Abstract:
An image locking system for DNA micro-array synthesis provides a feedback system to stabilize or lock the image with respect to an image capture device, such as a camera and/or microscope. The image locking system includes the use of detection or reference marks. When a shift in image position is detected, a correction signal is sent to one of two mirrors, moving the image to correct for the shift in image position. The system comprises a first light beam directed towards a micromirror device that forms an alignment pattern on a reaction cell and a second light beam directed towards the micromirror device that forms a micro-array image on an active surface of the reaction cell. A camera captures the alignment pattern and an alignment mark. A computer calculates a correction signal to realign the alignment pattern with the alignment mark when movement is detected.

Description:
STATEMENT OF GOVERNMENT RIGHTS 
     This invention was made with United States Government support awarded by the following agencies: DARPA DAAD 19-02-2-0026. The United States has certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to the field of DNA micro array and synthetic DNA strands manufacturing. More particularly, the present invention relates to an image locking system for DNA micro-array synthesis. 
     BACKGROUND OF THE INVENTION 
     Researchers believe that thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way. However, traditional methods in molecular biology generally work on a “one gene in one experiment” basis, which means that the throughput is very limited and the “whole picture” of gene function is hard to obtain. In the past several years, a new technology, called DNA microarray, has attracted tremendous interests among biologists. This technology attempts to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously. 
     An array is an orderly arrangement of samples. It provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems, such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample. In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots. Microarrays require specialized robotics and imaging equipment that generally are not commercially available as a complete system. 
     DNA microarray, or DNA chips, are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously—a dramatic increase in throughput. 
     In the process of manufacturing DNA micro array and synthetic DNA strands, an image is repeatedly projected on the substrate. While the substrate is not moved during processing, the images need to be kept stable across different phases of exposure that may last a total of 4–8 hours. During this time, the optical system drifts from its reference state because, for instance, of changes in the environment. It is not practical to try to completely eliminate these drifts. As such, there is a need for a feedback system to stabilize or lock the image used in the DNA micro array and strands manufacturing. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, an image locking system for DNA micro-array synthesis provides a feedback system to stabilize or lock the image with respect to an image capture device, such as a camera and/or microscope. The image locking system includes the use of detection or reference marks. When a shift in image position is detected, a correction signal is sent to one of two mirrors, moving the image to correct for the shift in image position. 
     In an exemplary embodiment, the image locking system includes a reaction cell with an active surface on which a micro-array may be formed, a micromirror device, an alignment mark located at the reaction cell, a second light beam that is directed towards the micromirror device forming an alignment pattern on the reaction cell, a camera that captures an alignment image that comprises the alignment mark and the alignment pattern, a computer that identifies a change in the alignment image and calculates a correction signal to remove the change from the alignment image, and at least one actuator provided to adjust the alignment image in response to the correction signal calculated by the computer. The micromirror device is formed of an array of electronically addressable micromirrors wherein each micromirror can be selectively tilted between one of at least two positions whereby a first light beam directed towards the micromirror device forms a micro-array image on the active surface of the reaction cell. 
     In an exemplary embodiment, a method of forming an image locking system comprises projecting a first light beam towards a micromirror device that forms an initial alignment pattern, reflecting the initial alignment pattern along an optical path and onto a reaction cell, capturing an initial alignment image wherein the initial alignment image comprises an alignment mark and the initial alignment pattern projected onto the reaction cell, projecting the first light beam towards the micromirror device that forms a current alignment pattern, reflecting the current alignment pattern along the optical path and onto the reaction cell, capturing a current alignment image wherein the current alignment image comprises the alignment mark and the current alignment pattern projected onto the reaction cell, calculating the displacement between the initial alignment image and the current alignment image, and sending a correction signal to at least one actuator to remove the displacement between the initial alignment image and the current alignment image. 
     In an alternative embodiment, the method of forming an image locking system comprises projecting a first light beam towards a micromirror device that forms an initial alignment pattern, reflecting the initial alignment pattern along an optical path and onto a reaction cell, capturing an initial alignment pattern image of the initial alignment pattern projected onto the reaction cell, projecting the first light beam towards a micromirror device that forms a current alignment pattern, reflecting the current alignment pattern along the optical path and onto the reaction cell, capturing a current alignment pattern image of the current alignment pattern projected onto the reaction cell, calculating the displacement between the initial alignment pattern image and the current alignment pattern image, and sending a correction signal to at least one actuator to remove the displacement between the initial alignment pattern image and the current alignment pattern image. 
     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 
       In the drawings: 
         FIG. 1  is a diagrammatic representation of an illumination and optical system of a maskless array synthesizer according to an exemplary embodiment. 
         FIG. 2  is a schematic of an image locking system in accordance with and exemplary embodiment. 
         FIG. 3(   a ) is a diagrammatic representation of a reference mark on a reaction cell. 
         FIG. 3(   b ) is a diagrammatic representation of a projected alignment pattern with the reference mark on a glass slide. 
         FIG. 3(   c ) is a diagrammatic representation of locations of alignment marks. 
         FIG. 4(   a ) is a cross-section view of a reaction cell with image locking in accordance with an exemplary embodiment. 
         FIG. 4(   b ) is a diagrammatic representation of a captured image to be processed in accordance with an exemplary embodiment. 
         FIGS. 5(   a ), ( b ), and ( c ) are captured images to be processed. 
         FIG. 6  is a diagrammatic representation of an image projected on a substrate where the image includes several micro-mirrors. 
         FIG. 7  is a diagrammatic representation of an image projected on a substrate wherein the image of the mask appears as a dark line. 
         FIG. 8  is a diagrammatic representation of an exposure scheme for performance verification. 
         FIGS. 9(   a ) and ( b ) are diagrammatic representations of radiochromic film images formed continuously without image locking. 
         FIGS. 10(   a ), ( b ), and ( c ) are diagrammatic representations of radiochromic film images performed continuously with and without image locking in accordance with an exemplary embodiment. 
         FIGS. 11(   a ) and ( b ) are diagrammatic representations of a virtual mask layout. 
         FIG. 12  is a diagrammatic representation of an image of a microarray fabricated without using image locking. 
         FIGS. 13(   a )–( h ) are diagrammatic representations of images of a microarray fabricated without using image locking at 10 times magnification. 
         FIGS. 14(   a )–( h ) are diagrammatic representations of an image of a microarray fabricated without using image locking at 50 times magnification. 
         FIG. 15  is a diagrammatic representation of an image of a microarray fabricated using image locking. 
         FIGS. 16(   a )–( h ) are diagrammatic representations of images of a microarray fabricated using image locking in accordance with an exemplary embodiment at 10 times magnification. 
         FIGS. 17(   a )–( h ) are diagrammatic representations of an image of a microarray fabricated using image locking in accordance with an exemplary embodiment at 50 times magnification. 
     
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
       FIG. 1  illustrates a schematic of an optical system  10  of a gene synthesizer according to an exemplary embodiment. The system  10  includes a maskless array synthesizer  12  comprising a mercury (Hg) arc lamp  14 , a condenser  18 , a digital micro-mirror device (DMD)  20 , and a microarray reaction cell  22 . The digital micromirror device (DMD)  20  may consist of a 1024×768 array of 16 μm wide micro-mirrors. Preferably, these mirrors are individually addressable and can be used to create any given pattern or image in a broad range of wavelengths. Each virtual mask is generated in a bitmap format by a computer and is sent to the DMD controller, which forms the image onto the DMD  20 . 
     The maskless array synthesizer  12  can generate several μm of drift over several hours due to the thermal expansion of optics parts. The optical path between the DMD  20  and DNA cell  22  is about 1 meter. Due to the thermal expansion caused by the temperature and humidity fluctuation of surrounding environments and also due to ultraviolet (UV) exposure, a slight change of position or rotation of the primary spherical mirror and other optical parts may result. This slight change may cause several μm of drift of the projected image. Since the space between each digital micromirror is only 1 μm, this image drift can cause the projected image to be shifted to expose the UV light at the wrong oligonucleotide spots, generating defects in oligonucleotides sequences and their spatial distribution. An image locking system confines the image shift within a certain range to minimize image drift. 
       FIG. 2  illustrates a diagram of an image locking system  28 . The image locking system  28  comprises a laser  42 , a flat mirror  36 , a 1:1 ratio projection system  16 , a camera  40 , an x-actuator  48 , and a y-actuator  50 . The 1:1 ratio projection system  16  comprises a UV lamp  44 , a digital light processor (DLP) or digital micromirror device (DMD)  30 , a concave mirror  32 , a convex mirror  34 , and a reaction cell  38 . The 1:1 ratio projection system  16  forms a UV image of the virtual mask on the active surface of the glass substrate mounted in a flow reaction cell  38  connected to a DNA synthesizer. In an exemplary embodiment, the laser  42  is a He—Ne laser with a wavelength of 632.8 nm (red light) and does not disturb the photochemical reaction of oligonucleotide synthesis. The He—Ne laser beam from the laser  42  is projected to a reaction cell  38  using an “off” state (rotated −10°) of micromirrors without interrupting the current UV exposure system with UV light from the UV lamp  44  which is projected to the reaction cell  38  using an “on” state (rotated 10°) of micromirrors. The He—Ne laser  42  is at the opposite side of the UV lamp  44  with incident angle of −20° into the DMD  30 . 
     The system  28  can be a 0.08 numerical aperture reflective imaging system based on a variation of the 1:1 Offner relay. Such reflective optical systems are described in A. Offner, “New Concepts in Projection Mask Aligners,” Optical Engineering, Vol. 14, pp. 130–132 (1975). The DMD  30  can be a micromirror array available from Texas Instruments, Inc. The reaction cell  38  includes a quartz block  47 , a glass slide  49 , a projected image  51 , and a reference mark  53 . The UV lamp  44  can be a 1000 W Hg Arc lamp (e.g., Oriel 6287, 66021), which can provide a UV line at 365 nm (or anywhere in a range of 350 to 450 nm). In an alternative embodiment, the lamp  44  may be a visible wavelength lamp. 
     The laser  42  projects a laser beam onto flat mirror  36  which reflects the beam onto DMD  30 . DMD  30  has a two-dimensional array of individual micromirrors which are responsive to the control signals supplied to the DMD  30  to tilt in one of at least two directions. A telecentric aperture may be placed in front of the convex mirror  34 . 
     The camera  40  is a charge coupled device (CCD) camera used to capture an image of alignment marks. The captured image is transferred to a computer  46  for image processing. When a misalignment is detected, correction signals are generated by the computer  46  and sent to actuators  48  and  50  as the feedback to adjust the mirror  32 , so that the correct alignment is reestablished. In at least one alternative embodiment, three electro-strictive actuators (instead of actuators  48  and  50 ) are used to provide minimum incremental movement of 60 nm and control the rotations and movement of the mirror  32 . The displacement of the projected image at the glass slide is highly sensitive to the rotations and movement of the mirror  32 . 
       FIG. 3(   a ) illustrates the alignment mark  53  patterned on the quartz block  47  in the reaction cell  38 . The quartz block  47  includes an outlet  55  and an inlet  57  through which fluid may flow through the reaction cell  38 . Such a reaction cell is described in U.S. Pat. No. 6,375,903 entitled “Method and Apparatus for Synthesis of Arrays of DNA Probes.” A predefined micromirror pattern shown in  FIG. 3(   b ) is projected, being centered at the alignment mark  53 . In an exemplary embodiment, the projected image  51  is manually aligned at the beginning of synthesis, so that the center of the projected image  51  is roughly overlapped with the center of the alignment mark  53 . The CCD camera  40  is used to capture the image that is formed by a 20× (magnification) microscope lens, which is focused at the middle between the reference mark  53  and the projected image  51 . An image processing program in the computer  46  calculates the centers of the reference mark  53  and the projected image  51 , generating the amount and direction of any displacement, and sending its correction signals to the corresponding actuator(s)  48  and/or  50 . The reference mark  53  is patterned on the surface of the quartz block  47  as shown in  FIG. 3(   a ). The relative position of the projected image  51  to the reference mark  53  is shown at  FIG. 3(   c ). 
       FIG. 4(   a ) illustrates a cross-sectional view of the reaction cell  38 . The projected image  51  is focused on an inner glass slide surface  61  of the glass slide  49  where the oligonucleotides are grown. The reference mark  53  and the projected image  51  are not at the same focus plane. A microscope lens focuses at the middle plane between the reference mark  53  and the projected image  51 . As such, the image captured by the camera  40  is blurred, as shown in  FIG. 5(   c ). The gap between the glass slide surface  61  and quartz block surface  65  of the quartz block  47  is 100 μm. To locate the center position of each pattern, an 2D optical pattern recognition technique, which is based on correlation theory, is used. Correlation analysis compares two signals (or images) in order to determine the degree of similarity, where input signal is to be searched for a reference signal. Each correlation gives a peak value where the reference signal and input signal matches the best. If the location of this value is different from the previous value, it means that the image has been shifted, indicating the need of correction. 
     In an exemplary embodiment, an image processing procedure calculates the image displacement from the images captured by the camera  40 , by calculating the cross-correction signals between a captured input image described with reference to  FIG. 5(   c ), the reference mark  53  of FIG.  5 ( a ), and the projected image  51  of  FIG. 5(   b ). The cross-correlation is a measure of the similarity between two images, such as images from  FIGS. 5(   a ) and  5 ( c ) and such as images from  FIGS. 5(   b ) and  5 ( c ). Mathematically, the cross-correlation can be calculated as: 
                 c   gh     ⁡     (     X   ,   Y     )       =       ∫     -   ∞     ∞     ⁢       ∫     -   ∞     ∞     ⁢       g   ⁡     (     x   ,   y     )       ⁢     h   ⁡     (       x   +   X     ,     y   +   Y       )       ⁢     ⅆ   x     ⁢     ⅆ   y                 
or, using the Wiener-Khintchine Theorem, as:
   c   gh ( X,Y )= IFFT ( FFT 2( g ( X,Y ))· FFT 2(rot90( h ( X,Y )))) 
     The new locations of the reference mark and the projected image are marked by correlation peaks (i.e., the highest value of c gh (X,Y)). Based on the new locations, correction signals are computed and sent to the actuators to move the mirror. This correction procedure continues until the synthesis is completed. 
     In an exemplary embodiment, computer programs control the actuators and generate the correction signals by image processing. A log file of displacements can also be recorded and analyzed for measuring actual displacement indirectly and its direction for further refinement of the algorithm. Various mark shapes (e.g., crosses, chevrons, circles) can be used as the reference mark  53 . 
       FIG. 6  illustrates an image  71  projected on a substrate where the image includes several micro-mirrors  73 ,  75 ,  77 , and  79  according to another exemplary embodiment. A reference mark  74  is included on the substrate. In the field of microscope, the micro-mirrors  73 ,  75 ,  77 , and  79  appear as a bright image while the reference mark  74  can be dark so that the image of the mask will appear as a dark line  76  ( FIG. 7 ). As such, overlap of the micro-mirrors  73 ,  75 ,  77 , and  79  and the reference mark  74  can be observed. Image processing software can determine if the dark shadows are centered on the micro-mirror and if not, apply a correction. 
     Since each pixel is approximately 16 μm in size, it is necessary to keep the image locked to less than 200 nm. Since the distance from the concave mirror  32  ( FIG. 2 ) to the reaction cell  38  can be approximately 500 mm, the angle pointing accuracy is 0.4×10 −6  radians. Since the diameter of the optics is 200 mm, a piezoelectric or similar system can be used to generate the angular shift by applying a displacement of 80 nm. Typically, a nanopositioner can control displacements of even 10 nm. 
     Other designs are possible, involving different schemes for the detection of the displacements. The actuators  48  and  50  can be used to effectively align the optics. In another exemplary embodiment, diffractive marks can also be used, alleviating the need for microscopes. Partially transmitting marks (half toned) can be used for other schemes of detection. 
       FIGS. 8–10  illustrate the performance of an exemplary image locking system.  FIG. 8  illustrates image patterns for measuring drift. In  FIG. 8(   a ), a square shape reference frame  81  is exposed at time equal to zero (t=0). In  FIGS. 8(   b ), ( c ) and ( d ), each adjacent pixel of the reference frame  81  is progressively exposed every 10 min to create a line  83 . If there is a drift, the gap between the reference frame and the line  83  will change. 
       FIGS. 9(   a ) and ( b ) show the results of a projected image shift as an image is projected without image locking. In one experiment, the ambient temperature around the system was measured to be 23.56±1° C. and the humidity around 23.2%.  FIG. 9(   b ) shows a zigzag displacement is approximately half pixel&#39;s size (˜8 μm) for 490 minutes exposure. Such a shift can increase to about 50 μm for 24 hour&#39;s continuous exposure. 
       FIGS. 10(   a ), ( b ), and ( c ) show the results of exposing radiachromic film at room temperature for 200 minutes (pixels  1 – 20 ), and increasing the environmental temperature by 5° C. for 120 minutes (pixels  21 – 32 ). Then, the environmental temperature is reduced back to room temperature for 150 minutes (pixels  33 – 48 ). The humidity variation is 11.7% to 16.3% as the temperature change.  FIG. 10(   a ) illustrates the experimental results showing image drift without image locking. However, in  FIGS. 10(   b ) and ( c ) with image locking, the image is stable with drift in each direction smaller than 1 μm. 
       FIGS. 11(   a ) and ( b ) show an exemplary virtual mask layout used to verify the image locking performance.  FIG. 11(   a ) shows the entire mask (1024×768) and  FIG. 11(   b ) shows one of the sections of the mask that expands to the entire chip. From the upper left corner to the lower right corner, the features are composed of single pixel, 3×3, 5×5 (with interim mirrors off), 1:4 ratio, 5×5 (all pixels on), 3×3 (of 9×9 mirrors), 5×5 (of 3×3 mirrors), 9:36 ratio. 
       FIG. 12  illustrates a fluorescence image with the synthesized oligomers (25 mer in length) using the virtual mask layout described with reference to  FIG. 11 , hybridized with their complementary sequences (probes) that has cy3 cynano-nucleotide at its end. The chip is scanned in 2 μm resolution using an applied precision microarray scanner. The target oligomers have an additional 5Ts as a linker on the substrate glass for efficient hybridization. 
     The small features are not visible in  FIG. 12  because they have extremely low fluorescence signal intensity due to the lack of exposure, caused by the image drifting over time. Larger features have a relatively bigger overlapping area of exposure and those areas have target oligonucleotides to be hybridized even though the feature shape is distorted. However, small ones such as in the upper row in  FIG. 12  will have progressively smaller amount of exposure as the synthesis advances, resulting in very poor synthesis. 
       FIGS. 13 and 14  show the images of the same features as in  FIG. 12 , captured by a Nikon Fluorescence Microscope using 10× and 50× lens respectively. In these Figures, the hybridization signal intensities are not comparable to each other because their images are scaled to be seen so that the shapes, directions and amount of the drift can be brought out. Their actual intensity of smaller features are approximately 10,000-fold lower than the bigger ones. The single pixel that doesn&#39;t have any adjacent pixels is not detectable due to its extremely low signal and is not shown. In 100 cycles of synthesis, 5 to 6 pixels&#39; displacement occurred in the particular synthesis even though there is no enforced environment change.  FIG. 13(   f ) shows the directionality of the drift. Only horizontal features are left, indicating that there is some dominant directions of drift.  FIG. 14  shows more magnified images of those shown in  FIG. 13  by using a 50× lens instead of a 10× lens. 
       FIG. 15  is the scanned image of the DNA chip that was fabricated under the same conditions as the chip in  FIG. 12  (without image locking) but where the image locking system is engaged. All the features in the mask layout are visible, keeping their shape (square micromirror shape), even the single pixel. The synthesis images also have maximum hybridization signal intensities. 
       FIGS. 16 and 17  show fluorescence microscope capture images using 10× and 50× respectively. The lanes and the posts of the micromirros are clearly seen, indicating the firm image locking. 
     It should be understood that the invention is not limited to the embodiments set forth herein as illustrative, but embraces all such forms thereof as come within the scope of the following claims.