-GHz frequency-domain fluorometer,” Rev. Sci. Instrum. 57(10) October 1986, utilize high frequency cross correlation measurements of lifetime using very short periodic pulses from a laser, such as 5 picosecond pulses. Phase delays at frequencies higher than the fundamental pulse frequency are measured using cross correlation of high-order harmonics of the pulse repetition rate. 
         [0011]    All of the systems described above apply to direct or indirect measurements of the phase lag that is induced by the luminescent material. Even so, the techniques generally neglect the effect of extraneous phase lag that is introduced by the optical and electrical components of a luminescence lifetime measurement system. An extraneous phase lag may be defined as an appreciable phase lag that derives from something other than the phase lag arising from the luminescence lifetime phase shift phenomenon indicated as problem is generally defined as Δφ in context of Equations (3) and (4). 
         [0012]      FIG. 2  below shows a prior art sensor system  200  for use in generating and detecting luminescence to perform a lifetime measurement in the phase or frequency domain. 
         [0013]    A signal generator  202  generates an excitation signal  203 , e.g., as a voltage signal V s  or a current signal I s , according to a time-dependant function x(t). By way of example, the excitation signal  203  is a periodic excitation signal which may be a sine wave or square wave. The signal generator may be an analog or digital signal generator. The excitation signal  203  travels from the signal generator  202  to both a phase comparator  204  and source driver circuitry  206 . The source driver circuitry  206  amplifies the excitation signal  203  and provides a current source that follows the excitation signal in intensity. The current output from the source driver circuitry  206  is applied to an optical excitation source  208 , which produces excitation light  210  of varying intensity according to function x(t)′ that closely follows the function x(t) as processed by the source driver circuit  206 . The optical excitation source  208  is preferably a light emitting diode (LED), laser diode or vertical cavity surface emitting laser (VCSEL), but may be any other light source that can be modulated at a sufficiently high frequency. The modulated light  210  according to the function x(t)′ stimulates photon emission light  212  in a time-dependant emission function y(t). Photon emission light  212  occurs by the activity of excitation light  210  on luminescent material in a luminescent sensor  214 . The optical excitation source  208  is selected to emit light  210  in a bandwidth that includes a wavelength or wavelengths that induce corresponding photon emission light  212  in the luminescent sensor  214 . 
         [0014]    It will be appreciated that a portion of the excitation light  210  may continue through the luminescent sensor  214  into pathways shown generally in  FIG. 1  as the photon emission light  212 . A pair of complementary optical color filters  216 ,  218  may be used to tune sensor system  200  for rejection of undesirable wavelengths of light. The color of the optical excitation source  208  and excitation filter  216  are matched to the characteristics of the luminescent material in luminescent sensor  214 . Likewise, the emission filter  218  is also selected according to the color of the luminescence emission. By way of example, where the excitation light  210  emitted by optical excitation source  208  is blue or green, a corresponding blue or green excitation filter  216  is used to narrow the band of blue or green excitation light  210  impinging upon the luminescent sensor  214 . Where the photon emission light  212  is red, a red emission filter  218  is used to reduce or eliminate scattered excitation light  210  that passes through the luminescent sensor  214  from reaching a photo detector  220 . Thus, light  222  may be filtered in this manner to select for a narrow bandwidth that enhances the signal output and quantitation from photo detector  220 . The photo detector  220  may be, a photodiode, a photomultiplier tube, microchannel plate, avalanche photodiode, or other type of photo detector. The light  222  impinges on detector  220 , which converts the light  222  to a detection signal  224 , e.g., a voltage signal Ve or current signal Ie. The detection signal  224  embodies information from the emission function y(t). A preamplifier  226  operates upon detection signal  224  to provide an amplified output signal  228  having the form of function y(t)′. The amplified output signal  228  passes to phase comparator  204 . 
         [0015]    The phase of the emission function y(t) lags the phase of the excitation function x(t)′ by an amount relating to the luminescence lifetime of the luminescent material, according to Equation (3) above. The phase comparator  204  measures the phase difference between the excitation signal  203  and the amplified output signal  228 . If the phase lag in all electrical and optical components within sensor system  200  are negligible when compared to the phase lag introduced by the luminescent material, then the phase difference that is measured by the phase comparator can be taken as the phase lag of the luminescent material in the luminescent sensor  214 . This phase difference can be used, by way of example, to determine the lifetime using Equation 3 above or to determine analyte concentration using Equation (4) above. 
         [0016]    The foregoing description of sensor system  200  is a generic sensing arrangement for monitoring luminescent lifetime by measuring phase shift. Any one of the time or frequency domain lifetime measurement methods discussed above may use a configuration which is similar to the above diagram. 
         [0017]    The foregoing calculation methodology described in context of  FIG. 2  assumes, for example, that the excitation function x(t)=x(t)′ and the emission function y(t)=y(t)′; however, significant measurement error may result from this assumption. This occurs because the electrical components of sensor system  200  may introduce appreciable changes to the phase difference measurements. As discussed above in context of  FIG. 2 , the time domain and frequency domain lifetime measurement techniques of the prior art generally suffer from the fact that the measured lifetime represents not only the phase lag introduced by the luminescent material, but also the extraneous phase lag that intercedes from use of the various optical and electrical components in sensor system  200 . 
         [0018]    By way of example, a blue LED from Panasonic (DigiKey Inc part # P465-ND) has a given emission bandwidth at 110 MHz with −10 db attenuation relative to low frequencies, and may be used as optical excitation source  208 . At this frequency the phase lag due to the LED emission is significant when compared to nanosecond fluorescence lifetimes. If the physical distance between the optical excitation source  208  and the luminescent material in luminescent sensor  214  is sufficiently large, the phase lag due to transit time may also become significant. The detector  220  and preamplifier  226  add phase lag to the respective signals  224 ,  228 . The time and frequency domain lifetime measurement methods discussed above cannot separate the extraneous phase lag introduced by optical components, electrical components and distance from the luminescent material. 
         [0019]    A common strategy for correcting for the extraneous phase lag imparted by optical and electrical components is to measure a standard sample of known phase lag. For example, Lakowicz et. al. in “2-GHz frequency-domain fluorometer,” Rev. Sci. Instrum. 57(10) October 1986, report use of the fluorescent dye Bengal rose in water at pH=9 for a standard fluorescence lifetime of 75 picoseconds. Alternatively Lakowicz et. al. use a 25 picosecond quartz plate etalon as a standard. Since the characteristics of such standard samples are well known, the extraneous phase of the luminescent lifetime measurement system can be measured over a range of modulation frequencies. With this information in hand, the extraneous phase can be subtracted before calculating the lifetime according to Equation (3). 
         [0020]    The disadvantages of this technique are twofold. First, a standard sample is required, which is impracticable for many applications. Second, there is an assumption that the extraneous phase lag remains constant over time for any given instrument. In fact this is usually not the case. Environmental factors can cause the extraneous phase of the electronic and optical components to change. Large swings in temperature induce significant variations in phase lag of LEDs, photodiodes and associated electronics. A sensor instrument that is stationed outside the confines of a building must compensate for large variations in temperature. If the temperature induced changes in extraneous phase are not compensated for, accuracy of the measurement of lifetime, and hence quantification of analyte will suffer. Ageing of optical and electronic components may also lead to significant changes in extraneous phase lag. Another disadvantage of this system is a requirement for operator intervention where the operator must replace the normally used luminescent sensor with the standard luminescent sensor to calibrate the lifetime measuring system. 
         [0021]    Proposed solutions to the extraneous phase problem are complex and costly in implementation. Other methods of measuring and correcting for extraneous phase use wavelength selection to select for and detect the higher energy luminescence excitation. If only the higher energy luminescence excitation is detected, it contains only the extraneous phase of the electro-optical components. By way of example, Riedel in WO 01/22066 A1 describes use of a microprocessor to select and mechanically place different optical filters in front of the photo detector used to monitor luminescence emission. When a long wavelength-pass optical filter is selected, the detector measures only the luminescence emission of the material. When a short-pass filter is selected, the detector monitors only the higher energy excitation light. European Patent EP 0 702 226 B1 discloses a method for using a wavelength selecting element in front of a detector to directly monitor the higher energy excitation wavelengths, similar to Riedel above. These methods can be used to correct for extraneous phase, however at the expense of a more complicated optical configuration requiring mechanical selection of optical filters. 
         [0022]    It is therefore of interest to develop instrumentation that can as accurately as possible determine lifetime, with minimal disturbance from extraneous phase changes over wide range of temperatures, environmental conditions and age of components. 
       SUMMARY 
       [0023]    The instrumentalities described below overcome the problems outlined above and advance the art by providing a simplified solution to the extraneous phase lag problem. 
         [0024]    A frequency domain luminescence system is used to determine at least one of a phase lag and a luminescence lifetime. The system is improved by providing a second optical source in addition to a conventional optical excitation source that produces a luminescent response in a luminescent sensor. The second optical source emits in a spectrum that does not induce a luminescent response in the luminescent sensor. Light from the second optical source may be, for example, in preferred embodiments, an LED that emits light in the emission band of the luminescent material. Sensing of the light from this second optical source provides a good approximation of the extraneous phase lag in the system, particularly where the second optical source is selected to have an emission lag which closely approximates that of the optical excitation source. The second optical source may emit, for example, at 800 nm to 1100 nm. 
         [0025]    The system may operate in two modes including a calibration mode and a quantitation mode. In the quantitation mode, the system is effective to assess an analyte concentration where the analyte is in contact with the luminescent sensor. In the calibration mode, the system performs a self calibration to correct for extraneous phase lag. In either mode the relevant optical sources may be operated, for example, at a constant frequency ranges from 1 kHz to 1 MHz or from 1 MHz to 2 GHz where the driven signals may be, respectively in complementary nature, sine waves, square waves, periodic train of pulses, another patterns, or combinations thereof. The driven signal frequency may be much higher with subsequent heterodyning or downconversion of the detector signal. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0026]      FIG. 1  illustrates a luminescence lifetime phenomenon as a phase lag between excitation and emission events where the luminescence lifetime may be used for quantitation of an analyte concentration; 
           [0027]      FIG. 2  is a block schematic diagram of a prior art sensor system that does not correct for extraneous phase lag; 
           [0028]      FIG. 3  is a block schematic diagram showing a sensor system that corrects for extraneous phase delay according to the instrumentalities disclosed herein; 
           [0029]      FIG. 4  is a block schematic diagram that illustrates a switching configuration that places the sensor system in a calibration mode; 
           [0030]      FIG. 5  illustrates an alternative embodiment where a backprojection sensor is used in the sensor system; 
           [0031]      FIG. 6  is a graph showing ambient temperature variations that were used to test one embodiment of the sensor system; 
           [0032]      FIG. 7  shows experimental data confirming that sensed quantification phase lag results do vary with temperature variations as extraneous phase lag; 
           [0033]      FIG. 8  shows a calibration phase lag that similarly varies with temperature; 
           [0034]      FIG. 9  shows a corrected or true luminescence phase lag that is achieved by deducting the quantification phase lag from the calibration phase lag to correct for system temperature effects; and 
           [0035]      FIG. 10  is a flowchart that illustrates one embodiment of user-selectable control logic for the sensor system. 
       
    
    
     DETAILED DESCRIPTION 
       [0036]    There will now be shown and described a sensor system that incorporates a technique for eliminating extraneous emission from the measurements. This nonlimiting disclosure is by way of example to show implementation of preferred materials and methods. 
         [0037]      FIG. 3  is a schematic block diagram of sensor system  300 . Identical components with respect to sensor system  200  shown in  FIG. 2  retain identical numbering with respect to  FIG. 3 , except as noted below. Light indicated generally as light  210 ,  212 ,  222  travels on a first optical pathway. Sensor system  300  differs from sensor system  200  by the addition of emission band light source  302  and electronically controllable switches SW 1  and SW 2 . The emission band light source  302  inay be selectively energized by closure of SW 2  to emit light in a bandwidth that encompasses the emission spectrum or wavelength of the luminescent material in the luminescent sensor  214 . For example, where the emission spectrum is red, the emission band light source  302  may be a red LED, red laser diode, or red VCSEL. Switch SW 1  may be opened to cease emissions from optical excitation source  208  while SW 2  closed to drive emission band light source  302  according to function x(t), which is embodied in signal  203  and driven by source driver circuitry  206 . Emission band light source  302  may also be driven simultaneously with optical excitation source  208 , but this mode of operation is less preferred. Activation of emission band light source  302  causes emission of light in the emission band of luminescent sensor  214  to travel on pathway  304 , through luminescent sensor  214  to impinge upon detector  220 . Detector  220  is capable of detecting light on pathway  304 . 
         [0038]    The modulated light from the emission band optical source  302  is not absorbed and emitted by the luminescent behavior of the luminescent sensor  214 . Passage of light on pathway  304  merely scatters, diffuses, and transmits the light on pathway  304 . The emission band optical source  302  is preferably selected to have a phase response or emission delay characteristic which is similar to that of the optical excitation source  208  because this type of selection provides the best approximation of extraneous phase lag. 
         [0039]    The signal output from preamplifier  226  following emission on pathway  304  contains the extraneous phase lag of the electronic and optical components of the sensor system  300 , with no phase lag contribution from the luminescent sensor  214 . One particular advantage of the embodiment shown is that the phase comparator may by signal arrangement automatically open and close switches SW 1  and SW 2  in a predetermined way. Closure of SW 1  with opening of SW 2 , as shown in  FIG. 3 , represents a quantitation mode in which quantitation is performed with correction for extraneous phase lag. In quantitation mode, an analyte (not shown) is interacting with the luminescent sensor  214  in a conventional way for diffusion of an analyte into the luminescent sensor  214 . Conversely, opening of SW 1  with closure of SW 2  as shown in  FIG. 4  represents a calibration mode to assess the extraneous phase lag for use in the quantitation mode. Phase comparator  304  may switch between the respective modes to perform a real-time extraneous phase lag correction between successive measurements or periodically throughout a system of measurements. In this sense, “real time” means that there has been insufficient time for the analyte and/or environmental conditions to change the optical behavior of the luminescent sensor  214  between conduct of quantitation mode measurements and calibration mode measurements. This real-time phase correction provides a more accurate measurement of the luminescent lifetime or phase lag without requiring the use of standard samples or complex compensation mechanisms. 
         [0040]    It will be appreciated that a backprojection sensor may be substituted for luminescent sensor  214  as shown in  FIG. 3  with the same effect where pathway  304  is a reflective pathway.  FIG. 5  shows one such arrangement in backprojection system  500  where a conventional backprojection sensor  502  replaces luminescent sensor  214  of  FIGS. 3 and 4  to establish a reflective pathway  304 . 
         [0041]    In one embodiment, a blue LED is used as optical excitation source  208  in combination with a blue optical filter  216  to excite a luminescent oxygen sensor  214 . The luminescent oxygen sensor  214  emits red light which passes through a red emission filter  218  before striking a photodiode detector  220 . Determination of the true phase lag of the luminescent oxygen sensor  214  entails two separate measurements. First the phase lag through the system  300  using the blue LED optical excitation source  208  is measured at a modulation frequency of 11 kHz according to signal  203  and function x(t) with closure of SW 1  and opening of SW 2  to establish a quantitation mode. Next SW 1  is opened, disabling the blue LED optical excitation source  208 . SW 2  is closed for activation of a red LED emission band source  302  to establish a calibration mode. Another phase lag measurement is made when modulating the red LED emission band source  302  at 11 kHz according to signal  203  and function x(t). 
         [0042]    The phase lag due to the luminescent oxygen sensor is then simply calculated as follows. 
         [0000]      Δφ sensor =Δφ Q −Δφ C   (5) 
         [0000]    where phase comparator  306  uses Δφ sensor  as the true luminescence phase lag Δφ in Equation (2), (3) or (4), Δφ Q  is quantitation mode phase lag; and Δφ C  is the calibration mode phase lag. The phase lags Δφ Q , and Δφ C  need not be measured absolutely and in high accuracy if the relative phase difference between Δφ Q  and Δφ C  is accurate. Mathematically equivalent true phase lag calculations resulting from different methods used in the phase comparator  306 , e.g., using derivative methods or finite difference methods analyzing signals  203 ,  228  other than strictly by subtraction, do not affect this measurement so long as the operative principle of eliminating the calibration phase lag is observed. The ordering of quantitation and calibration modes may be in any order, such as by placing the calibration measurement before the quantitation measurement, by interspersing the calibration measurement throughout a plurality of quantitation measurements, or by averaging a plurality of calibration and/or quantitation measurements. 
       Comparative Example 
       [0043]    The utility of this two step measurement method is illustrated when the temperature of the electronics and optics drift with fluctuations in room temperature or by heating or cooling of electrical components by virtue of frequency of measurement. In one test apparatus, the two quantities Δφ Q  and Δφ C  were measured continuously in repeat intervals over 8000 seconds. The luminescent sensor  214  was kept in a constant temperature and oxygen environment, so as not to alter the phase lag and luminescent lifetime by changes in the analytical environment during the experiment. 
         [0044]    The phase measurement system  300  including a blue LED as optical excitation source  208  and a red LED as the emission band source  302 , the a photodiode detector  220 , preamplifier  226  and phase comparator  306  were exposed to ambient indoor conditions. The ambient temperature was monitored centrally with respect to sensor system  300 . The ambient temperature fluctuated due to warm-up of the electronics and influence of the laboratory air conditioning system, as shown in  FIG. 6 . Significant variations in temperature occurred over the 8000 second experimental period. 
         [0045]      FIGS. 7 and 8  show the measurements of the blue phase lag Δφ Q  ( FIG. 7 ) and the red phase lag Δφ Q  ( FIG. 8 ) in sensor system  300  over the same timeframe. The Δφ Q  and Δφ C  phase lag measurements were obtained alternately, the pair of measurements being made once every second. Both the sources  208 ,  302  were modulated at 11 kHz for these measurements. 
         [0046]    Comparing the Δφ Q  and Δφ C  phase lags shown in  FIGS. 7 and 8  to the temperature of  FIG. 6  over the experiment duration, it is shown that the perturbations in measured phase correlate with changes in temperature. The phase lag due to the luminescent oxygen sensor, Δφ O2 sensor , was calculated by subtracting the red phase lag Δφ C  from the blue phase lag Δφ Q . In this system, using Equation (3), a phase shift of 9.65 degrees at 11 kHz modulation corresponded to a luminescence lifetime of 2.46 microseconds. This is shown in  FIG. 9  where the value 9.65 approximates on average a horizontally-extending band of data representing the Δφ O2 sensor  value of Equation (5). 
         [0047]    It will be appreciated that the band of data shown in  FIG. 9  encompasses a range of noise extending generally between 9.5 and 9.7, but the best value may be calculated as an arithmetic average where this band is immune to the perturbations shown in  FIG. 7  and. If the manner of calculating without correction for extraneous phase lag, the noise would range from 8.1 to 8.3 over the experiment, as shown in  FIG. 7 . Any value in this range would be appreciably in error with respect to about 9.65 (see  FIG. 9 ). 
         [0048]    This example shows that correction of extraneous phase lag significantly reduced the variations due to environmental temperature effect on the measurement optics and electronics. As a result the lifetime of the luminescent oxygen sensor was more accurately measured, in turn giving a more accurate measurement of oxygen concentration in the analyte. 
         [0049]    The foregoing example demonstrates the use of optical phase correction with a luminescent oxygen sensor. Table 1 below identifies various oxygen-sensitive luminescent materials that may be used as the luminescent material in sensor  214  when sensor  214  is an oxygen sensor. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 LUMINESCENT DYES SUITABLE FOR OXYGEN SENSING. 
               
             
          
           
               
                   
                   
                   
                 Lifetime in 
               
               
                   
                   
                   
                 absence of 
               
               
                 Dye 
                 Excitation 
                 Emission 
                 oxygen 
               
               
                   
               
             
          
           
               
                 palladium octaethyl porphyrin 
                 390,536 
                 670 nm 
                 1.6 
                 msec 
               
               
                 (PdOEP) 
                   
               
             
          
           
               
                 Platinum octaethyl porphyrin 
                 382,532 
                 nm 
                 650 nm 
                 100 
                 μsec 
               
               
                 (PtOEP) 
               
               
                 platinum pentafluoryl- 
                 390,536 
                 nm 
                 660 
                 78 
                 μsec 
               
               
                 tetraphenyl porphyrin (PtTFPP) 
               
               
                 ruthenium(bathophenanthrolene) 
                 450 
                 nm 
                 620 nm 
                 5 
                 μsec 
               
               
                 PtOEPK Pt(II) 
                 450 
                 nm 
                 750 nm 
                 60 
                 usec 
               
               
                 (octaethylporphine) ketone 
               
               
                 [Porphyrin Products] 
               
               
                   
               
             
          
         
       
     
         [0050]    The excitation of these materials is ideally compatible with solid state light emitting diodes, or laser diodes, and the emission is preferably detectable using silicon photodiodes. The luminescent lifetime in the absence of oxygen influences the sensitivity of the oxygen sensor. Selection of the luminescent material depends upon conditions in the intended environment of use. For example, PdOEP has a very long lifetime and, consequently, is suitable for use with analytes having very low concentrations of oxygen. At high concentrations of oxygen, PdOEP is highly quenched and unsuitably dim to make an accurate lifetime measurement. 
         [0051]    Other well suited luminescent dyes are described by Papkovsky, D. B., “Luminescent porphyrins as probes for biosensors,” Sens and Act B 11 (1993) 293-300 and Papkovsky, D. B., “New Oxygen sensors and their application to Biosensing,” Sens and Act B 29 (1995), 213-218 and J. N. Demas, B. A. DeGraff, “Design and Application of Highly Luminescent Transition Metal Complexes,” Anal. Chem. vol 63 n17 829-37, 1991. 
         [0052]    Table 2 lists various oxygen-permeable polymer matrices that have been successfully used with oxygen-sensitive luminescent dyes. Generally, the luminescent sensor  214  includes a polymer matrix into which an analyte can diffuse, where a luminescent material is dispersed to substantial homogeneity in the polymer matrix. By way of example, the decision to use a particular polymer with a particular oxygen sensitive dye depends principally on the luminescence lifetime in the absence of oxygen and the oxygen permeability of the polymer. The pairing of a very long lifetime dye (e.g. PdOEP) with a highly permeable polymer, e.g. RTV-118 Silicone, may be suitable for low concentrations of oxygen, i.e. below 1 ppb dissolved in water. This same combination would probably not be suitable for higher concentrations of oxygen found in water near standard atmospheric pressure, composition and temperature because the sensor would be too highly quenched for accurate measurements. In general a “good” polymer and dye combination gives a dynamic range of 5 to 10 over the range of oxygen concentration expected in the analyte. Dynamic range is defined as the intensity or lifetime at the lowest oxygen concentration divided by the lifetime or intensity at the highest oxygen concentration. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 OXYGEN PERMEABLE POLYMER MATRICES 
               
             
          
           
               
                   
                 Physical 
                 Oxygen 
               
               
                 Polymer 
                 properties 
                 Permeability 
               
               
                   
               
               
                 polymethyl methacrylate (PMMA) 
                 hard, durable 
                 very low 
               
               
                 dimethly-siloxane co-block bisphenyl A 
                 hard durable 
                 moderate 
               
               
                 acrylic random copolymer 
                 flexible 
                 high 
               
               
                 fluorinated acrylic random copolymer 
                 flexible 
                 high 
               
               
                 fluorinated silane random copolymer 
                 flexible 
                 very high 
               
               
                 RTV-118 Silicone (General Electric) 
                 rubbery 
                 very high 
               
               
                 polystyrene 
                 hard 
                 moderate 
               
               
                 fluorinated polystyrene 
                 hard 
                 high 
               
               
                   
               
             
          
         
       
     
         [0053]    Oxygen sensors typically require modulation frequencies from 2 kHz to 1000 kHz, depending on the type of luminescent material that is used to accurately measure the luminescent lifetime. Other analytes may, for example, use excitation frequencies above those used for oxygen sensors. The use with excitation frequencies from 1 MHz to 2 Ghz is particularly useful for fluorescence sensors that measure glucose, pH, Ca2+ and other ions and chemical species. 
         [0054]      FIG. 10  is a flowchart that shows programmable modes of operation for sensor system  300  as shown in  FIG. 3  and described above. The operational logic of process  1000  may be implemented by program instructions or circuitry, for example, as provided in the phase comparator  306  or any other processing unit. The program instructions may be used on a single processor with associated memory or in a distributed processing environment. 
         [0055]    A quantitation action  1002  of measuring a quantitation phase lag in a luminescent sensor is followed by a test  1004  to ascertain whether system  300  has been instructed to skip calibration on a particular iteration form among N such iterations, for example, to perform one calibration in step  1006  for every three passes through action  1002 . Action  1002  occurs with the system  300  in quantitation mode, as described above. Calibration step  1006  involves using the sensor system  300 , generally, electro-optic equipment, in calibration mode to determine the calibration phase lag. Loop test  1008  inquires whether it is appropriate on the basis of P iterations to loop back to the quantitation action  1002  or proceed to step  1010  for correction of extraneous phase lag according to Equation (5). 
         [0056]    The programmable variations indicated in  FIG. 10  permit, for example, the use of a varying frequency applied in a pattern from signal generator  202  to drive the optical excitation source  208  during the quantitation action  1002  and again in the calibration step  1006 , but in different time domains with storage of resulting phase lag values ΔφQ and ΔφC in system memory for use in the step of correcting  1010  to produce Δφ sensor, i.e., the true luminescence phase lag. This value may be used to analyze the concentration of an analyte that is in contact with luminescent sensor  214 , by Equations (3), (4), or other calculations known in the art. 
         [0057]    The phase compensation scheme discussed above is applicable to any phase/frequency-based method for measuring luminescent lifetime or phase retardation of a periodic optical signal. For example, the lifetime measurement systems and methods that are described in U.S. Pat. No. 4,716,363, 5,646,734, or 4,845,368 may be modified as shown in  FIGS. 3 and 4  to correct for extraneous phase lag. Systems using phase comparators of any kind may benefit from the presently disclosed system and method. Phase comparators using a two-phase lock-in or Fourier transform method may also benefit from modification to include the system and method that is presently disclosed. 
         [0058]    The phase compensation scheme is also useful when with a servo-feedback-loop phase comparator. The phase measurement method used by Venkatesh Vadde and Vivek Srinivas “A closed loop scheme for phase-sensitive fluorometry”, American Institute of Physics, Rev. Sci. Instrum., Vol. 66, No. 7, July 1995, p. 3750 is a phase comparator that uses a servo feedback loop to optimize the determination of phase between the excitation signal and the luminescent emission. In this method the phase comparator uses a servo feedback loop that adds additional phase shift to the luminescence emission signal until the modified emission signal is 90 degrees out of phase with the excitation signal. The additional phase shift is subtracted from 90 degrees to obtain the phase shift between the excitation signal and the luminescent emission signal. The extraneous phase can be corrected in this example by making a second measurement using the red reference LED in place of the blue excitation LED, and subtracting the result from the prior measurement of the luminescent emission signal phase. 
         [0059]    The embodiments described above use primarily a constant modulation frequency emanating from signal generator  202  for determination of luminescent lifetime. Other equally suitable embodiments may utilize a variable modulation frequency with a constant or variable phase shift through the luminescent material. By way of example, U.S. Pat. No. 4,716,363 issued to Dukes, and U.S. Pat. No. 6,157,037 issued to Danielson teach the use of variable modulation frequency of the excitation signal The Dukes patent uses a phase comparator that demands a constant phase shift between the excitation signal and the luminescent emission. The phase comparator adjusts the modulation frequency of the excitation light source to achieve a certain constant phase shift, e.g. 45 degrees, between the excitation and the luminescent emission. The phase comparator used by Danielson demands a variable, frequency dependent phase shift between the excitation signal and the luminescent emission. The phase comparator simultaneously adjusts the excitation frequency and the phase shift requirement. In this case the preferred embodiment is to measure the calibration phase lag through the system over all anticipated frequencies in advance of switching to the quantitation mode. The phase offset as performed by the Dukes or Danielson method is continuously corrected using the previously measured calibration phase lag. If the modulation frequency does not exactly match a frequency at which a calibration phase lag measurement is made, then interpolation may be used to more accurately determine the phase correction. 
         [0060]    Another embodiment exists where the phase comparator uses downconversion. In this embodiment the modulation frequencies in the quantitation and calibration modes are higher than the frequency at which the phase comparator measures the phase lag. In a phase comparator using downconversion, the modulation frequencies of the excitation and the emission are converted to lower frequencies while preserving their phase relationship. By way of example, this embodiment may use heterodyning or downconversion of the modulated luminescence emission before determination of phase lag. By way of example, U.S. Pat. No. 5,196,709 teaches the use of downconverting the modulated luminescence emission to a lower frequency for determination of phase lag. European Patent Application EPA 1988-03-16 0259973/EP-A2 “Fluorometric sensor system using heterodyne technique” discusses the heterodyne technique. These systems may be modified to include instrumentalities as presently shown and described to correct for extraneous phase lag. 
         [0061]    It will be appreciated that optical excitation source  208  and emission band source  302  are selected to emit at different wavelengths to the uses described above. Although it is preferred that the emission band of optical source  302  persists at a wavelength which is inherent to the emission spectrum of luminescent sensor  214  but does not induce corresponding luminescent emission, this is not a strict requirement. By way of example, a choice of LEDs may be appropriately matched to the absorption and emission spectra of the luminescent material and the characteristics of the excitation and emission color filters  216 ,  218 . It is most convenient if the reference LED emits light at substantially the same color as the luminescent material emits. But LEDs often emit a relatively broad range of wavelengths, even if only weakly. The emission of some blue LEDs contain significant amounts of red light, so a blue LED could also be used to provide the light for measurement of a red phase lag. In this case, however, it may be necessary to use an optical filter in front of the blue LED that only allows red light to pass. Otherwise the blue LED would stimulate luminescence emission. A separate blue LED could also be used if it were coated with a sufficiently fast lifetime fluorescent material that emitted at substantially the same wavelength as the luminescent materials. 
         [0062]    Another embodiment replaces an emission band source  302  its wavelengths of light substantially different from the excitation light source and from the luminescent emission. For example a near IR LED or laser diode, with emission from 800 nm-1000 nm could be used in conjunction with a 600 nm-800 nm red emitting luminescent material. If the emission filter were selected to allow for light of 800 nm-1000 nm to pass, an LED with a substantially longer wavelength output than the luminescent material could be used. This has one advantage that IR LED and laser diodes are widely used and available at a low cost. 
         [0063]    Another embodiment does not sequentially measure quantization phase lag and then the calibration phase lag for every lifetime determination, but measures and records the calibration phase lag at a much lower occurrence. The recorded calibration phase lag may be subtracted from each quantitation phase lag measurement. 
         [0064]    This is particularly useful if the phase of the luminescent sensor  214  needs to be measured at a high data rate without interruption. By way of example, the calibration phase lag may be measured and recorded at intervals of time, or intervals of measurements, instead of at every measurement. 
         [0065]    A similar method uses a red LED as an emission band source that is modulated at a slightly different frequency than a blue or green LED that is used as excitation source  208 . The red LED is modulated at a sufficiently different frequency so that its signal may be digitally filtered or separated from the luminescent emission of the sensor. If the phase response is sufficiently flat in the region of measurement, the correction phase may be used directly. If not, then prior knowledge of the phase/frequency slope could be used to adjust the correction. Alternatively, if the shape of the phase shift with frequency is known, the actual phase shift could be found by interpolation or extrapolation. 
       REFERENCES 
       [0066]    The following references are incorporated herein by reference to the same extent as though fully disclosed herein:
   Arnaud, Forsyth, Sun, Zhang and Grattan, “Strain and temperature effects on Erbium-doped fiber for decay-time based sensing,” Rev. Sci. Instrum., 71, pp. 104-8 (2000);   Chang, Randers-Eichhorn, Lakowicz, and Rao.,  Biotechnology Progress  1998, 14, pp. 326-331;   Danielson, U.S. Pat. No. 6,157,037;   Demas, U.S. Pat. No. 4,845,368;   Demas, DeGraff, “Design and Application of Highly Luminescent Transition Metal Complexes,” Anal. Chem. vol 63 n17 829-37, 1991.   Dukes, U.S. Pat. No. 4,716,363;   Khalil, U.S. Pat. No. 5,043,286;   Lakowicz et. al., “2-GHz frequency-domain fluorometer,” Rev. Sci. Instrum. 57(10) October 1986;   Lin, Szmacinski, and Lakowicz, “Lifetime-Based pH Sensors: Indicators for Acidic Environments,”  Analytical Biochemistry  269, 162-167 (1999);   Papkovsky, D. B., “Luminescent porphyrins as probes for biosensors,” Sens and Act B 11 (1993) pp. 293-300;   Papkovsky, D. B., “New Oxygen sensors and their application to Biosensing,” Sens and Act B 29 (1995), pp. 213-218;   Rabinovich et al., U.S. Pat. No. 6,673,626 B1   Smith et. al. “Fluorescence energy transfer sensor for metal ions,” Proc. SPIE Vol. 2388, p. 171-181, Advances in Fluorescence Sensing Technology II; Joseph R. Lakowicz; Ed. May 1995;     Topics in Fluorescence Spectroscopy , ed J. Lakowicz, Vol. 4, Chap 10;   Vadde and Srinivas, “A closed loop scheme for phase-sensitive fluorometry”, American Institute of Physics, Rev. Sci. Instrum., Vol. 66, No. 7, July 1995, p. 3750; and   Venkatesh, U.S. Pat. No. 5,646,734.