Patent Publication Number: US-2010128269-A1

Title: Miniaturized surface plasmon resonance imaging system

Description:
CROSS-REFERENCE(S) TO RELATED APPLICATION(S) 
     This application claims the benefit of U.S. Provisional Application No. 60/814,253, filed Jun. 16, 2006, which is hereby expressly incorporated by reference. 
    
    
     STATEMENT OF GOVERNMENT LICENSE RIGHTS 
     The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of 1 U01 DE14971-03 awarded by the National Institutes of Health. 
    
    
     TECHNICAL FIELD 
     This invention generally relates to optical imaging and, more particularly, to a miniaturized optical imaging system suitable for Surface Plasmon Resonance imaging. 
     BACKGROUND 
     In Surface Plasmon Resonance (SPR) imaging, an optical imaging system is used to observe biomolecular binding events that have spatial structure. Generally, such a system includes a light source to illuminate a sensor substrate under conditions which produce SPR and a detector to image the light reflected from the sensor substrate. As known in the SPR art, the sensor substrate is typically provided with a resonance layer of metal, such as gold (e.g., 50 nm thick layer). The side of the gold layer opposite from the sensor substrate faces an aqueous sample (including biomolecules), which is typically contained in a flow cell (a detector sensor volume). Light is directed through the sensor substrate and the gold layer to the aqueous sample, and is reflected therefrom. The amount of light reflection varies depending on the change in the refractive index (RI) of the sensor substrate upon adsorption of target biomolecules to the sensor substrate. 
     Specifically, for certain wavelengths and angles of incident illumination, part of the incident energy couples into a surface plasma wave traveling between the gold layer and the aqueous sample. The loss of this energy is observed as a decrease in reflectivity. Because the coupling conditions vary widely with the refractive index of biomolecules, observations of reflectivity may be used as a sensitive measure of the sample&#39;s refractive index, and hence of the biomolecules contained in the sample. Therefore, SPR reflectivity measurements can be used to detect various target biomolecules, such as proteins. To make an SPR imaging system for detection of specific biomolecules, the side of the gold layer, with which the sample interfaces, may be chemically functionalized (for instance, by attaching antibodies to the surface), such that the target biomolecules will bind to the sensor substrate while other material will tend not to bind. If the functionalized layer on the gold layer is patterned such that different regions tend to bind different biomolecules, the changes in reflectivity may be analyzed to determine which of a number of different biomolecules are present in the (aqueous) sample, and in what concentration. In other words, in SPR imaging, any spatial variation in surface RI across the sensor substrate causes the reflected intensity to have spatial structure. SPR imaging can be used, for instance, in medical diagnostics, to analyze a fluid (such as blood or saliva) and determine the concentration of a certain set of biomolecules in that fluid. 
       FIG. 1A  illustrates a schematic diagram of an SPR imaging system  40  as disclosed in United States Patent Application Publication No. US2005/0134860 A1, published Jun. 23, 2005, which is hereby expressly incorporated by reference. The system  40  includes a light source  42 , at least one input optical element  44 , a sensor substrate  50 , at least one output optical element  52 , and a detector  60 . The at least one input optical element  44  is illustrated as a collimating lens, which is disposed between the light source  42  and the sensor substrate  50 . Preferably, the light source  42  is positioned at the focus of the collimating lens  44 . The at least one output optical element  52  is illustrated as a lens, which is disposed between the sensor substrate  50  and the detector  60 . Preferably, the lens  52  is capable of accepting light from the sensor substrate  50  at a range of angles corresponding to the range of angles at which light is emitted from the collimating lens  44 . The sensor substrate  50  may be provided by a surface of a prism  48 . Optionally, the SPR imaging system  40  further includes one or more wavelength/polarization-selection filters  46  between the at least one input optical element  44  and the sensor substrate  50 . As discussed above, one side of the sensor substrate  50  that faces the sample (i.e., the top side in  FIG. 1A ) is provided (e.g., coated) with a resonance layer such as a gold layer. 
       FIG. 1B  is a gray-scale plot that shows the transverse magnetic (TM) reflectivity of the SPR sensor substrate at various wavelengths, angles, and refractive indices. For a given refractive index (e.g., n=1.33), the plot shows the darkest region in the form of a curve descending from approximately 600 nm at 76 degrees to 1000 nm at 64 degrees. When the refractive index increases to 1.36, for example, the dark region (or the resonance position) moves higher in angle and wavelength. In SPR imaging, both the angle and wavelength are fixed (i.e., a single x-y point is being examined in  FIG. 1B ), and brightness changes are observed due to the changing refractive index. Thus, to sense refractive indices around 1.33, the wavelength and angle are set to some point on the dark curve for n=1.33. 
     Referring back to  FIG. 1A , light emitted from the light source  42  passes through the collimating lens  44 , passes through one side of the prism  48 , and strikes the gold-layered sensor substrate  50  at an incident angle appropriate for observation of SPR. The reflected light passes through the lens  52  and is focused onto the detector  60 , which records the image. It is proposed that the light source  42  may comprise a light-emitting diode (LED) array, and the angle of incidence may be varied by illuminating a selected one or more (e.g., a row) of the LEDs in the light source  42 .  FIG. 1A  illustrates one ray  43  emitted from each of three different LEDs, each ray  43  striking the sensor substrate  50  with a different incident angle. Illuminating an entire row of LEDs as opposed to a single LED results in increased light throughput. Alternatively or additionally, to change the incident angle, it is proposed that the light source  42  may be configured to be linearly movable by a manual or motorized positioner off the optical axis of the imaging system  40 , i.e., along the line representing the light source  42  in  FIG. 1A . Thus,  FIG. 1A  may be viewed as illustrating the same light source  42  being positioned at three different locations to emit a ray from each of the locations. Further, the one or more filters  46  may be used for selecting the polarization and source wavelength range, for instance if a white light source is used. If LEDs are used, which emit a narrow range of wavelengths, further wavelength filtering may not be necessary though polarization filtering may be. Though the filter  46  is illustrated to be between the collimating lens  44  and the sensor substrate  50 , it may be placed anywhere else in the optical path compatible with filter properties (such as the filter size and ability to accept light at non-normal incidence). The physical size and wavelength distribution of the light source  42  is adjusted such that the detector  60  operates just below saturation, so as to achieve the greatest signal-to-noise ratio (SNR). 
     Referring additionally to  FIG. 1C , it is proposed that the detector  60  is positioned (or tilted) according to the Scheimpflug angle. Particularly, the sensor substrate  50 , the output optical element (lens)  52 , and the receiving surface of the detector  60  are positioned such that the planes of each ( 56 ,  57 , and  58 ) intersect in a single line (or at a single point  59 , as in  FIG. 1C ). Because the sensor substrate  50  is tilted relative to the illumination light, the image also should be tilted by an amount given by a relation termed the Scheimpflug condition, so as to achieve best focus. Superficially, the Scheimpflug condition states that if object (the sensor substrate  50 ) and image (the detector  60 ) are tilted such that the object plane ( 56 ), the image plane ( 58 ), and the lens plane ( 57 ) meet in a single line, then the entire image will be in sharp focus. 
     The present invention extends the SPR imaging system described above, to provide a miniaturized and highly-portable SPR imaging system having particular applicability to medical diagnostics and life sciences research and development. 
     SUMMARY 
     This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. 
     In accordance with various exemplary embodiments of the present invention, a miniaturized Surface Plasmon Resonance (SPR) imaging system is provided, which includes a light source, a sensor substrate arranged to receive light at an incident angle from the light source, and a detector for detecting an image from the sensor substrate. The system further includes a folded light path structure arranged between the light source and the detector. The folded light path structure includes the sensor substrate, and is configured so as to receive the light from the light source, to redirect the received light to be incident on the sensor substrate at the incident angle (first redirection), and to further redirect the light reflected from the sensor substrate toward the detector (second redirection). The folded light path structure achieves a minimal instrumentation footprint, which in turn allows for the construction of a miniaturized, hand-portable SPR imaging system. 
     In accordance with one aspect of the invention, the miniaturized SPR imaging system further includes at least one input optical element between the light source and the folded light path structure, such as one or more of a collimation lens, wavelength-selection filter, and a polarizer. When a polarizer is included, the system may be advantageously configured to obtain a TE-polarized reference image for normalizing a TM-polarized image of the sensor substrate. The system may still further include at least one output optical element between the light path structure and the detector, which is capable of accepting light reflected from the sensor substrate. In one embodiment of the invention, the output optical element may include a pair of biconvex lenses and an achromatic lens. In accordance with a further aspect of the invention, wherein the output optical element comprises a lens, the light source, the detector, and the lens are arranged relative to each other so as to satisfy the Scheimpflug condition, thereby achieving sharp focus of the image of the sensor substrate. 
     In accordance with another aspect of the invention, the miniaturized SPR imaging system is further configured to be capable of adjusting focus and/or magnification of an image of the sensor substrate. 
     In accordance with a further aspect of the invention, the folded light path structure is configured such that the optical path resulting from the first redirection and the second redirection lies approximately in parallel with the sensor substrate. Also, the folded light path structure may include two symmetrical substructures on the light source side and the detector side, respectively. In one embodiment of the invention, such folded light path structure is formed with one main prism, one redirection optical element provided on each of the two sides of the main prism, and one deflection optical element also provided on each of the two sides of the main prism. 
     In accordance with yet another aspect of the invention, the folded light path structure includes one or more optical elements and the folded light path structure is further configured to adjust the one or more optical elements relative to the light source and the detector. For example, the one or more optical elements are adjustable at least linearly along one direction and angularly along the optical axis of the miniaturized SPR imaging system. Likewise, the light source and the detector may be adjustable at least linearly along one direction and angularly along the optical axis of the miniaturized SPR imaging system. 
     In accordance with a still further aspect of the invention, the incident angle of the light from the light source on the sensor substrate is adjustable. This could be accomplished, for example, by configuring the light source to selectively emit illumination light from two or more locations along a line perpendicular to the optical axis of the miniaturized SPR imaging system. 
     In accordance with further embodiments of the present invention, a miniaturized Surface Plasmon Resonance (SPR) imaging instrument is provided, including a miniaturized SPR imaging system described above. The SPR imaging instrument further includes a case that houses the miniaturized SPR imaging system, such that the sensor substrate is exposed to the outside of the case. The case also houses an electronic board for controlling the operation of the SPR imaging instrument. The SPR imaging instrument additionally includes a microfluidic card configured to be positioned adjacent to the exposed sensor substrate so as to subject an aqueous sample flowing therethrough to SPR imaging. Finally, the SPR imaging instrument includes a computer including a user interface to guide a user through the operation of the SPR imaging instrument. The computer is further loaded with suitable image processing and analysis software. 
     In accordance with one aspect of the invention, the microfluidic card comprises a window layer, a resonant (gold) layer formed on the window layer, and a flow layer formed on the resonant layer and including fluid paths for flowing the aqueous sample therethrough. The microfluidic card may be further configured to be mated with external fluidics that input and output the aqueous sample to be analyzed. 
     In accordance with another aspect of the invention, the miniaturized SPR imaging instrument further includes means for stabilizing the internal temperature of the case, such as a fan and/or a heater, to reduce and minimize wavelength drift or changes in a polarizer. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
       The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: 
         FIG. 1A  is a schematic diagram illustrating an SPR imaging system; 
         FIG. 1B  is a gray-scale plot illustrating reflectivity as dependent upon the wavelength of illumination, the angle of incidence of illumination, and the refractive index of the target sample; 
         FIG. 1C  is a schematic diagram illustrating imaging of tilted surfaces pursuant to the Scheimpflug condition; 
         FIG. 2A  is a schematic diagram illustrating a miniaturized SPR imaging system comprising a folded light path, in accordance with one embodiment of the present invention; 
         FIG. 2B  is a schematic diagram illustrating the “unfolded” light path of the miniaturized SPR imaging system of  FIG. 2A ; 
         FIG. 3  is a diagram illustrating various components of a miniaturized SPR imaging system provided on (or in) adjustable mounts, formed in accordance with one embodiment of the present invention; 
         FIG. 4A  is a perspective view of a complete SPR imaging instrument including a miniaturized SPR imaging system, external fluidics, and a microfluidic card, formed in accordance with one embodiment of the present invention; 
         FIG. 4B  is a schematic, partial cross-sectional view of the external fluidics provided on the microfluidic card, which in turn is provided on the miniaturized SPR imaging system, as in  FIG. 4A ; 
         FIG. 5A  is a sample SPR image obtained based on the use of a microfluidic card, in accordance with one embodiment of the present invention; 
         FIG. 5B  is a chart illustrating the binding curves of different sections within the sample SPR image of  FIG. 5A ; 
         FIG. 6  is a sample user interface panel for guiding a user through the operation of an SPR imaging instrument; 
         FIG. 7  is another sample user interface panel for guiding a user through the operation of an SPR imaging instrument; and 
         FIG. 8  is a sample user interface screen for guiding a user through the operation of image processing analysis software, in accordance with one embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION 
     In accordance with various embodiments, the present invention offers a miniaturized optical system with a small instrumentation footprint, particularly designed to allow a highly sensitive SPR (SPR) bio-assay to be conducted over a sensor substrate that can contain many different simultaneous assays. The system utilizes an optimized imaging technique to resolve the assay response for very small localized regions anywhere in its field of view. The optical path through the system is designed to be very rugged and compact, with few or no moving parts. There are provisions to allow the response of the system to be tuned to optimize the sensitivity of the assay for a given protocol chemistry or for a particular set of samples. 
       FIG. 2A  illustrates a schematic diagram of a miniaturized SPR imaging system  20  in accordance with an embodiment of the present invention. The system  20  includes a light source (not shown) on a source mount  21 , illumination optics  22  which may include, for example, wavelength/polarization-selection filters and collimating lens(es), and a folded light path structure  23 . In the illustrated embodiment, the folded light path structure  23  includes a first 90°-deflection prism  24   a , with its reflective (e.g., aluminum) hypotenuse plane sitting on a 90°-base prism  24   b , a first redirection equilateral prism  25   a , a main prism  26   a , a second redirection equilateral prism  27   a , and a second 90°-deflection prism  28   a . The folded light path structure  23 , as the name indicates, is configured to fold the light path so as to achieve a compact, miniaturized SPR imaging system with a minimal instrumental footprint. 
     In the illustrated embodiment, light rays (three are shown)  29  emitted from the light source propagate through the illumination optics  22  and strike the hypotenuse plane of the 90°-deflection prism  24   a , from which the rays  29  are reflected to enter the first redirection equilateral prism  25   a  to strike its plane  25   b , from which the rays  29  are reflected to enter the main prism  26   a  through one of its side planes  26   b . The rays  29  then strike a sensor substrate  26   c  of the main prism  26   a , and are totally internal-reflected therefrom to exit the main prism  26   a  through the other of its side planes  26   d , to enter the second redirection equilateral prism  27   a  to strike its plane  27   b . The rays  29  reflected from the plane  27   b  exit the second redirection equilateral prism  27   a  to enter the second 90°-deflection prism  28   a  to strike its hypotenuse plane  28   b , from which the rays  29  are reflected to exit the second 90°-deflection prism  28   a  to enter imaging optics  30 , such as lens(es), to be finally received by an image detector (not shown) on a detector mount  31 . Thus, an image of the sensor substrate  26   c  is formed on the detector. Note that the sensor substrate  26   c  of (or on) the main prism is provided with a metal layer, such as gold, on the side that faces an aqueous sample to be analyzed (i.e., the top side in  FIG. 2A ). In one embodiment, the sensor substrate  26   c  is directly coated with a gold layer. In another embodiment, a glass window (e.g., microscope slide)  32  is provided and index-matched to the sensor substrate  26   c , and the gold layer may be applied over the window  32 . 
     As should be apparent to those skilled in the art, the folded light structure  23  illustrated in  FIG. 2A  is merely an example, and other structures including fewer or more number of optical elements to achieve the same or different folded light paths are also within the scope of the present invention. 
       FIG. 2B  illustrates the “unfolded” light path corresponding to the folded light path illustrated in  FIG. 2A . Note that, unlike  FIG. 2A ,  FIG. 2B  does not show the first and second 90°-deflection prisms  24   a  and  28   a  nor the first and second redirection equilateral prisms  25   a  and  27   a .  FIG. 2B  is similar to  FIG. 1A , indicating that the miniaturized SPR imaging system  20  is a further extension of the SPR imaging system  40  shown in  FIG. 1A , and as such many preferable features of the SPR imaging system  40  may be used in the miniaturized SPR imaging system  20  also. 
     In the illustrated embodiment of  FIG. 2B , the light source  42  is preferably a collimated light source and may comprise one or more LEDs, for example,  880  nm surface-mount (SMT) LED array. As discussed in the background section above, by selectively activating one or a row of LEDs, the incident angle of illumination may be adjusted (three different incident angles are shown). The illumination optics  22  may comprise a lens  22  having the focal length F of 75 mm. The imaging optics  30  may comprise a series of lenses (three are shown), including two 50 mm Bi-Convex (BCX) lenses and a −75 mm achromatic lens. The detector  60  may be a 4.8 mm×3.6 mm CCD detector, and in the illustrated embodiment is tilted to satisfy the Scheimpflug condition to reduce focus errors. Specifically, as described in reference to  FIG. 1C  above, in  FIG. 2B  also, the image plane  58  of the detector  60  is tilted such that it intersects with both the object plane  56  and the lens plane  57  in a single line, to render the image in sharp focus. 
     The following describes in detail various subsystems of a miniaturized SPR imaging system formed in accordance with various exemplary embodiments of the present invention. Thereafter, implementation of a complete miniaturized SPR imaging instrument will be described. 
     Illumination Subsystem 
     As used herein, the illumination subsystem includes the light source  42 , the illumination optics  22 , and the folded light path structure  23 . The signal produced by an SPR imaging system is highly dependent on the wave vector of the illumination. Therefore, preferably, the illumination subsystem illuminates the entire sensor substrate  26   c  with light that has a uniform wave vector and radiance. Achieving uniform radiance helps optimize the signal-to-noise ratio (SNR) in the imaging system. Also, a non-uniform wave vector could result in different portions of the sensor substrate  26   c  having different response calibration, yielding complicated nonlinear results. Different assay chemistries require different wave vectors of illumination for optimal response. Therefore, it is further preferable that the illumination subsystem allows the illumination wave vector to be adjusted to achieve optimized sensitivity. 
     One way to adjust the wave vector is to fix the spectral content of the illumination and then vary angle of the illumination on the sensor substrate  26   c . In one embodiment of the present invention, this is achieved by using a light source  42  with a narrow spectral width and moving the light source  42  relative to the illumination (collimation) optics  22 . The illumination (collimation) optics  22  are chosen to produce a beam with a uniform wave vector for a range of light source positions relative to the optical axis of the imaging system. When the light source  42  is translated relative to the optical axis, as shown in  FIG. 2B , the output beam will point in a different direction. Therefore, the output beam will still have a uniform wave vector across its width, but the wave vector relative to the optical axis will vary. The translation of the light source  42  may be accomplished by linearly moving the point or line light source  42 , or by selectively activating a point or line light source in an array of light sources, as discussed above. This variable angle effect can also be achieved using other techniques such as, without limitation, directing collimated light into an acoustic modulator, a rotating prism, or a rotating mirror. 
     Another way to adjust the wave vector is to fix the angular content of the beam and vary the wavelength content. In one embodiment of the present invention, this is achieved by putting a broad wavelength source through an interference filter to narrow the spectral width of the output. Such interference filter may be provided as part of the illumination optics  22 . If the filter is tipped, the central wavelength of the output beam can be varied over a range of wavelengths. In this fashion, the output beam will still have a uniform wave vector across its width, but the wave vector will vary with wavelength. This spectral adjustment can also be accomplished using techniques such as, without limitation, rotating prisms or diffractions gratings, but these may not produce a very uniform wave vector across the beam. Using a technique such as, without limitation, a fiber optic to mix the output prior to collimation will help solve this issue. 
     The light source  42  may comprise, without limitation, one or more LEDs with narrow spectral width at 660 nm, 880 nm, or 920 nm; one or more LEDs with a broad spectral width, an incandescent source, a fluorescent source, a laser source, an optical fiber, among others, either alone as a single point source, as a line source (such as a row of LEDs or a rectangular fiber bundle, thereby improving flux with increased light throughput without significantly increasing the range of wave vectors in the illumination beam), or as a two-dimensional array. 
     The techniques for adjusting the light source  42  may include, without limitation, a single light source provided on a moving mount that is driven by a manual or motorized actuator, an array of light sources where each source can be individually controlled such that only the source(s) that produces the optimal wave vector is turned on, and a wavelength-selection filter (e.g., as part of the illumination optics  22 ) on a moving mount driven by an actuator, which causes the filter to tip relative to the optical axis. 
     Polarization Control 
     The signal produced by an SPR imaging system is highly dependent on the polarization state of the illumination beam. When the beam is TE (Transverse Electric) polarized relative to the sensor substrate  26   c , no SPR occurs and the image recorded by the detector  60  will represent the illumination transfer function of the entire imaging system. When the illumination beam is TM (Transverse Magnetic) polarized, an SPR imaging signal can be recorded by the detector  60 . However, this signal will also contain all the non-uniformities caused by the rest of the system throughput. In accordance with various exemplary embodiments of the present invention, the TE reference image is used as a “real time” reference to normalize the TM image so as to remove the non-uniformities, leaving only the SPR image signal from the sensor substrate  26   c . In one embodiment of the present invention, a liquid crystal element (as part of the illumination optics  22 ) is used to rotate the polarization state of the incident illumination by applying a voltage to the liquid crystal element. Other techniques for varying the polarization state include, without limitation, an actuator-driven stage to rotate a piece of linear polarizer. 
     Folded Light Path 
     In various exemplary embodiments of the present invention, the optical paths of the illumination and SPR imaging signal light are folded up in an approximately symmetrical fashion using identical components, as described in reference to  FIG. 2A  above. Other embodiments, where the optical paths are not necessarily symmetrical, are also within the scope of the present invention, depending on the specific application. One purpose of folding the light path is to produce a portable SPR imaging system with a minimized footprint. To that end, in various exemplary embodiments of the present invention, the light path is folded such that the optical axis lies along a plane (or planes) that is approximately parallel with the sensor substrate  26   c . See, for example, the embodiment of  FIG. 2A , where the optical axis from the light source mount  21  to the detector mount  31  is approximately parallel with the sensor substrate  26   c.    
     In this embodiment, the illumination beam is first folded by the first 90°-deflection prism  24   a  with its mirrored hypotenuse plane, to be perpendicular to the sensor substrate  26   c . While the prism  24   a  is used in this embodiment to simplify the mounting and cleaning of optics, the 90°-deflection may also be achieved with a mirror. Whatever deflection optic is used, it may be mounted on (or in) an adjustable mount that allows three axes of angular adjustment and one axis of linear adjustment. For example, referring additionally to  FIG. 3 , the miniaturized SPR imaging system  20  of  FIGS. 2A and 2B  may be mounted on a board  33 , with various components such as the deflection optics (not shown) in a linearly adjustable manner along the direction of a linear adjustor element  34 . Likewise, mounts for various components may be configured to be angularly adjustable so as to allow three axes of angular adjustment to the components, such as the deflection optics. These adjustments can be used to optimize the position of the illumination beam onto the sensor substrate  26   c . Note that, in  FIG. 3 , the sensor substrate  26   c  of the miniaturized SPR imaging system  20  is facing downward, and in that sense  FIG. 3  is an upside-down view of  FIGS. 2A and 2B . 
     After the deflection optic, the beam is redirected from the normal (i.e., perpendicular to the sensor substrate  26   c ) to the sensor substrate  26   c  so that it illuminates the backside of the sensor substrate  26   c  at a grazing angle. In the embodiment illustrated in  FIG. 2A , this redirection is accomplished using a redirection prism  25   a , but may also be achieved with the use of a mirror. In the illustrated embodiment, the geometry of the redirection prism  25   a  is chosen so that the nominal optical axis of the redirected beam intersects the sensor substrate  26   c  at 75° from normal. It should be appreciated by those skilled in the art that the incident angle may be at any nominal value and may be adjusted over any range. 
     One advantage of using a redirection prism  25   a  is that it can be bonded to the main prism  26   a . This provides a simple way to construct the miniaturized SPR imaging system  20 . In other embodiments, the main prism  26   a  may be constructed with a monolithic shape that has the characteristics of a redirection prism bonded to a main prism. 
     In the embodiment illustrated in  FIG. 2A , a second redirection prism  27   a  is provided such that the SPR imaging signal light reflected from the sensor substrate  26   c  is redirected to be normal to the sensor substrate  26   c . This redirection prism  27   a  may be bonded to the main prism  26   a  or formed directly as part of a monolithic shape. After exiting the second redirection prism  27   a , the SPR imaging signal light is deflected to be generally parallel to the sensor substrate  26   c . For the deflection, a second 90°-deflection prism  28   a  or a mirror may be used, which may further be mounted on (or in) an adjustable mount similar to the first 90°-deflection prism  24   a  on the illumination side. 
     Mounting the Main Prism and Sensor Substrate 
     In one embodiment of the present invention, the main prism  26   a  is bonded to one side of a large, flat glass window  32 , the other side of which is coated with gold. The sensor substrate  26   c  is index-matched to the window  32  so that the center of the sensor substrate  26   c  intersects the optical nominal axis of the illumination beam. The use of the window  32  is advantageous in that it provides a simple way of holding the main prism  26   a  in proper position. Further, the window  32  is easy to mount to the rest of the SPR imaging system  20 , and also serves as a flat support surface to ensure that the sensing surface (as provided by the gold layer) remains flat. Finally, the window  32  helps protect the rest of the SPR imaging system  20  by minimizing any sample leaks and also containing excessive amounts of index matching. 
     Imaging Optics and Detector 
     The imaging optics  30  are responsible for creating a crisp image of the sensor substrate  26   c  on the detector  60 . Because the sensor substrate  26   c  is tipped relative to the optical axis of the illumination side, the detector  60  also should be tipped in a configuration known as the Scheimpflug condition to achieve best focus, as discussed above. In accordance with various exemplary embodiments of the present invention, it may be further preferable to magnify the image by a factor of  1 . 5 , a condition that makes the SPR imaging system more linear than can be achieved by simply obeying the Scheimpflug condition. It is still further preferable that the image remains stable on the detector  60  even when the angle of the illumination beam is adjusted. 
     To achieve one or more of these preferable features, in one embodiment of the present invention as illustrated in  FIG. 2B , an objective lens (part of the imaging optics  30 ) is split into a pair of biconvex lenses to minimize aberrations and utilize commonly available lenses. This lens pair is followed by a negative lens (e.g., −75 mm achromatic lens) to act as a field flattener. The use of an achromatic element is preferable to further reduce aberrations. It should be understood by those skilled in the art that other lens combinations can also be used to accomplish the same goals, including but not limited to placing a field flattener in near proximity of the detector  60  to further correct field curvature and letting the field flattener be off center relative to the optical axis to better correct the non-linearity caused by the off axis imaging. 
     In one embodiment of the present invention, the detector  60  is a rectangular (e.g., 4.8 mm×3.6 mm) CCD array detector placed at the image plane  58  of the SPR imaging system. In various exemplary embodiments of the present invention, the detector  60  is mounted in such a way that it can be translated in three axes and rotated in two axes in order to achieve optimal focus. Preferably, the detector  60  is at least rotatable around the optical axis in order to account for any tip and tilt imparted by the mounts of the deflection prisms (e.g., the first and second 90°-deflection prisms  24   a  and  28   a ). At the same time, the illumination subsystem (comprising the light source  42 , the illumination optics  22 , and the folded light path structure  23 ) is preferably rotatable around its optical axis to keep everything aligned. 
     Stable Thermal Environment 
     To achieve a stable SPR imaging signal, it is preferable that the thermal environment for the optical path either remains constant or is corrected for by the software. Software correction typically works only over a relatively small temperature range, so a sound thermal design is preferable for a system to be stable over a relatively large temperature range. Temperature variation can cause the wave front to wander due to: mechanical variation in the mounting structure, wavelength drift of the light source  42 , wavelength drift of any optical filters (if used as the light source  42 ), and/or changes to the polarizer. 
     In one embodiment of the present invention, the temperature is stabilized slightly above ambient temperature using a fan to circulate the air inside a case (containing the optics) and a heater on a controller to add just enough heat. Another embodiment uses a circulating fan and a heat pipe to pull the heat from the imaging electronics out of the case. An external fan on a controller may also be used to control the amount of heat conducted out of the case. Those skilled in the art will appreciate that various other techniques to stabilize the internal temperature of the system are also within the scope of the present invention. 
     Hyperspectral Imaging 
     The embodiments discussed so far are intended to monitor an illumination beam with a stable wave vector over a period of time to establish an assay for a given location. This is a single point measurement scheme, and even with careful normalization against a “real time” reference image (e.g., the TE reference image) and careful stabilization of the system, it is prone to undesirable wavelength drift. This drift can be minimized or eliminated by moving from a single point measurement to a multipoint measurement. One way to accomplish this is to measure a range of wave vectors at each location in rapid succession. It is then possible to recover a more stable signal using signal processing techniques such as, without limitation, ratiometric techniques, minima hunting, correlation techniques, or regression techniques. 
     One technique of achieving such data from a range of wave vectors is to time share the detector  60 . If the illumination beam is fixed at an angle and a series of successive images are taken at different wavelengths, the spectra for each pixel on the detector  60  can be built up from the same corresponding pixel in the series of images. This is a technique referred to as hyperspectral imaging. A similar set of hyperspectral data can also be taken with a fixed wavelength and a rapid series of images at different illumination angles. In various exemplary embodiments of the present invention, the use of hyperspectral image processing improves the functionality of the SPR imaging system. 
     Implementation of the Complete Instrument 
     The miniaturized SPR imaging system with a small footprint, as described above, may be readily incorporated into a complete SPR imaging instrument  70 , as shown in  FIG. 4A . The SPR imaging instrument  70  generally includes a case  71 , which houses a miniaturized SPR imaging system  20  (not clearly shown in  FIG. 4A ). The instrument  70  further includes external fluidics  72  comprising a silicone manifold for inputting and outputting aqueous sample(s) to be analyzed, and a microfluidic card  74   a ,  74   b  that is configured to mate with both the miniaturized SPR imaging system  20  and the external fluidics  72 . Briefly, the card  74   a ,  74   b  is configured to flow aqueous sample(s), supplied from the external fluidics  72 , within the card along the paths in parallel with the plane of the card, such that the aqueous samples will flow adjacent to the sensor substrate  26   c  of the miniaturized SPR imaging system  20  to produce an SPR image signal. In the illustrated embodiment, the card  74   a  is provided as a spare, while the card  74   b  is positioned to mate with both the miniaturized SPR imaging system  20  (housed within the case  71 , underneath the card  74   a  in  FIG. 4A , with its sensor surface  26   c  facing upward) and the external fluidics  72  including a (plastic) plate  75  (placed on top of the card  74   a  in  FIG. 4A ). Further, the SPR instrument  70  includes a computer  76  with a user-interface for user control/operation of the SPR instrument  70 , such as a table PC. Finally, the SPR instrument  70  includes, within the case  70 , a digital-signal-processor (DSP) for controlling image acquisition and transfer, and an electronic board for control of light source(s)  42  and the valves and pumps of the external fluidics  72 . The case  71  provides light shielding so that the SPR instrument  70  may be used in normal room lighting. As one can appreciate, the SPR instrument  70  may be constructed in a compact, hand-portable form, in accordance with various exemplary embodiments of the present invention. 
     (a) Microfluidic Card 
     Referring additionally to  FIG. 4B , a microfluidic card  74   b  may include a window layer  32  (provided by a microscope slide, for example), a gold layer  78  formed thereon, and a flow layer  80  including fluidic paths  81  running therethrough. In one embodiment, the flow layer  80  contains a flow cell fabricated from multiple layers of laser-cut Mylar and adhesive. (Note that  FIG. 4B  is a schematic diagram for illustrating the relative positioning of various elements, and thus the elements are not to scale.) When mated with the miniaturized SPR imaging system  20 , the fluidic paths  81  within the flow layer  80  run generally in parallel with the sensor substrate  26   c  of the main prism  26   a . Further, when mated with the external fluidics  72 , tubes  82  running through the plate  75  of the external fluidics  72  may be inserted into fluidic ports  83  in the flow layer  32  of the card  74   b , to thereby input and output aqueous samples into and out from the fluidic paths  81 . In one embodiment of the present invention, a magnetic clamping mechanism may be provided for reproducible placement of the microfluidic card  74   b  on the SPR instrument  70 . Prior to such placement, preferably, the window layer  32  is index-matched to the sensor substrate plane  26   c  of the main prism, for example, by dropping index matching liquid therebetween. In a further embodiment of the present invention, the microfluidic card  74  is configured to be disposable after a single use. 
       FIG. 5A  illustrates, as an example of how the microfluidic card  74   b  may be used in SPR imaging, an SPR image of an immunoassay detection region composed of a nonfouling (i.e., cell or protein resistant) coating sub-region, such as a polyethylene glycol (PEG) surface  84   a , upstream of the gold-layered sensor substrate sub-region that is functionalized for phenytoin-bovine serum albumin, such as a phenytoin surface  84   b . The image shows a sample channel  85 , and two reference channels  86   a  and  86   b  that are used for the quantification of positive control samples and serve as on-card calibrators. The bright rectangles  87  between the channels  85 ,  86   a , and  86   b  are regions of adhesive on the gold layer  78 , which may be used to produce reference signals for the purpose of monitoring light intensity fluctuations.  FIG. 5B  shows the signal as a function of time due to the binding of phenytoin antibody in buffer, for example a binding curve  88  of phenytoin antibody in buffer (i.e., the target) to the phenytoin-bovine serum albumin functionalized gold layer under stopped flow conditions. The binding curve  89  for the nonfouling PEG surface  84   a  shows no detectable adsorption, as expected. The difference signal is shown as a curve  90 . Note that in the difference curve  90 , the wiggles at the start of the other two curves have been subtracted out. The offset of the three curves is arbitrary and for display purpose only. 
     (b) DSP Software 
     In one embodiment of the present application, a Texas Instruments TMS320C6410 DSP may be used to control the CCD image detector  60  that captures the reflected light and to transfer the resultant SPR images to the computer  76  (e.g., the tablet PC) for processing and analysis. The specific functions of the DSP software, which was developed in the Texas Instruments Code Composer Studio software development environment, include setting the image detector shutter speed, acquisition of images from the detector, averaging the images to remove temporal noise, and transmission of the averaged images via Ethernet to the tablet PC. 
     (c) Tablet PC Software 
     The computer (e.g., tablet PC) software is responsible for control of the SPR imaging instrumentation, processing and analysis of the averaged SPR images, and for providing a simple user interface to guide a user through the experimental procedure. In one embodiment, the instrumentation control and user interface software were developed in the National Instruments LabWindows/CVI software development environment, while the SPR image processing and analysis software was developed in the Mathworks Matlab environment. 
     In one embodiment of the present invention, the image acquisition software module connects to the DSP via two Ethernet channels. The first channel is used to launch the program on the DSP. The second channel is used for transmitting images from the DSP and sending to the DSP the desired image detector shutter speed and number of images for averaging. Each image received from the DSP may be stamped with the time it was received from the DSP, the number of images averaged, and the shutter speed, before being saved to disk for processing and analysis. In addition, the most recently acquired image may be displayed on the computer  76  screen at a reduced resolution. 
     The flow of fluids through the SPR imaging instrument  70  may be controlled by a set of six Kloehn syringe pumps, which is part of the external fluidics  72 . In one embodiment, each pump, which contains a 6-way valve, syringe, and stepper motor, is individually programmed via an RS-232 serial connection. The software sets the valve position and determines the distance and velocity for syringe activity in units of motor steps based on the syringe volume, fluid flow duration, fluid flow direction (aspirate or dispense), and fluid volume. 
     The intensity of the light source  42 , such as an LED light source, may be set via a codeword corresponding to the current to be sent to the LEDs. The codeword is transferred to the electronic control board via a serial connection using the RS-232 protocol. 
     The rotation angle for the electronic polarizer (as part of the illumination optics  22 ) may be set via a codeword transferred to the electronic control board via an RS-232 serial connection. In one embodiment, a codeword of zero corresponds to TM polarization and a codeword of  1974  corresponds to TE polarization. 
     In one embodiment, a set of four valves (part of the external fluidics  72 ) controls the flow path of fluids in the microfluidic card  74   b . The software sets each valve to either an open or closed state using a binary code, which is transferred to the electronic control board via an RS-232 serial connection. In one embodiment, three of the valves operate in unison, thus requiring a two-bit code. 
     (d) User Interface 
     In accordance with various exemplary embodiments of the present invention, the SPR imaging instrument  70  offers a graphical user interface consisting of multiple panels, to guide users through the experimental procedure. The main panel  91  of the user interface, which is shown in  FIG. 6 , consists of a set of buttons  92  and a message box  93 . The message box  93  informs the user when to perform each task in the experimental procedure. 
     In the illustrated embodiment, five of the buttons  92  correspond to a fixed set of pump and valve operations, thereby eliminating the need for the user to be aware of fluid flow rates and volumes. The sixth button  94  adjusts the polarizer and light source for the acquisition of dark, TE-polarization, and TM-polarization reference images. Finally, a “STOP” button is available to immediately terminate all pump activity. 
     For advanced control of the SPR imaging instrument  70 , as shown in  FIG. 7 , two additional interface panels (“Manual System Control” panel  95  and “Instrumentation Setup” panel  96 ) may be provided. These panels may be used to allow manual control of the “Camera Setup”  97 , such as grab rate, shutter speed, and image averaging, the “Display”  98 , such as display rate and full-resolution view of an image, the “Polarizer Setup”  99 , such as TE/TM polarization, the light source or “LEDs”  100  (on/off), the “Valves”  101  (open/closed), and the “Pumps”  102 , such as syringe size, flow rate, flow direction and flow duration. 
     (e) Image Processing and Analysis 
     As discussed above, one embodiment of the image processing and analysis software was implemented in Mathworks Matlab, to provide on-the-fly experiment results for the user. The user interface for the image processing and analysis software is shown in  FIG. 8 . The three buttons  104  on the left side correspond to the three main modules: an initialization procedure (“Initialize”), an on-the-fly analysis routine (“Run”), and a setup panel for advanced control of the initialization and analysis routines (“Setup”). The other three buttons  106  allow the user to save data and load or save the region of interest (ROI) locations. 
     The initialization procedure automatically finds the directory containing the current set of SPR images, initializes the regions of interest (e.g., rectangular areas used for data analysis) in the images, loads the dark, TE and TM reference images, and sets up the graphs for the data display. 
     In one embodiment, the analysis routine, which checks for images periodically based on a timer, gives the user a choice of four ways to visualize data. The first method is a plot of the mean spatial intensity of each region of interest over time. The user also has the option of specifying whether the images are corrected for background, dark, and illumination noise. Second, the images can be subtracted from a reference image to show differences in intensity from a known point in time. Two other ways to visualize the data are via horizontal and vertical line profiles. Third, specifically, a single row or column of each image is displayed as a line plot depicting intensity as a function of spatial location. The same line from each progressive image is appended to the display using a different color to allow temporal analysis of the line. Alternatively, multiple rows or columns can be averaged together for the line profiles. The fourth data visualization method is a histogram of pixel intensities that is destructively updated for each image. 
     As described hereinabove, the present invention, in accordance with various exemplary embodiments, offers a miniaturized SPR imaging system with a minimal footprint, which in turn makes possible a compact and portable SPR instrument containing such miniaturized SPR imaging system. In addition to being compact, the miniaturized SPR imaging system of the present invention includes a robust optical path that may be adjustable (in terms of focus, magnification, illumination wavelength, illumination incident angle, etc.) so as to achieve optimal illumination onto the sensor substrate and optimal imaging onto the detector. The miniaturized SPR imaging system is also designed to be mechanically robust in order to hold its adjustment. Further features may be provided to allow the image to be stable for a range of conditions of the light source illumination, including changes in the wavelength, angle of incidence, and polarization. 
     The SPR instrument in accordance with the present invention enables rapid, convenient desktop monitoring of biochemical binding interactions, for instance for immunoassays. The miniaturized SPR imaging system described herein is useful for many applications, including those requiring (1) detection and/or quantification of biological binding events; (2) detection and/or quantification of other binding or adsorption processes; or (3) refractometry of substances or surfaces which have a spatial distribution. Thus, it has particular applicability to medical diagnostics and life sciences research and development. While this invention is generally directed to a miniaturized high-performance SPR imaging system, it will be appreciated by those skilled in the art that the optical imaging system disclosed herein could be useful for many applications. Other angle-dependent optical sensing techniques such as ellipsometry and Brewster angle microscopy will likewise benefit, as will imaging or illumination systems in which facile adjustment of illumination conditions is needed. 
     While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.