Abstract:
An analytical instrument for making measurements based on detection of an SPR resonance minimum or a refractometer transition shadowline on a detector array is improved by configuring a diaphragm of the instruments illumination system to include a first aperture, a second aperture, and an opaque region between the first and second apertures, wherein the opaque region of the diaphragm casts a shadow on the detector array to provide a reference minimum from which a relative location of the resonance minimum or transition shadowline is measurable. By establishing a reference minimum on the detector array as a reference location for relative measurement, the instrument compensates for signal drift and other instrument variations. The diaphragm may include additional apertures and opaque regions for generating additional reference minima over the extent of the detector array.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to analytical instruments for optically measuring a parameter of a test sample by analyzing detector signal information provided by pixels of a light-sensitive detector array, wherein the measurement value depends upon a location of a defining feature of light received by the detector array. The present invention may be applied, for example, to analytical instruments for measuring molecular binding interactions using the principle of surface plasmon resonance (SPR), wherein the defining feature is a resonance minimum (resonance shadow) cast on the detector array. As a further example, the present invention may be applied to critical angle refractometers, wherein the defining feature is a transition “shadowline” between an illuminated region and a darkened region on the detector array. 
       BACKGROUND OF THE INVENTION 
       [0002]    Snell&#39;s law describes what happens when light is directed through a high refractive index prism (e.g. Sapphire—refractive index 1.76) to a surface of the prism in contact with a low refractive index medium, for example a sample fluid. In conventional refractometry, light rays below the critical angle exit the prism bending toward the prism surface, while light rays above the critical angle are totally internally reflected back through the prism. Photons that are totally internally reflected create an electric field at the interface. Here, light is not coming out of the prism, but an electric field extends beyond the reflecting surface. This field oscillates with the usual characteristics of an electromagnetic mode. The electrical component perpendicular to the interface decays exponentially; this is called an evanescent wave. The evanescent wave is bound to the surface. 
         [0003]    In critical angle refractometers configured to measure refractive index of a sample, the location of a shadowline corresponding to the critical angle is detected to enable calculation of the sample refractive index. 
         [0004]    In SPR spectroscopy instruments, a thin metal layer is added between the prism or slide surface and a fluid compartment contacting the thin metal layer. Free electrons in the metal layer can act as a resonator. Energy for the resonance comes from the evanescent wave produced by the totally internally reflected photons. When certain conditions are met, as determined by the wavelength and angle of incident illumination, and by the refractive indices of the prism, metal and fluid layers, then coupling/resonance occurs between the plasma oscillations of the free electrons in the metal and the bound electromagnetic field of the totally internally reflected photons. This coupling is the result of the momentum of the incoming light equaling the momentum of the plasma electromagnetic field. Photons are “absorbed” and converted to surface plasmons. Because the photons are not reflected, a localized “shadow” occurs in the reflected light and a resonance minimum (resonance shadow) may be detected to measure changes occurring at the surface. 
         [0005]    Reichert Inc., assignee of the present invention, currently manufactures SPR instruments that are optically configured to illuminate a spot on a gold layer with rays incident over a range of angles from about 58 to 85 degrees. When the contacting fluid sample is physiological saline solution, the “shadow” or SPR minimum corresponds to light incident to the interface at about 66 degrees. Mass and/or composition changes at the interface between the gold layer and the sample cause changes in the local refractive index near the gold layer, thereby changing the resonance angle. For example, if a protein layer is added to the gold/aqueous interface, then the resonance angle would be about 66.6 degrees, and a sharp dip in reflectivity is observed for this illumination angle. 
         [0006]    In Reichert&#39;s SR7000 and SR7000DC instruments, the gold surface is illuminated by rays over a range of angles that encompass these shifts in the resonance angle. This range of angles is continuously monitored with a linear photodiode detector array having a plurality of photosensitive pixels each providing a signal indicative of light intensity received thereby. Analysis of the pixel signals determines the illumination angle at which the resonance minimum occurs. 
         [0007]    In any given instrument, there is a tendency for signal drift over time due to temperature and light source variations. As a result, the pixel location of the resonance minimum corresponding to a particular illumination angle will change slowly over time, even though the illumination angle and interface chemistry may be the same. 
         [0008]    Also, for SPR measurements, the gold layer is typically applied to a removable sensor slide which is coupled to the prism surface by a coupling fluid, such as oil having a known refractive index. This introduces slight variations from measurement to measurement because the coupling fluid layer may not have a uniform thickness for each measurement, such that the sensor slide may be slightly inclined relative to the prism surface to different degrees from measurement to measurement. 
         [0009]    These drawbacks reduce the accuracy and repeatability of SPR measurements made by a particular instrument. 
         [0010]    Similar drawbacks exist for critical angle refractometers concerning the location of a detected transition shadowline. For example, the AR6 and AR7 series automatic refractometers are subject to signal drift caused by temperature and light source variations. 
       SUMMARY OF THE INVENTION 
       [0011]    It is therefore an object of the present invention to improve accuracy and repeatability of analytical measurements of the types mentioned above. 
         [0012]    An analytical instrument formed in accordance with the present invention generally comprises a measurement interface associated with a test sample; an illumination system for illuminating the measurement interface with light having rays incident to the measurement interface over a range of illumination angles, the illumination system including at least one light source and a diaphragm between the at least one light source and the measurement interface; and a detector array arranged to detect light coming from the measurement interface, the detector array including a plurality of photosensitive pixels each providing a signal indicative of light intensity received thereby. 
         [0013]    The invention is characterized in that the diaphragm has a first aperture, a second aperture, and an opaque region between the first and second apertures, such that the opaque region casts a shadow on the detector array to provide a reference minimum in light intensity at a location on the detector array. The location of the reference minimum on the detector array, like the location of an SPR resonance minimum or a critical angle shadowline on the array, is subject to signal drift over time as a result of instrument use. Therefore, the reference minimum may be used as a reference from which a relative location of an SPR resonance minimum or a critical angle shadowline on the detector array may be measured to cancel the effects of signal drift and other variations in the measurement system. 
         [0014]    An alternative embodiment of the invention is characterized in that the diaphragm has at least one further aperture spaced from the second aperture to define at least one further opaque region for creating another reference minimum on the detector array. In such an alternative embodiment, signal drift behavior over an extended pixel range that includes the normal measurement range may be evaluated and compensated for in the reported measurement value. 
         [0015]    The invention encompasses a method of compensating for signal drift in an analytical instrument having an illumination system for illuminating a measurement interface associated with a test sample. The method generally comprises the steps of configuring the illumination system to cast a shadow on a detector array arranged to detect light coming from the measurement interface, wherein the shadow is located between illuminated regions of the detector array, to provide a reference minimum on the detector array, the location of the reference minimum being subject to signal drift over time as a result of instrument use; and measuring the location of a feature of the detected light relative to the location of the reference minimum. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING FIGURES 
         [0016]    The nature and mode of operation of the present invention will now be more fully described in the following detailed description of the invention taken with the accompanying drawing figures, in which: 
           [0017]      FIG. 1  is a schematic side view representation of an analytical instrument known in the prior art; 
           [0018]      FIG. 2  is a plan view of a diaphragm used in an illumination system of the prior art instrument shown in  FIG. 1 ; 
           [0019]      FIG. 3  is an enlarged view showing an example of a measurement interface configured for SPR measurements; 
           [0020]      FIG. 4  shows a detector scan of the prior art instrument of  FIG. 1  configured for SPR measurement of a water sample placed on a slide, in which light intensity is plotted as a function of pixel number; 
           [0021]      FIG. 5  is a schematic side view representation of an analytical instrument formed and operating in accordance with an embodiment of the present invention; 
           [0022]      FIG. 6A  is a plan view of a diaphragm used in an illumination system of the instruments shown in  FIGS. 5 and 8 ; 
           [0023]      FIG. 6B  is a plan view of a diaphragm formed in accordance with an alternative embodiment of the present invention; 
           [0024]      FIG. 6C  is a plan view of a diaphragm formed in accordance with another alternative embodiment of the present invention; 
           [0025]      FIG. 7A  shows a detector scan of the instrument of  FIG. 5  configured for SPR measurement of a water sample placed on a sensor slide, in which the diaphragm of  FIG. 6A  is used and light intensity is plotted as a function of pixel number; 
           [0026]      FIG. 7B  shows a detector scan similar to that shown in  FIG. 7A , however the diaphragm of  FIG. 6B  is used; 
           [0027]      FIG. 7C  shows a detector scan similar to that shown in  FIG. 7A , however the diaphragm of  FIG. 6C  is used; 
           [0028]      FIG. 8  is a schematic side view representation of an analytical instrument formed and operating in accordance with another embodiment of the present invention; 
           [0029]      FIG. 9  is an enlarged view showing an example of a measurement interface of the instrument of  FIG. 8  configured for critical angle refractometric measurements; 
           [0030]      FIG. 10A  shows a detector scan of the instrument of  FIG. 8  configured for critical angle refractive index measurement of a water sample placed on a slide, in which light intensity is plotted as a function of pixel number; and 
           [0031]      FIG. 10B  is a scan similar to that of  FIG. 10A , however the scan corresponds to a configuration wherein multiple reference minima are generated. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0032]    Reference is made initially to  FIGS. 1-3  for description of an analytical instrument  10  formed in accordance with the prior art. Analytical instrument  10  comprises a light source  12  illuminating a pinhole aperture  14  for generating an illumination beam  16  propagating along an optical path. Instrument  10  further comprises a polarizer  20  located in the optical path for polarizing illumination beam  16 . The polarized illumination beam  16  then passes through a pair of focusing lenses  22 ,  24  and is thereby converted from a divergent beam to a convergent beam. A diaphragm  26  is positioned in optical path  18  immediately after the second focusing lens  24  to act as a field stop. As shown in  FIG. 2 , diaphragm  26  includes a single elongated slit aperture  27  surrounded by an opaque region  29 . Light transmitted through aperture  27  of diaphragm  26  is received by high-index prism  28  through a light entry surface  30  of the prism. The convergent illumination beam  16  may be focused at a point just below a sample surface  32  of prism  28 , such that the beam is once again divergent as it approaches a measurement interface  34  of instrument  10 . Alternatively, the convergent beam  16  may be focused at a point somewhere above measurement interface  34 . As may be understood, measurement interface  34  may be illuminated with either a divergent or convergent beam, as long as the illumination beam includes rays incident over a range of angles designed to include resonance angles expected to be measured with instrument  10 . For example, the range of angles from about 58 to 85 degrees has been found suitable for a variety of measurement applications in the Reichert instruments mentioned above. 
         [0033]      FIG. 3  shows a configuration wherein the measurement interface  34  is a surface plasmon resonance (SPR) interface. In this configuration, a sample  13  is applied to a sensor slide  15  having a glass substrate  17  coated with a thin gold layer  19 . Sensor slide  15  is coupled to sample surface  32  of prism  28  by a layer of oil  21  having an index of refraction that is less than or equal to the index of refraction of prism  28  and preferably matches the index of refraction of either prism  28  or glass substrate  17 . Other configurations may be used to provide an SPR interface in accordance with techniques known to those skilled in the art. 
         [0034]    Light reflected from measurement interface  34  leaves prism  28  through exit surface  36  and passes through a cylindrical collection lens  38  before it is received by a detector array  40 . Detector array  40  includes a plurality of photosensitive pixels each providing a signal indicative of light intensity received thereby. A linear or two-dimensional solid-state array may be used as detector array  40 . The shaded region K in the reflected beam represents a resonance minimum corresponding to a sharp drop in light intensity due to surface plasmon resonance. The pixel signal information from detector array  40  is processed by signal processing electronics  42  to determine the illumination angle at which surface plasmon resonance occurs, thereby providing analytical information about sample  13 . 
         [0035]      FIG. 4  illustrates a representative scan of detector array  40  for a water sample placed on sensor slide  15 . The resonance minimum K is observable graphically in the scan at approximately pixel number  256  along the array. In accordance with the prior art, the pixel number corresponding to resonance minimum K provides an absolute basis for determining the illumination angle at which surface plasmon resonance occurs. Those skilled in the art are aware that various algorithms are available for determining which pixel (or fractional sub-pixel) on detector array  40  corresponds to the location of resonance minimum K. A weighted centroid algorithm is currently preferred for this task. 
         [0036]    As mentioned in the background section above, signal drift and small inclination differences of sensor slide  15  may cause instrument  10  to yield varying measurement results when constant measurement results are expected. 
         [0037]    An analytical instrument  110  formed in accordance with an embodiment of the present invention is shown in  FIG. 5 , and is operating with an SPR configuration as illustrated in  FIG. 3 . Instrument  1   10  is largely similar to instrument  10  of the prior art, but comprises a diaphragm  126  that differs from diaphragm  26  depicted in  FIG. 2 . Diaphragm  126  is shown in  FIG. 6A  as including a first aperture  127  which may be in the form of an elongated slit similar to aperture  27  of diaphragm  26 , a second aperture  131  which may be in the form of a circular hole or other geometric shape, and an opaque region  133  between first aperture  127  and second aperture  131 . As used herein, the term “aperture” refers to a light-transmitting region, and may be embodied by open space or by light-transmitting material. Second aperture  131  may be shorter in length than first aperture  127 . Opaque region  133  casts a shadow on detector array  40  to provide a reference minimum R in light intensity at a pixel location on the detector array. Apertures  127  and  131  may be designed such that first aperture  127  transmits rays incident to the measurement interface at angles within the range of illumination angles in which the resonance angle (or critical angle, in the case of refractometric measurement) is expected to be found, and second aperture  131  transmits rays incident to the measurement interface at angles outside this range of illumination angles. 
         [0038]      FIG. 7A  shows a scan of detector array  40  of instrument  110  depicted in  FIG. 5  wherein diaphragm  126  of  FIG. 6A  is used. Instrument  110  is configured for SPR measurement of a water sample placed on a sensor slide. The pixel locations of resonance minimum K and reference minimum R are indicated. The pixel location of resonance minimum K relative to reference minimum R is indicated by the difference ARK in  FIGS. 5 and 7A . 
         [0039]    As may be understood, the absolute pixel location of reference minimum R is subject to the same fluctuations as the absolute pixel location of resonance minimum K resulting from signal drift over time and differences in sensor slide inclination related to the coupling fluid layer  21 . In accordance with the present invention, the pixel location of resonance minimum K may be determined relative to the pixel location of reference minimum R. The pixel location of resonance minimum K relative to reference minimum R is substantially constant over time for a given sample because the absolute locations of K and R are subject to the same signal drift and system fluctuations. Thus, by configuring diaphragm  126  to provide reference minimum R at a previously unused portion of detector array  40 , relative measurement of resonance minimum K is possible so that signal drift is canceled out. The absolute pixel location of reference minimum R, to which the absolute pixel location of resonance minimum K may be compared for relative measurement, may be determined using the same algorithm used to determine the absolute pixel location of resonance minimum K, or using a different algorithm. 
         [0040]    An alternative embodiment may be realized by substituting modified diaphragm  226  shown in  FIG. 6B  for diaphragm  126  shown in  FIG. 6A . Diaphragm  226  has a second aperture  231  that is shaped as a rectangle rather than a circle, and is separated from first aperture  127  by an opaque region  233 .  FIG. 7B  shows a representative scan where diaphragm  226  is used in place of diaphragm  126 . 
         [0041]    Another alternative embodiment may be realized by substituting modified diaphragm  326  shown in  FIG. 6C  for diaphragm  126  shown in  FIG. 6A . Diaphragm  326  has a second aperture  331 , a third aperture  335  positioned such that first aperture  127  is between second aperture  331  and third apertures  335 , a fourth aperture  339 , and a fifth aperture  343 . The apertures are spaced apart by opaque regions  333 ,  337 ,  341 , and  345  as shown in  FIG. 6C .  FIG. 7C  shows a representative scan where diaphragm  326  is used in place of diaphragm  126 . As is apparent, a plurality of reference minima R are generated, one in a lower pixel number region before the measurement region containing resonance minimum K and three in a higher pixel number region after the measurement region containing resonance minimum K. Where multiple reference minima are generated, signal drift behavior over the entire scanned array may be evaluated to indicate localized signal drift effects, whereby appropriate signal drift compensation may be applied to a given measurement. For example, a linear or non-linear compensation function may be determined and applied to each measurement. 
         [0042]      FIG. 8  shows an instrument  210  that is substantially similar to instrument  110 , but is configured as shown in  FIG. 9  for critical angle refractive index measurement of a sample  11  placed directly on sample surface  32  of prism  28 . A scan of detector array  40  is provided in  FIG. 10A  for a measured water sample. Illumination impinging upon detector array  40  is characterized by a transition shadowline S between an illuminated region and a darkened region, wherein the pixel location of shadowline S is dependent upon the refractive index of sample  11 . In accordance with the present invention, the pixel location of shadowline S may be measured relative to reference minimum R to reduce the influence of signal drift and other systemic variations on the measurement. The relative pixel location of shadowline S is labeled ΔRS in  FIGS. 8 and 10A .  FIG. 10B  shows a representative scan similar to that of  FIG. 10A , wherein the illumination configuration is altered by replacing diaphragm  126  in  FIG. 8  with a diaphragm  326  formed in accordance with  FIG. 6C  to provide a plurality of reference minima. 
         [0043]    The present invention may be implemented in Reichert&#39;s SR7000 or SR7000DC SPR spectrometer, or in Reichert&#39;s AR6 and AR7 series automatic refractometers, by modifying the existing diaphragm to provide an additional aperture, and by programming the processing software to determine the pixel location of the reference minimum and measure the pixel location of the resonance minimum relative to the reference minimum or transition shadowline, as the case may be. 
         [0044]    While not depicted in the drawing views, it is contemplated to provide a dual channel illumination system whereby two separate illumination spots are formed side-by-side at measurement interface  34  and detected on a pair of side-by-side detector arrays.