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.
Snell'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.
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.
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.
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.
In Reichert'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.
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.
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.
These drawbacks reduce the accuracy and repeatability of SPR measurements made by a particular instrument.
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.
It is therefore an object of the present invention to improve accuracy and repeatability of analytical measurements of the types mentioned above.
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.
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.
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.
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.
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:
Reference is made initially to
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.
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.
An analytical instrument 110 formed in accordance with an embodiment of the present invention is shown in
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.
An alternative embodiment may be realized by substituting modified diaphragm 226 shown in
Another alternative embodiment may be realized by substituting modified diaphragm 326 shown in
The present invention may be implemented in Reichert's SR7000 or SR7000DC SPR spectrometer, or in Reichert'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.
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.