Abstract:
A surface plasmon resonance imaging apparatus provides an improved optical assembly allowing fixed source and detector operating with a horizontal test surface for a more compact design. In a preferred embodiment, a mechanical linkage of planar mirrors provides a single point adjustment of angle of incidence and angle of refraction while maintaining a constant optical axis of the source and detector.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application is a continuation-in-part of U.S. application Ser. No. 10/411,583 filed Apr. 10, 2003 hereby incorporated by reference. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002] This invention was made with United States government support awarded by the following agency: DOD ARPA F30602-01-2-0555. The United States has certain rights in this invention. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0003]    The present invention relates to instruments for chemical and biological analyses employing surface plasmon resonance and, in particular, to an instrument having an improved light path that may be easily adjusted.  
           [0004]    In surface plasmon resonance, a sensor comprised of a thin metallic film is illuminated by light of an appropriate wavelength and angle of incidence on a “reflecting” side of the film. The energy from the light couples to electrons of the metal of the film creating a resonant condition (surface plasmon resonance) that is highly sensitive to surface conditions on a “sensing” side of the film opposite the side that is illuminated.  
           [0005]    Probe molecules may be attached to the sensing side of the metallic film to selectively bind with target molecules in a solution to be analyzed. This binding, through the agency of the electron resonance in the film, causes a drop in reflectance of the reflecting side of the film. Detection of the decrease in reflected light thus provides a sensitive measurement of the binding of target molecules to the probe molecules, in turn providing a sensitive indication of the presence of target molecules in the solution being analyzed.  
           [0006]    By placing a variety of different probe molecules on the sensing surface of the film, many different target molecules may be rapidly assessed. Importantly, the target molecules need not be labeled with fluorescent dye or the like prior to analysis.  
           [0007]    Current surface plasmon resonance (SPR) equipment can be bulky and difficult to use. Adjustment of the optical system may require separate movement of an analyzing camera and/or light source and possible rotation of the sensing surface. Typically, the sensing surface is oriented vertically, which allows ready access to the optical system for such adjustment, but this vertical orientation can cause problems sealing the flow cells holding the solution to be analyzed against the sensing surface. SPR equipment with a horizontal sensing surface has been constructed to improve access to the sensing surface and improved sealing of the flow cell. The resulting displacement of the camera and light source below the sensing surface, however, complicates adjustment of the camera and light source and undesirably increases the overall height and bulk of the instrument.  
         BRIEF SUMMARY OF THE INVENTION  
         [0008]    The present invention provides a surface plasmon resonance instrument with a horizontal sensing surface but with an optical mechanism that allows the light source and camera to be placed horizontally on either side of the instrument. The optical mechanism may be relatively compact and employ simple planar mirrors to allow one-step adjustment of the angle of incidence and reflectance of light on the sensing surface.  
           [0009]    Specifically, the invention provides a horizontal SPR instrument for use with a sample cell having a metallic film with probe molecules attached to a first side of the film, exposable to material flow across the first side of the film, and having a transparent support attached to a second side of the film opposite the first side. The SPR instrument has a support frame with an entrance providing a path for receiving an analyzing light beam along the first fixed axis and an exit providing a path for transmitting a modified light beam along a second fixed axis. A holder supports sample cells with its metallic film oriented horizontally, and an optical assembly adjustably directs the analyzing light beam from the first axis to one of a range of incident angles against the second side of the film and conducts reflected light at a corresponding reflection angle from a second side back along the second axis.  
           [0010]    Thus, it is one object of the invention to provide an SPR instrument with a horizontal sampling surface and with a fixed source and detector to provide simplified construction and adjustment of the instrument.  
           [0011]    The first and second fixed axes may be substantially aligned and horizontal.  
           [0012]    It is another object of the invention to permit the design of a low profile horizontal SPR instrument by displacing the light source and detector to the sides of the sample cell.  
           [0013]    The device may include a single operator communicating with the optical assembly to simultaneously change the angle of incidence and reflection by the same amount.  
           [0014]    Thus, it is another object of the invention to provide an instrument that may be easily adjusted without the need to separately move the camera and or light source with its attendant optical components.  
           [0015]    The device may include a coupling prism having a first prism face adjacent to the second side of the film and receiving from the optical assembly, at a second prism face, the analyzing light beam directed toward the second side of the film, and providing to the optical assembly, through a third prism face, the modified light beam reflected from the second side of the film.  
           [0016]    Thus, it is another object of the invention to improve light coupling to the film through the use of a prism element.  
           [0017]    The optical assembly may include a correction mechanism adjusting the position of the analyzing light beam incident on the second prism face and correcting the offset of the modified light beam from the third prism face caused by refraction of the light by the prism with different angles of incidence and reflection.  
           [0018]    Thus, it is another object of the invention to provide a mechanism that corrects for refractive effects of the prism that may cause misalignment of the optical path.  
           [0019]    The optical assembly may be constructed of movable planar mirrors.  
           [0020]    Thus it is another object of the invention to provide an optical assembly for an SPR instrument that does not require complex curved mirrors or other lens elements.  
           [0021]    The optical assembly may include a first mirror, receiving the analyzing light beam through the entrance along the first axis and directing the analyzing light beam at a third fixed angle to a second mirror that moves about a point near the second side of the film in the holder. The optical assembly may further include a third mirror also movable about the point near the second side of the film in the holder to receive the reflected modified light beam from the second side of the film and directing the modified light beam at a fourth fixed angle to a fourth mirror which in turn transmits the modified light beam to the exit along the second axis.  
           [0022]    It is thus another object of the invention to create a folded optical path in which the movement of mirrors, rather than the source and detector, provides changing angles of incidence and reflection.  
           [0023]    The second and third mirrors may be mounted on four-bar linkages to adjust a normal angle of the second mirror to substantially halfway between the third fixed angle and an angle of an axis between the second mirror and the second surface on the film and to adjust the normal angle of the third mirror halfway between the fourth fixed angle and an angle of an axis between the third mirror and the second surface on the film.  
           [0024]    Thus it is another object of the invention to provide a simple mechanism for providing complex orbiting and tilting motion of mirrors necessary to conduct light between a fixed light source and detector for a variety of needed angles of incidence and reflectance.  
           [0025]    The SPR instrument may include an angulation mechanism communicating with the four-bar linkages for swinging the second and third mirrors simultaneously by equal angles about the point on the second side of the film. This mechanism may be a pair of cams adapted to move in unison along the vertical axis, each cam engaging an opposite arm of one of the four-bar linkages of the second and third mirrors to urge them simultaneously to different angulations.  
           [0026]    It is thus another object of the invention to provide a simple mechanism for moving the mirrors on the four-bar linkages.  
           [0027]    The device may include a tracking mechanism communicating with the first and fourth mirrors to slide the first and fourth mirrors horizontally to direct the light to and from the second and third mirrors, respectively, with movement of the second and third mirrors.  
           [0028]    Thus it is another object of the invention to ensure proper optical alignment of the mirrors with angulation of the beams.  
           [0029]    The tracking mechanism may be a vertical arm extending from a first mirror to follow the horizontal position of the second mirror, and a second vertical arm extending from the fourth mirror to follow the horizontal position of the third mirror.  
           [0030]    It is thus another object of the invention to provide a simple mechanism for tracking the horizontal component of motion of the second and third mirrors.  
           [0031]    When the device includes a coupling prism, the first and second vertical arms may provide a profiled camming surface, communicating with the second and third mirrors, respectively, where the profile modifies the relative horizontal location of the second and first mirrors and the relative horizontal location of the fourth and third mirrors with angulation of the second and third mirrors about a point near the second side of the film.  
           [0032]    It is thus another object of the invention to provide a simple mechanism for correcting refractive effects of the prism beyond what can be obtained with simple linkages.  
           [0033]    These particular objects and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0034]    [0034]FIG. 1 is a simplified perspective view of the SPR instrument of the present invention showing a fixed light source and detector flanking an optical assembly providing angled incident and reflective light about a horizontal sensing surface;  
         [0035]    [0035]FIG. 2 is an elevational view of the elements of the optical assembly of FIG. 1 showing positioning of upper and lower planar mirrors to provide two different angles of incidence and reflection;  
         [0036]    [0036]FIG. 3 is a view similar to FIG. 2 showing one four-bar linkage such as holds lower mirrors of FIG. 2 for orbit about the sensing surface as moved by a roller assembly;  
         [0037]    [0037]FIG. 4 is an elevational fragmentary view similar to that of FIG. 2 showing a tracking mechanism for moving the upper mirrors of FIG. 2 with motion of the four-bar linkages of FIG. 3; and  
         [0038]    [0038]FIG. 5 is an enlarged view of a downward arm of the tracking mechanism of FIG. 4 showing a curvature that causes additional motion to correct for refractive effects. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0039]    Referring now to FIG. 1, a horizontal surface plasmon resonance instrument (HSPR)  10 , works with a light source  12  providing an analyzing light beam  14  to the analyzer unit  15  where it is modified to become modified light beam  32  and received by camera  30 . Both analyzing light beam  14  and modified light beam  32  are coaxial along fixed horizontal axis  44  allowing the light source  12  and camera  30  to be fixed and mounted conveniently to either side of the analyzer unit  15 .  
         [0040]    The light source  12  may be, for example, a monochromatic coherent or incoherent source including a lamp or laser, filter, polarizer, and lens system of types well known in the art. The light source directs the analyzing light beam  14  toward the analyzer unit where the analyzing light beam  14  enters an entrance area  17  to be received by the optical assembly  18  held by a support frame  16  of the analyzer unit  15 . The optical assembly  18  redirects the analyzing light beam  14  to create a first incident beam  20 , directed upward against the lower side of a sample cell  22  at an incident angle (by convention measured with respect to a vertical axis  66  normal to a lower surface of the sample cell  22 ).  
         [0041]    Referring momentarily to FIG. 2, the sample cell  22  may include, for example, a gold film  34  adhered to the top side of a transparent support  40  or the like as is well known in the art. The top surface of the film  34  may receive probe molecules  36 , intended to react with materials that will be passed over the surface of the film  34  as indicated by arrow  38  via a flow cell or the like. The incident light beam  20  passes upward through the transparent support  40  striking a center  54  of the lower surface of the film  34 .  
         [0042]    Referring again to FIG. 1, a reflected beam  24 , at a reflection angle equal and opposite to the incident angle, is reflected off the lower surface of the film  34  and is again received by the optical assembly  18  to be redirected as a modified light beam  32  to an exit area  28  as the modified light beam  32  received by the camera  30 .  
         [0043]    The camera  30  is preferably a digital camera such as employs a charged coupled sensor or the like to produce an electronic image signal that may be analyzed to detect reduced reflection caused by the surface resonance effect. For this reason, the camera  30  is focused on the lower surface of the film  34  to obtain an image therefrom. Similarly, the light source  12  may be focused on the lower surface of the film  34  to provide an even illumination across the area of the film  34 .  
         [0044]    Referring now to FIG. 2, the optical assembly  18  includes two upper mirrors  46  and  64  aligned generally along the axis  45  and slidable there along on either side of the sample cell  22 , and two lower mirrors  48  and  60  mounted generally to orbit about a center  54  located in the center of the lower surface of the film  34 , on opposite sides of the center  54 .  
         [0045]    The analyzing light beam  14  from the light source  12  is received by mirror  46 , angled at approximately 45° to the axis  45  to redirect analyzing light beam  14  from the horizontal axis  44 , vertically downward to second mirror  48 . The second mirror  48  redirects the analyzing light beam  14  toward the center  54  as the incident beam  20 .  
         [0046]    The incident beam  20  is reflected from the lower surface of the film  34  to become a reflected beam  24 . Reflected beam  24  is received by third mirror  60  which redirects the reflected beam  24  vertically upward to the fourth mirror  64 . The fourth mirror  64  is angled at 45 degrees to axis  45  (but 90 degrees to the mirror  46 ) to redirect the received beam along the axis  45  as the modified light beam  32 .  
         [0047]    As noted, mirrors  48  and  60  may move in orbits  52  symmetrically about the center  54  to provide a range of different angles of incidence and reflection observing the rule that the angle of incidence of the incident beam  20  must be equal to the angle of reflectance of the reflected beam  24  for the range of angles. As they move in orbits  52 , mirrors  48  and  60  also rotate  49  about their center pivots  56  and  56 ′, respectively, so as to constantly direct the incident beam  20  and reflected beam  24  toward the center  54 .  
         [0048]    As mirrors  48  and  60  move, mirrors  46  and  64  may slide in horizontal motion  50  so as to continue to align with mirrors  48  and  60 .  
         [0049]    Referring still to FIG. 2, the transparent support  40  of the sample cell  22  may abut a base face  44   a  of an optical prism  42 , the prism  42  having base face  44   a , a right face  44   b , and a left face  44   c  together whose planes describe an equilateral triangle. The prism  42  provides improved coupling of the light of incident beam  20  and reflected beam  24  to the film  34 , passing beam  20  approximately perpendicularly through face  44   b  and passing beam  24  approximately perpendicularly through face  44   a  to reduce interface reflections and refraction of the prism  42 . Deviations of the angle of passage from perpendicular, as the angles of incidence and reflectance change, cause some refractive effects as will be discussed below. The prism  42  may be a part of the transparent support  40  and replaceable with the film  34  in an alternative embodiment.  
         [0050]    Referring now to FIGS. 2 and 3, the orbits  52  and rotations  49  of mirrors  48  and  60  is provided by a four-bar linkage  70  and  70 ′ supporting mirrors  48  and  60 , respectively to provide necessary the orbits  52  and rotation  49 . Each four-bar linkage  70  and  70 ′ is duplicated on both front and rear sides of the sample cell  22  and mirrors  48  and  60  so as to be displaced from the light path while providing the necessary support for the mirrors  48  and  60 .  
         [0051]    Each four-bar linkage  70  and  70 ′ includes a lower bar  72 ,  72 ′ and an upper bar  74 ,  74 ′. Each of the lower bars  72 ,  72 ′ pivots about a common pivot point  75  on the support frame  16 , aligned with center  54  as shown in FIG. 2. The remaining ends of lower bars  72 ,  72 ′ pivotally attach to the center pivots  56 ,  56 ′ of the mirror  48 . The upper bars  74 ,  74 ′ are generally of different length than lower bar  72 ,  72 ′ and extends from secondary pivot point  76 ,  76 ′ on a side wall of the support frame  16 , removed from common pivot point  75 . The remaining ends of upper bars  74 ,  74 ′ attach to outer edges of mirrors  48  and  60  at edge pivots  78 ,  78 ′.  
         [0052]    The length and orientation of the elements of four-bar linkages  70  and  70 ′ are adjusted according to methods well known in the art, to provide the necessary rotations  49  during the orbits  52  of the mirrors  48  and  60  so that a normal  80 ,  80 ′, of the mirrors  48  and  60 , respectively, approximately bisects an angle formed between a first line intersecting center pivot  56 ,  56 ′, and center  54  and a second line extending vertically from center pivots  56 ,  56 ′.  
         [0053]    Simultaneous and equal movement of four-bar linkages  70  and  70 ′ and thus mirrors  48  and  60  is accomplished by means of a pair of rollers  82  and  84  which ride against the lower edges of lower bars  72  and  72 ′ and which are held by a vertical slide  86 , which with upward motion, such as may be provided by a micrometer mechanism or the like (not shown), causes upward motion of the rollers  82  and  84  raising lower bars  72  and  72 ′ equally and oppositely about common pivot point  75 . The slide  86  is supported on a side wall of the support frame  16 . It will be understood that motion of the slide  86  provides a single point of adjustment, thereby providing complete control of the angulation without the need to move the light source  12 , or camera  30 , or make other adjustments in the optical assembly.  
         [0054]    Referring again to FIGS. 2 and 4, mirrors  46  and  64  must move horizontally to transmit or receive light from their respective mirrors  48  and  60  with orbital motion of mirrors  48  and  60 . This is accomplished by means of a roller  88  positioned at the center pivots  56  and  56 ′ on lower arms  72  and  72 ′. Only lower arm  72  is shown for clarity, however, it will be understood from the following description that the same mechanism is applied in mirror symmetric fashion with respect to the mirror  64 .  
         [0055]    The roller  88  engages an inner camming surface  92  of a vertical arm  90 . The vertical arm  90  in turn extends downward from a sliding mirror support  93  supported for horizontal motion  50  on a slideway  51 . The mirror support  93  holds the mirror  46  allowing it to move with the vertical arm  90  against a helical compression spring  94  urging the mirror support  93  rightward. The helical compression spring  94  biases the inner camming surface  92  leftward against the roller  88  to move therewith. With upward angulation of the lower bar  72 , roller  88  rides against the camming surface  92  sliding the sliding mirror support  93  leftward so that the mirror  46  is always positioned above mirror  48  with orbit  52  of the mirror  48 . As mentioned, a similar arrangement with mirror symmetry is used for mirrors  60  and  64 .  
         [0056]    Referring now to FIG. 5 at different angulations of the incident beam  20 , a slight refractive effect will occur within prism  42  causing a displacement  96  of the center of the incident beam  20  with respect to the center  54 . This can be corrected by a slight motion of mirror  46  to advance or retard it in its following of mirror  48 . This advance and retard is provided by a slight tapering of camming surface  92  which may overlay a minor arbitrary functional dependence of the horizontal position of the mirror  46  as a function of the orbital angle of mirror  48 . A similar arrangement with mirror symmetry is used for mirrors  60  and  64 .  
         [0057]    It will be understood that motion of the slide  86  provides a single point of adjustment, thereby providing complete control of the angulation of the incident beam  20  and the reflected beam  24  and all the mirrors  46 ,  48 ,  60 , and  64  without the need to move the light source  12 , or camera  30 , or make other adjustments in the optical assembly.  
         [0058]    It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims.