Abstract:
An optical alignment system for a device for synthesizing DNA polymers provides for a patterned substrate for simple alignment of the system. The substrate may also be used for synthesis allowing precise alignment of the synthesis substrate during each synthesis operation. The patterning of the substrate may be used to promote separation of reaction sites.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       FIELD OF THE INVENTION  
         [0001]    This invention pertains generally to the field of biology and particularly to techniques and apparatus for the manufacture of arrays of polymers useful in the analysis and sequencing of DNA and related polymers.  
         BACKGROUND OF THE INVENTION  
         [0002]    The identification of DNA sequences is useful in the mapping of plant and animal genes as well as in other research and commercial applications.  
           [0003]    One method of identifying DNA sequences uses an array of oligonucleotide probes constructed using photolithographic techniques. Each probe of the array is designed to hybridize with a particular DNA target, the latter of which may be coupled to a fluorescent target. By observing where the DNA hybridizes, the identity of the DNA may be deduced. This technique is described generally in Pease, et al., “Light-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis,”  Proc. Natl. Acad. Sci. USA , 91:5022-5026 (May 1994).  
           [0004]    The probes are constructed on a substrate coated with photolabile protecting groups. Exposure by light passing through a photolithographic mask causes certain locations on the substrate to become reactive. DNA monomers are washed over the substrate and attached at the reactive sites. The exposed ends of the monomers are also protected by a photolabile material which in turn may be made reactive by selective illumination.  
           [0005]    This process may be repeated with different monomers or short oligomers until arbitrary DNA polymers are built up at the various reaction sites. By changing the photolithographic mask, different DNA sequences may be synthesized at different locations in the array.  
           [0006]    Photolithographic masks are cumbersome and expensive. For this reason, in an alternative approach, an array of switchable optical elements such as a two-dimensional array of electronically addressable micro mirrors may be used instead of the masks. Projection optics focus an image of the micro mirrors on the substrate where the nucleotide addition reactions are conducted. Under the control of a computer, each of the micro mirrors is selectively switched between a first position at which it projects light on the substrate through the optical system and a second position at which it deflects light away from the substrate. The cost of the masks and the time consuming process of exchanging mask is eliminated  
           [0007]    Careful alignment of the masks or micro mirrors (henceforth collectively termed “pattern generator”), the projection optics, and the substrate is required for reliable high-density synthesis of DNA probes. This complex and time-consuming process may need to be repeated over time as the system is used. Complicating the alignment process is the extremely small size of the details in the projected image and the fact that the light energy is typically in the ultraviolet range.  
         BRIEF SUMMARY OF THE INVENTION  
         [0008]    The present invention provides a simple and precise method of aligning the pattern generator, the projection optics, and the substrate by superimposing a projected image from the pattern generator on a prepatterned substrate or a substrate-like target. This alignment system makes it practical to align each substrate prior to synthesis and thereby allows the substrates to have alignment sensitive features, for example, reaction inhibiting materials positioned between reaction sites to provide for greater reaction spatial definition.  
           [0009]    In one embodiment, the pattern on the substrate is one that creates a “moiré” pattern with the image of the pattern generator. Regular moiré patterns can produce an apparent magnification of alignment problems allowing alignment by unaided visual inspection. Alternatively, the moiré pattern may be used with electronic light sensors to provide automatic alignment. In an alternative embodiment, a prepatterned-target may be used and the alignment of the system performed in a separate step before synthesis operations at which time the target is replaced by a substrate.  
           [0010]    It is thus one object of the invention to provide a simple mechanism for optical alignment of systems for DNA probe synthesis. It is another object of the invention to allow precise alignment of the substrate such as allows the substrate to incorporate alignment sensitive features.  
           [0011]    Further objects, features, and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]    [0012]FIG. 1 is a block diagram of an optical system for DNA probe synthesis suitable for use in the present invention, the optical system providing reflective optics projecting an image of a micro mirror array onto a substrate, showing servo controlled optical mountings allowing alignment of these components according to visual inspection or signals from electronic light sensors;  
         [0013]    [0013]FIG. 2 is a fragmentary perspective view of the surface of the micro mirror array showing individual mirrors separated by lanes and showing incident light and two directions of reflection for one mirror depending on the state of the mirror;  
         [0014]    [0014]FIG. 3 is a plan view of a pre-patterned substrate or target showing target regions and separation regions corresponding to the mirrors and lanes, respectively, of FIG. 2 with the separation regions treated for increased reflection;  
         [0015]    [0015]FIG. 4 is a figure similar to that of FIG. 3 showing target regions treated for increased reflection;  
         [0016]    [0016]FIG. 5 is a schematic cross-section through the pre-patterned substrates or target of FIGS. 3 and 4 showing a method of providing increased reflection at a specific angle through the use of an optical grating;  
         [0017]    [0017]FIG. 6 is a moiré pattern generated by a superposition of an image of the mirror array of FIG. 2 and the pattern of FIG. 3 showing an optical misalignment such as produces “keystone” distortion in the projected image;  
         [0018]    [0018]FIG. 7 is a figure similar to that of FIG. 6 showing an optical misalignment such as produces a left-right in-plane offset in the projected image;  
         [0019]    [0019]FIG. 8 is a figure similar to that of FIG. 6 showing an optical misalignment such as produces a relative rotation in the projected image;  
         [0020]    [0020]FIG. 9 is a figure similar to that of FIG. 6, showing an optical misalignment such as produces a “barrel” distortion in the projected image;  
         [0021]    [0021]FIG. 10 is an image similar to that of FIG. 6 showing a misalignment such as produces over magnification of the projected image;  
         [0022]    [0022]FIG. 11 is a plot showing variation in one axis of misalignment versus light intensity from the moiré pattern such as forms the basis for automatic alignment of the optical system of FIG. 1; and  
         [0023]    [0023]FIG. 12 is a cross-sectional fragmentary view of a substrate having synthesized DNA polymers and showing an intervening lane having a rejection surface for preventing synthesis in the lane region. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0024]    Referring now to FIG. 1, a DNA synthesis device  10  includes a light source  12 , typically being a mercury arc lamp or the like, but alternatively including lasers, solid state, and gas discharge sources to produce an ultraviolet or near ultraviolet source beam  14 . The beam  14  may be passed through a filter  16  selected to pass only the desired wavelengths (e.g., the 365 nanometer mercury line). Other devices for filtering or monochromating the light source e.g., diffraction gratings, dichromic mirrors, and prisms may also be used rather than a filter and are generically referred to as “filters” herein.  
         [0025]    The filtered beam  14  is received by a condenser system  18  forming a uniform beam  20  of specified divergence. The divergence (or conveyance angle is such to satisfy the requirement of spatial coherence of the illumination typically σ=0.7. A number of standard optical devices may be used including, for example, a prismatic/kaleidoscopic collimator such as that described in co-pending application Ser. No. 60/353,491 filed Jan. 31, 2002, and assigned to the same assignee as the present invention and hereby incorporated by reference.  
         [0026]    The illumination beam  20  strikes a pattern generator which imposes a pattern of light and dark areas on the illumination beam. In the preferred embodiment, the pattern generator is an array of micro mirrors, which is described in detail immediately below. Other types of pattern generators include static devices such as conventional photolithographic masks and reflective targets, and dynamic devices such as micro shutters, micro mirrors operated by bimorph piezoelectric actuators, LCD shutters, and reflective LCD devices.  
         [0027]    Referring also to FIG. 2, as mentioned in the preferred embodiment, the pattern generator is an array  22  of micro mirrors  29 , each micro mirror  29  being substantially square, but not limited to edges of ten to twenty micrometers. The micro mirrors  29  are arranged in the array  22  in rows and columns and are available in various sizes including, but not limited to 640×800, 640×840, 800×600, 1024×768, and 1024×1260. Each micro mirror  29  is capable of reflecting the light in the normal usable wavelengths including ultraviolet and near ultraviolet light in an efficient manner without damaging itself.  
         [0028]    Generally, the array  22  of micro mirrors  29  may be the Digital Light Processor (DLP) commercially available from Texas Instruments, Inc. Such arrays are discussed in the following article and patents: Larry J. Hornbeck, “Digital Light Processing and MEMs: Reflecting the Digital Display Needs of the Networked Society,” SPIE/EOS European Symposium on Lasers, Optics, and Vision for Productivity and Manufacturing  1 , Besancon, France, Jun. 10-14, 1996; and U.S. Pat. Nos. 5,096,279, 5,535,047, 5,583,688 and 5,600,383, hereby incorporated by reference.  
         [0029]    Each micro mirror  29  is operable independently, under electronic control such as from the output of a general-purpose digital computer  23 , to deflect along its diagonal over a range of approximately 10-12°, thus causing a deflection of 20-40° in the light. In this way, the micro mirrors  29  are capable of imparting a pattern of light and dark squares onto the collimated beam  20  Specifically, and referring still to FIG. 2, incident ray  30  (of the collimated beam  20 ) arrives at the micro mirrors  29  at an angle of approximately 20° with respect to a normal to the plane of the array  22 . “Absorbed” rays  32  will be reflected from the micro mirrors  29  in a first position of the micro mirrors  29  (e.g., minus 10° with respect to the plane of the array  22 ) and directed out of the system to an absorber (not shown). Projected rays  34  are reflected from the micro mirrors  29  in a second position (e.g., plus 10° with respect to the plane of the array  22 ) toward a telecentric projection system  36 . The micro mirrors  29  are separated by generally non reflecting lanes  50 .  
         [0030]    The projection system  36  is comprised of a concave spherical mirror  38  and an opposed convex mirror  40 . Both mirrors  38  and  40  are preferably spherical although aspherical shapes are possible as well. The mirrors  38  and  40  have enhanced UV coating for high reflectivity. The beam formed from projected rays  34  from the array  22  is received by one side of mirror  38  and reflected to mirror  40  which in turn reflects the beam  34  to the other side of mirror  38  from which it is directed to the active surface of a glass substrate enclosed in a flow cell  42 . The mirrors  38  and  40  are focused to produce an image of the array  22  of micro mirrors  29  within the flow cell  42 .  
         [0031]    In the preferred embodiment, the concave mirror  38  may have a diameter of 152.4 millimeters and a spherical mirror surface radius of 304.8 millimeters and the convex mirror may have a diameter of 25 millimeters and a spherical mirror radius of 152.94 millimeters. Ideally, the radius of curvature of the concave mirror is close to twice that of the convex mirror. Such reflective optical systems are well known and conventionally used in optical lithography in “MicroAlign” type systems. See, e.g., A. Offfner, “New Concepts in Projection Mask Aligners,”  Optical Engineering , Vol. 14, pp. 130-132 (1975), and R. T. Kerth, et al., “Excimer Laser Projection Lithography on a Full-Field Scanning Projection System,”  IEEE Electron Device Letters , Vol. EDL-7(5), pp. 299-301 (1986), hereby incorporated by reference.  
         [0032]    The mirror  38  may be mounted on an XY table  44  for moving the mirror  38  in two perpendicular directions perpendicular to the mirror&#39;s radius of symmetry. The XY table  44  may be motorized, with motors communicating with a motor controller  49  to be described below or may be movable manually by means of vernier screws. In the preferred embodiment, mirror  38  is mounted in a tilt stage capable of precise rotations in the two directions perpendicular to the mirror axis. These rotations provide precise alignment and location of the image.  
         [0033]    Similarly, mirror  40  may be mounted on a focus stage  46  having a motor or screw adjustment for moving mirror  40  toward and away from mirror  38  for focusing purposes. If motorized, the motor communicates with motor controller  49  which may perform automatic adjustments of these motor controlled movements or may accept manual input via keypad  70  providing, for example, a constant velocity motion of any axis when a corresponding button is pressed.  
         [0034]    The flow cell  42  may be mounted on an XY φ  table  48  or similar positioning device for moving the flow cell  42  in either or both of two perpendicular directions perpendicular to the axis of the beam  34  and to rotate the flow cell  42  about the axis of the beam  34 . Again, these movements may be motorized with the motors communicating with the controller  49  as will be described, or may be manually adjustable as will be described. The flow cell is connected to a supply of basic DNA monomers or polymers from a reagent supply system  43  of a type well known in the art.  
         [0035]    Referring to FIGS. 1 and 3, the projection system  36  will project an image of the micro mirrors  29  on a planar substrate  52  contained within the flow cell  42  as registered by mechanical guides  45 . The image (not shown) will have bright portions corresponding to the areas of the mirrors  29  when those micro mirrors  29  are reflecting light along the normal to the array  22  of micro mirrors  29  and dark portions corresponding to the mirrors  29  that are tipped to direct light to an absorber. Lanes  50  between mirrors will generally be dark.  
         [0036]    The substrate  52  may provide the reaction site for DNA synthesis or may be a target for alignment purposes only (henceforth also referred to as substrate  52 ) includes “reaction sites”  54  corresponding to regions where the images of the micro mirrors  29  may be formed and “reaction separation areas”  56  corresponding to regions where the images of the lanes  50  may be formed. The surface of the substrate  52  of the reaction separation areas  56  may be patterned to cause reduced light propagation at a given direction than the reaction separation regions  56 . In this case, the term propagation should be understood to mean generally the quality of directing light along an arbitrarily defined detection direction and thus should include reflection, refraction, diffraction, and transmission.  
         [0037]    Referring again to FIG. 1, the light propagated by reaction separation areas  56  and reaction sites  54  may be received either by a viewer  58  or an appropriately placed light sensor  60  or  60 ′ (which could for example be a photoelectric cell, or a camera and/or image processing circuitry, or the like) where location of sensor  60  indicates a detection of reflected or scattered light and sensor  60 ′ indicates a detection of transmitted light. The electronic sensors  60  also communicate with controller  49  as will be described.  
         [0038]    Referring now to FIG. 4, an alternative substrate  52  may be used having a pattern providing greater propagation in the reaction separation areas  56  and lesser propagation in the reaction sites  54  may also be produced.  
         [0039]    These variations in propagation may be produced by a variety of means including, but not limited to, a coating process coating an opaque, absorptive or reflective material in various regions or by a diffraction process in which lines are ruled in the surfaces of the substrate  52  to provide for a desired selective reflectivity.  
         [0040]    Referring now to FIG. 5, in this latter case, an optical grating  62  may be ruled in the surface of the substrate  52  in regions where selective reflection is desired. The grating  62  provides for grooves and ridges separated in a direction normal to the surface of the substrate  52  by one-half wavelength of the incident light. Light reflected from this ruled surface from the grooves and ridges will destructively interfere along an axis  64  perpendicular to the surface of the substrate  52 . Whereas at an angled axis  66 , the light from the grooves and ridges will constructively add providing a reflectivity profile  68  that is maximum at off-axis angles. Thus light to an eye  58  or sensor  60  for detecting reflection off-axis may be maximized.  
         [0041]    The propagated light may be used to align the substrate  52  with the image of the micro mirrors  29  through microscopic examination of differences in the locations of reaction sites  54  and in the superimposed image of the micro mirrors  29 . More simply, however, a moiré interference pattern may be observed in which macroscopic interference features are generated by the periodicity of the overlapping image in the reaction sites  54 .  
         [0042]    Referring now to FIG. 6, a keystone distortion of the image such as shortens (in this example) a top edge of the array image with respect to the pattern of FIGS.  3  and  4  produces a set of inwardly curving moiré fringes such as would be visible to the naked eye. Such keystone distortion can be caused by a substrate  52  lying in a plane that is not parallel to the array  22  of micro mirror  29 , and is minimized by the telecentric optics of the present invention, but may be at issue in other optical projection systems. The substrate  52  may be tipped appropriately through shims or adjustments, the like to correct this distortion.  
         [0043]    Referring now to FIG. 7, an XY misalignment of the substrate  52  with respect to the image (in this case leftward offset) will create a set of horizontal bands reducing the total light propagated. This variation in total light may be detected visually and manual adjustment made, or may be detected by sensors  60  and used as an input to controller  49  to maximize (or minimize) propagated light and thereby correct for such displacement.  
         [0044]    Referring to FIG. 11, the total light propagated from the substrate of FIG. 3 from the pattern of FIG. 7 is shown plotted against x-axis displacement in a solid line (light function  74 ) and the total light for the substrate of FIG. 4 is shown as a dotted line (light function  74 ′). The controller  49  following a maximizing (or minimizing) rule can thus automatically correct for x or y-axis displacement between the substrate  52  and the image of the array  22 . Such algorithms, well known in the art, make small perturbations in the controlled axis (e.g. x) and detect whether there has been an increase or decrease in the measured quality (propagation of light) and then move an increment in a direction that improves the measured quality, repeating this process until a peak or valley is found. A similar approach can be used to correct for y-axis displacement. Generally, but not shown, lack of focus may also affect the amount of light propagated. Thus, lack of focus can be corrected using a similar peak (or valley) automated correction mechanism.  
         [0045]    Referring now to FIG. 8, a rotation of the substrate  52  with respect to the image creates a tipped cross of interference patterns and can be corrected by rotation of the substrate until the cross disappears.  
         [0046]    Referring to FIG. 9, a warping of the image (pincushion or barrel distortion) is manifest by circular zones of interference fringes.  
         [0047]    Referring to FIG. 10, magnification errors may also be detected by the presence of virtual magnified images of reaction sites  54 . An elimination of these magnification regions to produce an essentially uniform gray field indicates a 1-to-1 magnification.  
         [0048]    The target may be treated with a fluorescent material or backed by fluorescent material to make the measurements of these distortions apparent to the naked eye. In this way, as mentioned, a human operator may control a set of axis controls  70  attached to the controller  49  to manually move the optical elements of mirror  38 ,  40  and XY φ  table  48  appropriately based on understanding of the patterns of FIGS. 6 through 10. Alternatively, certain of these adjustments may be made automatically by the controller  49  attempting to minimize a light function  74 ′ or maximize a light function  74  received from sensor  60  or  60 ′ based on a variation of a parameter  76  which may be one of the dimensions of distortion. For example, correction of magnification may attempt to minimize function  74  as a function of position of mirror  40  along its axis. Such servo control techniques are well known in the art. Alternatively, more sophisticated machine recognition systems may be used to mimic that of a human operator observing the moiré patterns for multi-axis correction.  
         [0049]    Referring now to FIG. 12, the ability to accurately locate the substrate  52  allows it to be pre-patterned not simply for the purpose of alignment but to allow the pattern to do double duty in the synthesis process. For example, the substrate  52  may include a topical coating  80 , such as a repellant coating, positioned in the reaction separation areas  56  so as to reject the bonding of the monomers  82  except in reaction sites  54  providing greater contrast between reaction sites and other sites.  
         [0050]    It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but that modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments also be included as come within the scope of the following claims. For example, although the flow cell  42  is moved in the preferred embodiment, the micro mirror array  22  may be moved to equal effect. Further, the substrate, as mentioned, may be a target fit into the flow cell, used for alignment, and then replaced by a substrate for DNA synthesis on the substrate.