Abstract:
An imaging system for collecting images of signals associated with a sample tile comprising a stage supporting the sample tile, a ring illuminator system emitting a uniform excitation energy upon an entirety of the sample tile causing at least a first signal to be generated from the sample tile, and an image collecting device collecting a first image of the first signal. The image collecting device further collecting a second image of a second signal emitted from the sample tile, wherein the second signal being different than the first signal.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of U.S. patent application Ser. No. 11/781,515 filed Jul. 23, 2007, which is a continuation of U.S. patent application Ser. No. 11/580,100 filed Oct. 12, 2006, which is a divisional of U.S. patent application Ser. No. 11/188,243 filed Jul. 22, 2005, now U.S. Pat. No. 7,135,667, which is a divisional of U.S. patent application Ser. No. 10/384,995 filed Mar. 10, 2003, now U.S. Pat. No. 6,970,240. The disclosures of the above applications are incorporated herein by reference. 
     
    
     FIELD 
       [0002]    The invention relates generally to imaging biomolecular or synthetic arrays. 
       BACKGROUND 
       [0003]    Substrate-bound biomolecular or synthetic arrays, such as oligonucleotide arrays, also known as micro arrays, enable the testing of the hybridization of different sequences in a sample to many different probes. These arrays can be composed of hundreds of thousands of probes deposited or synthesized within specific regions, defined as features, on a substrate. 
         [0004]    To analyze such arrays, the sample is labeled with one or more detectable markers, such as fluorescent or chemiluminescent markers, that hybridize with the probes at each feature on the substrate. The markers emit luminous signals, for example a fluorescent signal or a chemiluminescent signal, that are imaged and the images are analyzed. 
       SUMMARY 
       [0005]    In various configurations, an apparatus is provided for imaging an array of a plurality of features associated with a sample tile. The apparatus includes a stage that supports the sample tile in an illumination region, and an illumination source having a plurality of LEDs adapted to emit light. At least a portion of the light illuminates the illumination region. Additionally, the apparatus includes an image collecting device adapted to selectively collect images of a signal. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0006]    The present invention will become more fully understood from the detailed description and accompanying drawings, wherein; 
           [0007]      FIG. 1  is a perspective view of an imaging apparatus for collecting images of fluorescent and chemiluminescent hybridized markers in a biomolecular or synthetic sample; 
           [0008]      FIG. 2  is a perspective view of an illuminator shown in  FIG. 1 ; 
           [0009]      FIG. 3  is a schematic of a cross-section of the imaging apparatus shown in  FIG. 1 , illustrating illumination patterns of the illuminator shown in  FIG. 2 ; 
           [0010]      FIG. 4  is a schematic of a cross-section of the imaging apparatus shown in  FIG. 1 , illustrating the path of the chemiluminescent signals emitted from an array of features; and 
           [0011]      FIG. 5  is a flow chart for the basic operation of the imaging apparatus shown in  FIG. 1 . 
       
    
    
     DETAILED DESCRIPTION 
       [0012]    The following description is merely exemplary in nature and in no way is intended to limit the invention, its application, or use. 
         [0013]      FIG. 1  is a perspective view representative of various configurations of an imaging apparatus  10  for collecting images of fluorescent and chemiluminescent hybridized markers in a biomolecular or synthetic sample. The imaging apparatus  10  includes a base  14 , a frame  18  connected to the base  14 , and a mid-support  22  coupled to the frame  18 . Additionally, the imaging apparatus  10  includes a transport  26  and an elevator  30  that are controlled by a controller (not shown) to orient a stage  34  under an illuminator  38  that illuminates a sample tile  42  positioned on the stage  34 . The sample tile  42  is a support, such as glass, ceramic, or plastic, to which at least one feature of a sample (not shown) is associated, i.e. placed, synthesized, or attached. The feature can be, for example, any feature of the sample where a fluorescent and/or chemiluminescent marker has hybridized with a probe attached to the sample tile  42 . For example, the feature can be a co-spotted oligonucleotides labeled with one fluorescent marker and one chemiluminescent marker. 
         [0014]    In various configurations, the sample tile  42  includes an array of associated features having, for example, hundreds or thousands of features. In some configurations, the sample tile  42  includes a microarray having a larger plurality of associated features, for example, tens of thousands or hundreds of thousands of features. For the sake of convenience and clarity, exemplary configurations will be described below referencing an array of features, but it will be understood that the array could include as few as one feature, or the array could include as many as hundreds of thousands of features, or more. 
         [0015]    In various configurations the array of features is a nucleic acid microarray. Such microarrays are becoming an increasingly important tool in bioanalysis and related fields. Nucleic acid microarrays have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis. Current methods of manufacturing nucleic acid microarrays, and methods of their use as diagnostic assays have been described in U.S. Pat. Nos. 6,413,722, 6,215,894, 6,040,193, 6,040,138, and 6,387,675. 
         [0016]    Furthermore, the imaging apparatus  10  in various configurations includes a first lens  46 , a second lens  50 , a first filter  54 , and an image collecting device  58 . The first and second lenses  46  and  50  can be any lenses suitable for optical imaging performance, for example medium format photographic lenses. In some configurations not illustrated, a single lens is used for optical imaging performance. In various configurations, the first filter  54  is a longpass filter adapted to pass light having longer wavelengths, for example light having a wavelength greater than about 670 nm., or the first filter  54  is a bandpass filter adapted to pass light having wavelengths included in a certain range of wavelengths, for example light having wavelengths that are between about 670 nm and about 700 nm. 
         [0017]    The image collecting device  58  and the second lens  50  are positioned in relation to each other such that a primary imaging surface  62  of image collection device  58  is at the focal plane of the second lens  50 . The controller utilizes the transport  26  and the elevator  30  to position the stage  34  such that the tile  42  is at a focal plane of the first lens  46 . The transport  26  moves the stage  34  along an x-axis, while the elevator  30  moves the stage  34  along a z-axis. Both the transport  26  and the elevator  30  are controlled by software via the controller, which interfaces with a computer workstation (not shown). Through the workstation, a user enters a command, e.g. “load sample”, which is communicated to the controller. The controller interprets the command and utilizes at least one motor (not shown) to move the stage along the x-axis and z-axis to the commanded position. In various configurations the workstation is separate from the imaging apparatus  10 . In other various configurations the imaging apparatus  10  includes the workstation. In other various configurations, the imaging apparatus  10  includes various computer workstation components, such as memory and a processor, while other computer workstation components, such as a graphical user interface, are separate from the imaging apparatus  10 . 
         [0018]    In various configurations, the controller and the transport  26  move the stage  34  to pre-set x-axis positions when loading the tile  42  and imaging the features of the array. For example, in some configurations, the controller is configured to instruct the transport  26  to move the stage  34  to a “loading the sample” position, an “imaging position # 1 ” under illuminator  38 , and an “imaging position # 2 ” under the illuminator  38 . The elevator  34  is controlled by the controller to position the stage  34  at the focal plane of the first lens  46 . The elevator  30  moves the stage  34  along a z-axis, while the first and second lenses  46  and  50  remain stationary to achieve an optimum focus of the array for the image collecting device  58 . An algorithm processes image data collected by image collecting device  58  to determine the position for optimum focus of the array. Therefore, an image of the array is auto-focused for the image collecting device  58  without adjusting the first and second lenses  46  and  50 . 
         [0019]    For example, image collecting device  58  collects imaging data and communicates the data to the workstation where the algorithm determines the clarity of the image. That is, the algorithm analyzes the contrast of the image. If the image does not have a desired contrast, the algorithm instructs the controller to adjust the position of the stage along the z-axis. Then another image is collected and the data is communicated to the workstation where the algorithm again analyzes the contrast. This process is repeated until the contrast is maximized, i.e. an optimum focus is achieved. In various configurations, the fluorescent signals emitted by each fluorescent marker are used by the algorithm to auto-focus the array. In some configurations, the elevator  30  is adapted to rotate the stage  34  in the x-y plane, and the transport  26  is adapted to move the stage  34  along the y-axis. 
         [0020]    When the stage  34  is positioned under the illuminator  38 , at the focal plane of the first lens  46 , the image collecting device  58  collects at least one image of the array of features associated with the tile  42 . For example, if the sample tile  42  is in an environment illuminated using the illuminator  38 , the image collecting device  58  collects illumination data relating to the intensity of light emitted by the fluorescent marker in each feature. Or, for example, if the sample tile  42  is an environment absent light that will interfere with the chemiluminescent signals, the image collecting device  58  collects illumination data relating to the intensity of light emitted by the chemiluminescent markers in each feature. The image collecting device  58  can be any device suitable for collecting image data emitted from the array of features. For example, in some configurations, image collecting device  58  is configured to be a CMOS detector array. In some configurations the image collecting device  58  comprises a charge-coupled device (CCD). 
         [0021]      FIG. 2  is a perspective view representative of various configurations of the illuminator  38  (shown in  FIG. 1 ). The illuminator  38  comprises a light source configured to excite the fluorescent marker in each feature by flooding the entire tile  42  (shown in  FIG. 1 ) with light. That is, the illuminator  38  distributes light over the entire tile  42 , exciting the fluorescent markers in all features associated with the tile  42  at the same time. Additionally, the illuminator  38  substantially evenly distributes light over the tile  42 , such that approximately the same amount of light is distributed over the entire tile  42 . The evenly distributed flood illumination provides approximately 360° of light to each feature, thereby allowing more accurate evaluation of the feature by exciting a greater percentage of the fluorescence of each feature, possibly the entire fluorescence of each feature. More specifically, artifacts, i.e. irregularities, in the top surface are less likely to block the excitation light from reaching all areas of the top surface of each feature. Furthermore, flooding the tile and associated array with light from approximately 360° allows a shape and a size of each feature in the array to be easily determined. 
         [0022]    In various configurations, the illuminator  38  includes an opening  66  configured to allow images, i.e. fluorescent and/or chemiluminescent light signals, emitted from each feature to pass through the opening  66 . The signals are then re-imaged by the first and second lenses  46 ,  50  (shown in  FIG. 1 ), filtered by the first filter  54  (shown in  FIG. 1 ), and collected by the image collecting device  58  (shown in  FIG. 1 ). Although the illuminator  38  and opening  66  are shown in  FIG. 2  as having a rectangular shape, the illuminator  38  and opening  66 , in various configurations, can have any geometric shape suitable to flood illuminate the tile  42 . In various configurations, for example, the shape of the illuminator  38  matches the shape of the tile  42 . For example, in configurations in which the tile  42  is rectangular, the illuminator  38  and opening  66  are also rectangular. In configurations in which the tile  42  is round, the illuminator  38  and opening  66  are likewise round. 
         [0023]    Additionally, in various configurations, illuminator  38  can have a continuous ring form, comprising a single continuous body  64  that provides the opening  66 , as shown in  FIG. 2 . Additionally, In various configurations illuminator  38  can have a discontinuous ring form having a plurality of disconnected sections (not shown) that provides the opening  66 . For example, illuminator  38  could have discontinuous ring form comprising two disconnected essentially semi-circular sections, or four disconnected straight sections that form a rectangular ring disconnected at the corners. 
         [0024]    In various configurations, the illuminator  38  includes a plurality of LEDs  70 , wherein each LED  70  is associated with one of a plurality of second filters  74  and one of a plurality of diffusers  78 . For convenience, the second filters  74  and diffusers  78  are shown in  FIG. 2  as having different sizes, but are not required to be of different sizes to practice the invention. In some configurations, second filters  74  and diffusers  78  have the same size and same geometric shape, but in some configurations, the second filters  74  and diffusers  78  have different sizes and geometric shapes. Each LED  70  is enclosed in one of a plurality of recesses  82  that are covered by second filters  74  and diffusers  78 . However, in some configurations, illuminator  38  includes a plurality of any suitable excitation light sources other than LEDs  70 , for example, tungsten or xenon bulbs, a laser light source, and/or a fiber optic light source. 
         [0025]    The LEDs  70  are configured to emit a wavelength of light at an intensity level that excites a fluorescent marker in each feature. For example, in some configurations, the illuminator  38  includes LEDs  70  that emit light having a wavelength of about 635 nm to excite fluorescent markers that emit red light. In some configurations, the illuminator  38  includes LEDs  70  that emit light having a wavelength of about 470 nm used to excite fluorescent markers that emit blue light. Other wavelengths may be used to excite fluorescent markers having other excitation requirements. In various configurations the Illuminator  38  includes LEDs  70  that emit light having various wavelengths. For example, various LEDs  70  emit light having a wavelength of 635 nm, while other LEDs  70  in illuminator  38  emit light having a wavelength of 470 nm, and other LEDs  70  may emit light having other wavelengths. This would allow the use of multi-color fluorescent markers in the array of features. 
         [0026]    In various configurations, imaging apparatus  10  is configured to allow the illuminator  38  to be removed and replaced with an illuminator  38  comprising LEDs that emit light having a different wavelength. Thus, if tile  42  associated with an array of features having fluorescent markers that emit red light is removed and replaced with a tile  42  associated with an array of features having fluorescent markers that emit blue light, the illuminator  38  can be removed and replaced accordingly. 
         [0027]    Furthermore, in some configurations, each of the LEDs  70  is oriented in the recesses  82  so that light provided by each LED  70  is directed toward one or more desired areas of the tile  42 . For example, each LED  70  can be oriented so that light emitted from each LED is generally directed to the center of the tile  42 , or each LED  70  can be oriented so that light emitted from each LED is directed to different sections of the tile  42 . In various configurations, a front face  84  of the illuminator  38  is angled inward to allow the LEDs  70  to point downward and slightly inward toward a focal point in the center of the tile  42 . 
         [0028]    In some configurations, the diffusers  78  diffuse light emitted from each LED  70  to substantially evenly distribute the light from each LED  70  over the entire tile  42 . That is, diffusers  78  have a divergence angle selected so that light emitted from each LED  70  illuminates the entire tile  42 . Therefore, the light emitted from each LED  70  overlaps with the light emitted from each of the other LEDs  70 . Thus, the intensity of light provided by the illuminator  38 , over the entire tile  42  is a function of the number of LEDs included in the illuminator  38  and the selected intensity of the LEDs  70 . In some configurations, a single diffuser (not shown) is used. In various configurations the single diffuser has the same shape as the front face  84  of illuminator  38 . The single diffuser covers each LED  70  and simultaneously diffuses the light emitted from each LED  70 . In various other configurations at least two diffusers (not shown) are used to diffuse light emitted by the LEDs  70 . 
         [0029]    The second filters  74  eliminate light emitted by the LEDs  70  having a wavelength that would reflect off the array, the tile  42 , or the stage  38  and undesirably pass through the first filter  54  to the image collecting device  58 . For example, in some configurations, the first filter  54  passes light having a wavelength greater than about 640 nm, and the second filter  74  passes only light having a wavelength of less than about 635 nm. In some configurations, the second filters  74  are shortpass filters adapted to pass light having shorter wavelengths, for example light having a wavelength less than about 635 nm. In some configurations, the second filter  74  is a bandpass filter adapted to pass light having wavelengths included in a certain range of wavelengths, for example light having wavelengths that are between about 550 nm and about 635 nm. In various configurations, the apparatus  10  includes a single second filter (not shown) for eliminating light emitted by the LEDs  70 . In various other configurations, the apparatus  10  includes two or more second filters (not shown), whereby each of the second filters  74  filters light emitted by at least one of the LEDs  70 . 
         [0030]    In various configurations, EPI illumination is utilized, in place of the illuminator  38 , to illuminate the array and excite the fluorescent markers. An EPI based system would have a dichroic beam splitter (not shown) between the first lens  46  and the first filter  54 . Light emitted from the EPI illuminator would be shaped and imaged onto the sample tile  42  through the first lens  46 . LEDs, a lamp or a laser could be used as the illumination source. Any suitable illumination source can be utilized to illuminate the array and excite the fluorescent markers. For example, off axis illumination and electro luminescent panels can be utilized. 
         [0031]    Referring to  FIG. 1 , the first filter  54  is positioned between the first lens  46  and the second lens  50  when it is desirable to filter out light reflecting off the array from the illuminator  38  having certain wavelengths. Therefore, light emitted from the illuminator  38  that overlaps with the fluorescent emissions of the array of features is separated from the fluorescent emissions and substantially prevented from reaching the image collecting device  58 . The first filter  54  can be removed when filtering is not desired, for example, when chemiluminescent emissions are to be imaged, all light that can interfere with the enzymatically generated chemiluminescent signals must be substantially removed from the environment surrounding the imaging apparatus  10 . In some configurations, the removal and insertion of the first filter  54  is automated by the controller and a mechanism (not shown) suitable for inserting the first filter  54  between the first and second lenses  46  and  50 , and removing the first filter  46  when desired. 
         [0032]    In some configurations, a filter wheel having a plurality of filters is used as a first filter  54 , wherein each filter of the filter wheel filters out light of a different wavelength, or within a different bandwidth. Positioning of the filter wheel is automated by the controller and a mechanism suitable to rotate the wheel such that a desired filter, or no filter, is positioned between the first and second lenses  46  and  50 . The first filter  54  works in combination with the second filter  78  to allow only fluorescent emissions of the array to be collected by the image collecting device  58  when the illuminator  38  is illuminated. 
         [0033]      FIG. 3  is a schematic of a cross-section of various configurations of the imaging apparatus  10  (shown in  FIG. 1 ), illustrating the flood illumination of the illuminator  38  (shown in  FIG. 2 ) and the path of the fluorescent signals emitted from an array of features. Each LED  70  emits light directed at the tile  42  and the associated array. The light emitted by each LED  70  is filtered by the second filter  78  so that only light having a desired wavelength, or within a desired range of wavelengths, illuminates the tile  42  and associated array. Additionally, light emitted from each LED  70  is diffused by the diffuser  78  to provide a substantially uniform intensity of light over the entire tile  42 , as indicated by LED illumination pattern lines  86 . Therefore, the light emitted from each LED  70  overlaps with the light emitted from at least one of the other LEDs  70 , as generally indicated at overlap area  90 . 
         [0034]    The light emitted by the LEDs  70 , filtered by the second filters  74 , and diffused by the diffusers  78 , excites the fluorescent markers in each feature of the array, resulting in the emission of fluorescent signals  94 . The fluorescent signals  94  pass through the opening  66  in the illuminator  38  and enter the first lens  46 , where they are re-imaged. The signals  94  are then filtered by the first filter  54 , which filters out any light from the LEDs  70  that has reflected off of the array of features, the tile  42  and/or the stage  34 . The filtered signals  98  then pass through the second lens  50  where they are re-imaged again. After passing through the second lens  50 , the fluorescent signals  94  are collected by image collecting device  58 , and the collected image data is transmitted to a computer based system (not shown), where the data is processed and analyzed. 
         [0035]      FIG. 4  is a schematic of a cross-section representative of various configurations of imaging apparatus  10  (shown in  FIG. 1 ), illustrating the path of the chemiluminescent signals emitted from an array of features. To collect images of chemiluminescent signals  102  emitted by the feature in the array, the first filter  54  (shown in  FIG. 3 ) is removed from between the first and second lenses  46  and  50 , and the illuminator  38  is turned off. The chemiluminescent signals must be imaged in a substantially light free environment. That is, an environment substantially free from any light that will interfere with the chemiluminescent signals emitted from the array. 
         [0036]    In various configurations the chemiluminescent signals are enzymatically generated. Methods for generating chemiluminescent signal in biomolecular array, for example nucleic acid microarrays, have been described in U.S. Pat. Nos. 5,625,077, 5,652,345, 5,679,803, 5,783,381, 6,022,964, 6,133,459, and 6,124,478. 
         [0037]    The chemiluminescent signals  102  emitted from the array pass through the first and second lenses  46  and  50 , where the chemiluminescent signals  102  are re-imaged by each lenses  46  and  50 . After passing through the lenses  46  and  50 , the chemiluminescent signals  102  are collected by image collecting device  58 . The collected image data is then transmitted to the computer based system, where the data is processed and analyzed. In various configurations, each feature may have more than one chemiluminescent marker hybridized with probes associated with the tile  42 . In which case, the first filter  54  would not be removed in order to filter out light emitted from one of the chemiluminescent markers of the features while allowing wavelengths of different chemiluminescent signals to pass and be imaged by the image collecting device  58 . The first filter  54  would then be removed and replaced with a different first filter  54  that would allow other chemiluminescent signals to be imaged. 
         [0038]    Referring now to both  FIGS. 3 and 4 , in various configurations, the filtered fluorescent signals  98  collected by image collecting device  58  are used to auto-focus the array of features for the image collecting device  58 , as described above in reference to  FIG. 1 , for example corrections for chromatic aberrations are made. Additionally, the filtered fluorescent signals  98  collected by image collecting device  58  are used for gridding the array of features. That is, the filtered fluorescent signals  98  are used to identify the location of each feature within the array. Furthermore, the filtered fluorescent signals  98  collected by image collecting device  58  are used to normalize the array. More specifically, the filtered fluorescent signals  98  are used to normalize the chemiluminescent signals  102  collected by the image collecting device  58 . 
         [0039]      FIG. 5  is a flow chart  200  representative of various method configurations for operating an imaging apparatus  10  for imaging an array of features. To begin, a user positions the tile  42  and associated array of features onto the stage  34 , as indicated at  202 . The controller instructs the transport  26  to move the stage  34  along the x-axis to a first position under the illuminator  38 , where the array is illuminated by the illuminator  38 , as indicated at  204 . Next the first filter  54  is positioned between the first and second lenses  46  and  50 , as indicated at  206 . The array is then auto-focused for the image collecting device  58  by moving the stage  34  along the z-axis, via the elevator  30 , as indicated at  208 . A normalizing image of the fluorescent signals  98  emitted by each feature in a first portion of the array is then collected, as indicated at  210 . Next, the first filter  54  is removed from between the first and second lenses  46  and  50 , and the illuminator  38  is turned off, as indicated at  212 , thereby providing a substantially light free environment for imaging the chemiluminescent signals emitted by each feature. Then an image of the chemiluminescent signals  102  emitted by each feature in the first portion of the array is collected by the image collecting device  58 , as indicated at  214 . 
         [0040]    Next, in various configurations, depending on the size of the array, the stage  30  is moved to a second position under the illuminator  38 , first filter  54  is re-positioned between the lenses  46  and  50 , and the illuminator  38  is turned on, as indicated at  216 . Then, a second auto-focus procedure is performed, a normalizing fluorescent image of a second portion of the array is collected, the first filter  54  is again removed from between the first and second lenses  46  and  50 , and illuminator  38  is again turned off, as indicated at  218 . An image of the chemiluminescent signals  102  emitted by each feature in the second portion of the array is then collected by the imaging device  58 , as indicated at  220 . This process is repeated, as needed, until images of the chemiluminescent signals  102  for the entire array have been collected, as indicated at  222 . 
         [0041]    Thus, the imaging apparatus of the present invention automatically acquires multiple images of an array of fluorescent/chemiluminescent co-hybridized features, thereby acquiring image data for the entire array using a single apparatus. Additionally, the present invention allows better alignment between the fluorescent and the chemiluminescent image data because the optics are the same for the collection in both channels. Furthermore, the illuminator substantially evenly distributes excitation light over the entire array, thereby providing more consistent image data for multiple images across the entire array. 
         [0042]    While the invention has been described in terms of various configurations, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the claims.