Microarray dispensing with real-time verification and inspection

[0001] The present invention relates to automated microarray dispensing devices, more specifically it relates to automated microarray dispensing devices with automated quality inspection capability. This application is a continuation in part application of U.S. patent application Ser. No. 09/611,256 filed Jul. 6, 2000, now U.S. Pat. No. 6,558,623, which is incorporated by reference herein.

[0002] Microarrays, also known as biochips, have recently become important in the study of genomics. The use of a microarray involves laying down an ordered array of genetic elements onto a solid substrate such as a slide. Depending on the application, a microarray may consist of genomic DNA, reverse-transcribed cDNA, or smaller chains of oligonucleotides as well as any preparatory substrates. The microarray is useful because it allows genetic analysis to take place on a massively parallel scale, wherein thousands of genes and markers can be scored in one experiment.

[0003] A microarrayer, also known as a DNA array printer, is a high-capacity system used to print a microarray onto slides. Typically, a microarrayer is a specially built robotic platform designed to transfer solutions from the well of some type of microplate onto another surface for analysis. This process of depositing the liquid spot onto the slide is known as “spotting”.

[0004] Recently, microarrayers have become extremely popular in laboratories because they add to the efficient productivity of the laboratory to be able to print samples onto slides accurately and rapidly. Affymetrix, Inc., with offices in Santa Clara, California, makes an automated arrayer called the 417 ARRAYER (Part No. 0-0001 and Part No. 0-0009). BioRobotics, with offices in Boston, Massachusetts, produces two versions of an automated arrayer called the MICROGRID and MICROGRID II. GeneMachines, with offices in Menlo Park, Calif. makes an arrayer called the OMNIGRID (Model No. OGR-02). Packard Instrument Company with offices in Meriden, Conn. makes an automated arrayer called the BIOCHIP ARRAYER.

[0005] Although there are some differences between each of the above listed microarrayers, they are all similar in that they each spot microarrays in an automated fashion. However, there are significant problems with the prior art devices that detracts from their efficient operation.

[0006] A first problem arises due to the fact that as blank slides are cycled through prior art microarrayers, they can become askew or positioned improperly underneath dispensing tips. This problem results in spots being positioned improperly on the slides. A second problem can arise even if the slide is positioned correctly under the dispensing tips. It is possible for the spot to be deposited in the correct position, but be of poor quality and therefore useless as far as experimentation purposes.

[0007] Up to now, the only way to deal with these problems was to have a human operator visually monitor and inspect the microarrayer during its operation or inspect the samples after they come off the machine. This solution is an unacceptable waste of human effort. The BIOCHIP ARRAYER made by Packard Instrument Company has attempted to deal with the problem of monitoring the spotting process. However, it has only limited verification functionality with its integrated camera. This means that it verifies whether or not a spot has been dispensed, without any quality inspection to analyze whether that spot was good or bad.

[0008] What is needed is a better microarrayer with automated quality inspection capability.

[0009] The present invention provides a microarrayer for spotting solution onto a receiving surface in an automated microarray dispensing device. Elements of the present invention include: at least one dispense head for spotting the receiving surface, at least one light source capable of illuminating the receiving surface, at least one camera operating in conjunction with the at least one light source. The at least one camera is capable of acquiring and transmitting surface image data to a computer. The computer is programmed to receive the surface image data and analyze it. The computer will then generate post analysis data based on the analysis of the surface image data. The post analysis data is available for improving the spotting of the solution onto the receiving surface. In a preferred embodiment, the surface image data includes information relating to receiving surface alignment, information relating to spot quality, and receiving surface identification information. In a preferred embodiment, the analysis of the information relating to receiving surface alignment enables the computer to make automatic adjustments to the relative positions of the at least one dispense head and the receiving surface to increase the accuracy of the spotting. In a preferred embodiment, the analysis of the information relating to spot quality identifies a spot as pass or fail. An operator is then able to rework the spot. In a preferred embodiment, the analysis of the receiving surface identification information enables the computer to track each receiving surface. In a preferred embodiment the receiving surface is a plurality of slides.

[0042] A detailed description of a preferred embodiment of the present invention can be described by reference to FIGS. 1–38B. During the operation of the present invention, solution from reservoir plate 5 is automatically deposited onto an array of fifty blank slides 4A1–4E10 located on locating plates 4A–4E (see FIG. 1). An operator is able to select via a computer interface whether dispense tip 42 (located underneath dispense head 40) or a 4×6 array of dispense tips 7 (located underneath dispense head 6) will be used to make the deposits onto slides 4A1–4E10. In the preferred embodiment, dispense tips 7 and 42 are quill type dispense tips. Locating plates 4A–4E are mounted on linear actuator 15 so that they can move along the x-axis. Linear actuator 26 is mounted on linear actuator 21 so that linear actuator 26 can move along the y-axis. Dispense heads 6 and 40 are mounted on linear actuator 26 so that they can move along the z-axis. Camera 12 with strobe light 13 is focused so as to permit recording of the deposition process and functions to permit verification of slide identification information and to permit verification of proper deposition of solution on the slides. Periodically, during the cycle, the dispense tips are cleaned in sonic cleaner 9, rinsed in rinse fountain 10, and then dried in vacuum manifold 11. After the solution has been deposited onto the slides, the operator can retrieve locating plates 4A–4E containing slides 4A1–4E10 from the position shown in FIG. 2.

[0043] FIGS. 3–32 illustrate the sequence of operation of a preferred embodiment of the present invention.

[0044] In a preferred embodiment of the present invention, the operation of the components is controlled by PC control system 300, as shown in FIG. 37. FIGS. 38A–38E show a flowchart representing preferred programming of PC control system 300 and corresponds to the sequence illustrated in FIGS. 3–32.

[0045] As shown in FIG. 3, an operator places five locating plates 4A–4E each having ten clean, blank slides 4A1–4E10 on platform 2. In a preferred embodiment of the present invention, slides 4A1–4E10 are made by Sequonem with offices in San Diego, Calif. A preferred slide is shown in FIG. 16B. It has ninety-six etched dispense positions 60 and has its own unique 2D bar code 65 for identification purposes.

[0046] As shown in FIG. 4, linear actuator 15 moves platform 2 so that slide 4A1 is underneath the dispense head 6. Dispense head 6 is positioned directly above slide 4A1. Using camera 12 and the strobe light 13, an image is acquired of slide 4A1. The camera reads the bar code and inspects the positioning and alignment of slide 4A1 on locating plate 3A. The software then analyzes the position data and stores the information. The information stored and will be used later to adjust the positions of slide 4A1 and dispense head 6 to ensure accurate placement of the solution on the slide.

[0047] After camera 12 has acquired the image of slide 4A1, linear actuator 26 is moved via linear actuator 21 to the position shown in FIG. 5 so that dispense head 6 is directly above slide 4A2. Using camera 12 and the strobe light 13, an image is acquired of slide 4A2. As with slide 4A1, camera reads the bar code and inspects the positioning and alignment of slide 4A2 on locating plate 4A. The software then analyzes the position data and stores the information. The information stored and will be used later to adjust the positions of slide 4A2 and dispense head 6 to ensure accurate placement of the solution on the slide.

[0048] Linear actuator 26 is then moved via linear actuator 21 to the position shown in FIG. 6 so that dispense head 6 is directly above sonic cleaner 9.

[0049] As shown in FIG. 7, mounting plate 25 moves downward via linear actuator 26 so that dispense tips 7 are dipped in sonic cleaner 9 for a programmable time period while the cleaner is turned on. When finished, mounting plate 25 moves upward as shown in FIG. 8.

[0050] Then, as shown in FIG. 9, platform 31 moves to the left via pneumatic slide 30, thereby moving rinse fountain 10 and vacuum manifold 11 to the left. Linear actuator 26 is moved via linear actuator 21 back so that it is directly above rinse fountain 10.

[0051] As shown in FIG. 10, mounting plate 25 moves downward via linear actuator 26 so that tips 7 are dipped in rinse fountain 10 for a programmable time period while the fountain is turned on.

[0052] After rinsing, mounting plate 25 moves upward via linear actuator 26, as shown in FIG. 11. Platform 31 moves to the right via pneumatic slide 30, thereby moving rinse fountain 10 and vacuum manifold 1 to the right.

[0053] As shown in FIG. 12, dispense tips 7 are then lowered into the vacuum manifold 11 via linear actuator 26 and the vacuum is turned on for a programmable time period, thereby drying dispense tips 7. This cleaning cycle can be set by the user, via the computer interface, to be repeated as many times as necessary.

[0054] Mounting plate 25 is then raised via linear actuator 26 as shown in FIG. 13. Linear actuator 26 is then moved via linear actuator 21 so that dispense head 6 is directly above reservoir plate 5, as shown in FIG. 14.

[0055] Mounting plate 25 is then lowered via linear actuator 26 so that tips 7 are dipped into the solution contained in reservoir plate 5, as shown in FIG. 15. While in reservoir plate 5, dispense tips 7 pick up some of the solution to be dispensed.

[0056] Linear actuator 26 then moves to the first dispense position shown in FIG. 16A so that dispense head 6 is above slide 4A1. Based on the earlier positioning data regarding slide 4A1 (see discussion of FIG. 4), linear actuator 21 makes minute positioning adjustments to linear actuator 26 and linear actuator 15 makes minute positioning adjustments to platform 2 in order to accurately position dispense head 6 over slide 4A1 at the first dispense position.

[0057] FIG. 16B shows a top view of a blank slide 4A1. In this preferred embodiment, slide 4A1 has 96 positions 60 arranged in an 8×12 array. At each position 60, slide 4A1 is etched so as to be better able to retain a drop of solution deposited at the spot.

[0058] Dispense head 6 is then lowered via linear actuator 26 so that tips 7 are in contact with slide 4A1 at the first dispense position, as shown in FIG. 17A. As tips 7 contact slide 4A1, spots 62 are placed on slide 4A1 via surface tension, as shown in FIG. 17B.

[0059] Dispense head 6 is raised via linear actuator 26, as shown in FIG. 18. Based on the earlier positioning data regarding slide 4A1, linear actuator 21 makes minute positioning adjustments to linear actuator 26 and linear actuator 15 makes minute positioning adjustments to platform 2 in order to accurately position dispense head 6 over slide 4A1 at the second dispense position.

[0060] Dispense head 6 is then lowered via linear actuator 26 so that tips 7 are in contact with slide 4A1 at the second dispense position, as shown in FIG. 19A. As tips 7 contact slide 4A1, more spots 62 are added to slide 4A1, as shown in FIG. 19B.

[0061] Dispense head 6 is raised via linear actuator 26, as shown in FIG. 20. Based on the earlier positioning data regarding slide 4A1, linear actuator 21 makes minute positioning adjustments to linear actuator 26 and linear actuator 15 makes minute positioning adjustments to platform 2 in order to accurately position dispense head 6 over slide 4A1 at the third dispense position.

[0062] Dispense head 6 is then lowered via linear actuator 26 so that tips 7 are in contact with slide 4A1 at the third dispense position, as shown in FIG. 21A. As tips 7 contact slide 4A1, more liquid spots 62, are added to slide 4A1, as shown in FIG. 21B.

[0063] Dispense head 6 is raised via linear actuator 26, as shown in FIG. 22. Based on the earlier positioning data regarding slide 4A1, linear actuator 21 makes minute positioning adjustments to linear actuator 26 and linear actuator 15 makes minute positioning adjustments to platform 2 in order to accurately position dispense head 6 over slide 4A1 at the fourth dispense position.

[0064] Dispense head 6 is then lowered via linear actuator 26 so that tips 7 are in contact with slide 4A1 at the fourth dispense position, as shown in FIG. 23A. As tips 7 contact slide 4A1, more liquid spots 62 are added to slide 4A1, as shown in FIG. 23B.

[0065] Dispense head 6 is raised via linear actuator 26, as shown in FIG. 24. Camera 12 and strobe 13 scans slide 4A1 and acquires images and inspects for spot quality. It is at this point that PC control system 300 (FIG. 37) identifies slide 4A1 as pass or fail. (Preferred computer controlled techniques for making this determination are discussed in a following section.)

[0066] As shown in FIG. 25, based on the earlier positioning data regarding slide 4A2 (see discussion regarding FIG. 5), linear actuator 21 makes positioning adjustments to linear actuator 26 and linear actuator 15 makes positioning adjustments to platform 2 in order to accurately position dispense head 6 over slide 4A2 at the first dispense position for slide 4A2.

[0067] The four-stage liquid dispense cycle (explained above with respect to slide 4A1 in discussion regarding FIGS. 17A–24) is repeated so that at the end of the four-stage cycle, slide 4A2 contains spots 62, as shown in FIG. 26B. At the end of the four stage cycle, dispense head 6 is raised via linear actuator 26, as shown in FIG. 26A. Camera 12 and strobe 13 scans slide 4A2 and acquires images and inspects for spot quality. It is at this point that the control system identifies slide 4A2 as pass or fail.

[0068] As shown in FIG. 27, linear actuator 21 moves linear actuator 26 so that dispense head 6 is above slide 4A3.

[0069] The sequence outlined in the discussion regarding slides 4A1 and 4A2 (depicted in FIGS. 4–26 ) is repeated with regards to slides 4A3 and 4A4. To summarize, by utilizing light provided by strobe 13, camera 12 will first record the positions of slides 4A3 and 4A4. Then, dispense tips 7 are dipped in sonic cleaner 9. Dispense tips 7 are then rinsed in rinse fountain 10. Then, dispense tips 7 are dried in vacuum manifold 11. This cycle is repeated as needed. Then, liquid is picked up by dispense tips 7 when dispense tips 7 are lowered into reservoir plate 5. Then, liquid is spotted onto slide 4A3 by dispense tips 7 in a four-stage liquid dispense cycle. Likewise, liquid is spotted onto slide 4A4 in a four-stage liquid dispense cycle so that liquid has been spotted on both slides, as shown in FIGS. 28B and 28C.

[0070] The process is then repeated for the remaining six slides 4A5–4A10 until all the slides on locator plate 4A have been spotted, as shown in FIG. 29.

[0071] Linear actuator 15 then moves platform 2 so that slide 4B1 of locator plate 4B underneath dispense head 6, as shown in FIG. 30.

[0072] In a similar fashion, dispense tips 7 continue to spot all ten slides on locator plates 4B–4E, until the last slide 4E10 has been spotted, a

[0073] After slide 4E10 has been spotted and camera 12 and strobe 13 has scanned slide 4E10 for spot quality, linear actuator 15 moves platform 2 to the position shown in FIG. 32 so an operator can remove locator plates 4A–4E.

[0074] The computer controlled pass-fail determination technique determines individual spots as pass or fail based on several criteria. For each slide, the camera system scans a region to look for a spot. In a preferred embodiment the criteria that are applied to that inspection region are spot presence, spot size in area, spot location, and spot geometry. Additional criteria can be added through software configuration. Each of the criteria can have upper and lower limits designated which define the acceptable values for that particular criteria. Any value that falls outside of the limits for any criteria qualifies that spot and slide as failed. The actual inspection values are determined by analyzing the grayscale intensity of each pixel. The total number of pixels falling above and below a threshold are tallied to give values for each of the inspection criteria.

[0075] As explained above, after each slide has been spotted, camera 12 and strobe 13 scans the slide and acquires images and inspects for spot quality. It is at this point that the control system identifies the slide as pass or fail. In a preferred embodiment of the present invention, an operator monitoring the spotting process via monitor 305 (FIG. 37) has the option of correcting a slide that has failed.

[0076] For example, FIG. 29 shows locating plate 4A after all slides 4A1–4A10 have been spotted. At this point, an operator can scan locating plate 4A. A good plate shows up green as in all slides pass. A plate with at least one bad spot on one of the slides shows up red. The user can then zoom in on the bad slide and the good and bad spots show up green and red respectively as pass or fail. From there, the user can decide whether or not to rework the bad spots.

[0077] FIGS. 39A and 39B show where an operator has decided to rework slide 4A6 that has a spot that has failed quality inspection. Dispense head 40 is lowered via pneumatic slide 41 so that dispense tip 42 is lower than dispense tips 7. Solution from reservoir plate 5 is then deposited on the slide at the location of the failed spot. If there are other spots that failed, the operator can likewise rework those spots in a similar fashion.

[0078] Although in the description given above regarding the rework process, the operator reworked failed slides after locating plate 4A had been entirely spotted, it is also possible to rework failed slides at other stages during the spotting process. For example, it may be desirable to wait until all slides 4A1–4E10 on plates 4A–4E have been spotted (FIG. 31) before reworking them. This allows an operator to be free to do other activities while the initial spotting is taking place. Then, after all slides have been spotted, he can come back and do all the reworking at one sitting.

[0079] Alternatively, it may be desirable to rework each slide immediately after it has been spotted, as shown in FIG. 24.

[0080] The previous section described a preferred embodiment where an operator can decide whether or not to rework a spot based on a computer determination of pass or fail. In another preferred embodiment the rework decision is made automatically by the computer based on whether or not the spot has passed or failed. In this preferred embodiment, the computer makes a determination whether or not a spot has passed or failed using the computer controlled pass-fail determination technique earlier described. If, based on its analysis, the computer determines that the spot has failed, the computer will automatically take steps to rework the spot. For example, dispense tip 42 will extract solution from reservoir plate 5. Then, the computer will lower dispense head 40 via pneumatic slide 41 so that dispense tip 42 is lower than dispense tips 7, as shown in FIGS. 39A and 39B. Solution from reservoir plate 5 will then deposited on the slide at the location of the failed spot.

[0081] In a preferred embodiment, linear actuators 26, 21 and 15 are industrial grade precision ground ball screw linear actuators, as shown in FIG. 33. These linear actuators are manufactured by Parker Automation (Model #s:404XR and 406XR series). They are each controlled by a smart servomotor (SmartMotor, Model #2320 VRE, manufactured by Animatics, with offices in Santa Clara Calif.), which is a fully self-contained closed loop servo system. Each of these smart servomotors contains the motor, encoder, amplifier, and controller all in one small package mounted to the linear actuator. Linear actuator 15 (the x-axis positioning device) has an overall travel distance of 600 mm with an accuracy within +/−0.032 micrometers Linear actuator 21 (the y-axis positioning device) has an overall travel distance of 400 mm with an accuracy within +/−0.032 micrometers. Linear actuator 26 (the z-axis positioning device) has an overall travel distance of 100 mm with an accuracy within +/− micrometers. In this preferred embodiment, this extreme accuracy is needed to accommodate very small spot size and spacing between spots. The linear actuators have pitches of 5mm per revolution giving a positioning accuracy of 0.032×10⁻⁶ meters.

[0082] Linear actuator 15 controls the positioning of platform 2 containing slides 4A1–4E10 along the x-axis of motion making all slides presentable to the dispense head 6. Linear actuator 21 controls the positioning of the dispense head along the y-axis of motion making all slides 4A1–4E10, sonic cleaner 9, rinse fountain 10, vacuum manifold 11, and reservoir plate 5 presentable to dispense head 6. Linear actuator 26 controls the positioning of dispense head 6 along the z-axis of motion allowing dispense head 6 to be lowered to and raised from all slides 4A1–4E10, sonic cleaner 9, rinse fountain 10, vacuum manifold 11, and reservoir plate 5.

[0083] Cleaning station 33 consists of sonic cleaner 9, rinsing fountain 10, and a drying vacuum manifold 11. In a preferred embodiment, sonic cleaner 9 is an ultrasonic cleaner manufactured by Prosonic, Inc. (part no. E0028). Sonic cleaner 9 can contain either a cleaning solution or simply purified water. Dispense tips 7 are dipped in the sonic cleaner 9, where the ultra sonic oscillations of the cleaning solution clean the tips.

[0084] Rinsing fountain 10 and the vacuum manifold 11 are placed on a pneumatic slide 30. Pneumatic slide 30 is used to select which operation is to be performed, rinsing or drying. The reason for this slide is so that both operations can be performed at a single position along the y-axis. This allows for both operations without having to increase the overall travel of linear actuator 26 along the y-axis.

[0085] Rinsing fountain 10 pumps in purified water and drains it out to a waste bin. Dispense tips 7 are dipped in this purified water to rinse away any debris or cleaning solution that may remain on the tips after cleaning.

[0086] Drying vacuum manifold 11 is a block with an array of holes in it that match the array of dispense tips 7. The tips are inserted into the block, each of these holes are connected to a manifold which is connected to a vacuum generator and air supply. The vacuum pulls away any remaining liquid or debris left on the dispense tips after rinsing.

[0087] As shown in FIG. 35B, dispense head 6 is a 4×6 grid Micro Quill Holder (part no. 11946-0) made by Major Precision of Arizona. A 4×6 array of primary dispense tips 7 are held in dispense head 6. As shown in FIG. 35A, dispense head 40 is a Micro Quill Holder also made by Major Precision. Dispense tip 42 is held in dispense head 40. Dispense tips 7 and 42 are spring loaded within the dispense heads 6 and 40.

[0088] As shown in FIGS. 1, 34A and 34B, dispense head 6 is rigidly mounted to mounting plate 25, whereas dispense head 40 is mounted to pneumatic slide 41 (manufactured by Robohand, Inc., with offices in Montroe, Conn., part no. MPS1-2). Mounting plate 25 is capable of moving up and down along the z-axis via linear actuator 26. Furthermore, dispense head 40 is capable of independent additional movement up and down along the z-axis via pneumatic slide 41, as shown in FIGS. 34A–34B.

[0089] During normal operation, such as that depicted in the sequence illustrated in FIGS. 3–32, dispense head 6 is used to spot slides 4A1–4E10. F front view of dispense tips 7 in contact with slide 4A1. Dispense tips 7 will be used to spot slide 4A1 at positions 60, as shown in FIG. 34C. Note that when dispense head 6 has been selected, dispense head 40 is raised via pneumatic slide 41 so that dispense tips 42 do not interfere with the spotting process.

[0090] If, however, the operator wishes to spot slide 4A1 at positions 61 (FIG. 34C), dispense head 40 will be lowered via pneumatic slide 41 so that dispense tips 42 are in contact with slide 4A1 and dispense tips 7 are out of the way, as shown in FIG. 34B.

[0091] In a preferred embodiment, camera 12 and strobe 13 are mounted to the side of linear actuator 26 as shown in FIG. 1. Camera 12 is a self-contained camera with image processing and Ethernet capabilities manufactured by DVT Corporation with offices in Norcross, Ga. (series 600 model). Using light provided by strobe 13, camera 12 can snap pictures while in dynamic motion, process the image for results, pass the results off to the PC control system, and prepare for the next image acquisition. The camera uses a 55 mm Telecentric lens which provides the proper field of view and magnification for reading of 2D bar code 62 (FIG. 34C) and for image inspection. Strobe light 13 is preferably Model DL2449, manufactured by Advanced Illumination, with offices in Rochester, Vt. In the preferred embodiment, the image acquisition time is ˜40 ms and the image processing time is ˜50 ms. The system can also be equipped with a flouresence device along with the camera for further genomic expression analysis.

[0092] As shown in FIG. 36, vibration isolated base 80 is provided to minimize any possible affects that high frequency environmental vibrations might have on the dispensing process. This base is a pneumatic system which acts as a shock absorber to the system.

[0093] In a preferred embodiment, the base is manufactured by Newport, Inc. with offices in Irvine, CA, model # CM-225.

[0094] FIG. 37 depicts a block diagram of PC control system 300 and other components of a preferred embodiment of the present invention. PC Control System 300 includes CPU 301 with associated memory (RAM 302 and ROM 303). It also includes a touch screen monitor/interface 305 that allows for operator monitoring, intervention and control of the present invention. In the preferred embodiment, the computer system is a PC based computer equipped with an ethernet card and running windows software. The programming is preferably written in VISUAL BASIC. (VISUAL BASIC is a federally registered trademark of Microsoft Corp., a Delaware Corporation) PC Control System 300 is equipped with CMS (Central Monitoring System). The CMS gives PC Control System 300 it's own IP (Internet Protocol) address and ethernet connectivity. This allows for remote monitoring and control via Intranets as well as Internet provided that the bandwith is available for proper functionality. The software is highly comfigurable to allow increased flexibility for customers with varying slide types, slide sizes, slide orientations, spot size, spot spacing and many other variables.

[0095] As previously stated, linear actuators 26, 21 and 15 are industrial grade precision ground ball screw linear actuators. As shown in FIG. 37, PC control system 300 sends signals to smart servomotors 26A, 21A and 15A to control linear actuators 26, 21 and 15, respectively. PC control system 300 controls sonic cleaner 9 and rinse fountain 10. Compressed air source 310 provides compressed air to pneumatic slides 30 and 41 via valves 310 controlled by PC control system 300. Vacuum generator 320 provides a vacuum to vacuum manifold 11 via valve 317. As previously explained camera 12 and strobe 13 work in conjunction to provide sensory data to PC control system 300. This input is used to accurately position the dispense heads over the slides to ensure optimum spotting and to verify the quality of the spotting as “pass” or “fail” using multiple criteria as to placement at intended location as well as spot size (too big or too small).

[0096] A second preferred embodiment of the present invention is shown in FIG. 41. In the second preferred embodiment, dispense head 106 is connected via mounting plate 125 to linear actuator 126 so that dispense tips 107 can be raised and lowered along the z-axis into solution in microplate 190. Camera 112 and strobe 113 are rigidly mounted to the side of linear actuator 126 so that they remain stationary with respect to the side of linear actuator 126 along the z-axis. Linear actuator 126 is mounted to linear actuator 121 so that it can move along the y-axis.

[0097] Platform 182 is mounted to linear actuator 180 so that it can move along the x-axis. Locating plate 4A is placed on top of platform 182. Platform 102 is mounted to linear actuator 115 so that it can move along the x-axis. Microplate 190 is place on top of platform 102. In this preferred embodiment platform 102 has the capacity to hold ten microplates 190.

[0098] Solution in microplate 190 is removed via dispense tips 107. Linear actuator 126 then moves along the y-axis so that dispense tips 107 are above locating plate 4A. The solution is then spotted in a fashion similar to that described for the earlier preferred embodiments. Camera 112 with strobe 113 is focused so as to permit recording of the deposition process and functions to permit verification of slide identification information, permit verification of proper deposition of solution on the slides, and to verify slide alignment. As explained above, slide image data is transferred via camera 112 to a PC control system where the data is analyzed. The results of the analysis are then available for improving the spotting of the solution onto the slides. For example, spots that have failed to meet the threshold limits can be reworked. Also, the computer can automatically make adjustments to the relative positions of the slides and dispense tips based on the slide alignment analysis. Periodically, during the cycle, the dispense tips are cleaned in sonic cleaner 109, then rinsed in the rinse fountain and dried in vacuum manifold 111. Operation of the First Preferred Embodiment with the Second Preferred Embodiment

[0099] The first preferred embodiment (described in the sequence illustrated in FIGS. 3–32) can be used in conjunction with the second preferred embodiment to spot slides. For example as shown in FIG. 32, locating plate 4A can be removed from the microarrayer via an operator after it has been spotted with a base solution. Locating plate 4A can be transferred to the microarrayer depicted in FIG. 41. It can be placed on platform 180. DNA from microplate 190 can then be spotted on top of the base solution spotted already on slides 4A1–4A10.

[0100] Although the present invention was described as being used with the preferred microarrayer depicted in the sequence described by reference to FIGS. 3–32, those of ordinary skill in the art will recognize that it is possible to use camera 12, strobe 13 and a PC control system in conjunction with a variety of microarrayer designs. For example, in the background section of this application, several microarrayers were mentioned. It would be possible to one of ordinary skill in the art to modify a prior art automatic microarrayer to include camera 12 and strobe 13. Camera 12 and strobe 13 would then work in conjunction to provide sensory data to PC control system 300, as described above. Also, as explained above, the input would be used to accurately position the dispense heads over the slides to ensure optimum spotting and to verify the quality of the spotting as “pass” or “fail”.

[0101] The previous embodiments showed one dispense tip 42 extending downward from dispense head 40. It was explained how the single dispense tip 42 is used for reworking (correcting) defective spots. It is possible, however, to modify dispense head 40 so that multiple dispense tips can extend downward from dispense head 40. A preferred embodiment is shown in FIG. 40 in which five dispense tips 42A–E extend down below dispense head 40. In this preferred embodiment, dispense tips 42A–E are retractably connected to dispense head 40. As shown in FIG. 40, dispense tips 42A–D are retracted inside dispense head 40. The rightmost dispense tip 42E is extended below the other dispense tips and is spotting slide 4A1. In a preferred embodiment, dispense tips 42A–E are mounted to a pneumatic slides 43.

[0102] An advantage of this embodiment is that each dispense tip 42 can be configured to dispense a different volume of solution. For example, in a preferred embodiment, dispense tip 42A would dispense 1 nL of solution, dispense tip 42B would dispense 2 nL of solution, dispense tip 42C would dispense 4 nL of solution, dispense tip 42D would dispense 8 nL of solution, and dispense tip 42E would dispense 16 nL of solution.

[0103] After initially spotting the slides as explained above, camera 12 and strobe 13 would work in conjunction to provide sensory data to PC control system 300 reporting the quality of the spots. The spots would then be classified as pass or fail. If a spot has failed, the software in conjunction with PC control system 300 would determine the amount of solution required to correct the failed spot. Then, during the reworking sequence, the dispense tip that dispenses the most correct volume would be extended down from dispense head 40 and the other dispense tips would be retracted upward inside dispense head 40, as shown in FIG. 40.

[0104] A third preferred embodiment of the present invention is seen by reference to FIG. 42. FIG. 42 shows a perspective view of microarrayer 250. In the third preferred embodiment, dispense head 202 is connected via mounting plate 213 to linear actuator 214 so that dispense tips 208 can be raised and lowered along the z-axis. Camera 215 and strobe 216 are also mounted to mounting plate 213 and can be raised and lowered along the z-axis by linear actuator 214. Linear actuator 214 is mounted to linear actuator 217 so that it can move along the y-axis.

[0105] Platform 218 is mounted to linear actuator 219 so that it can move along the x-axis. 140 slides 220(1)–220 (140) are mounted on top of platform 218. Microplates 204(A–C) and pre-spot slide holder 205 having pre-spot slide 206 are placed on top of platform 218.

[0106] To remove solution from microplate 204(A), linear actuator 219 moves platform 218 along the x-axis and linear actuator 217 moves linear actuator 214 along the y-axis so that dispense head 202 is positioned over microplate 204(A), as shown in FIG. 44. Linear actuator 214 then lowers dispense head 202 so that dispense tips 208 are dipped into the wells of microplate 204(A). Linear actuator then raises dispense head 202. Dispense tips 208 contain solution removed from microplate 204(A).

[0107] The third preferred embodiment includes pre-spot slide 206. Pre-spot slide 206 allows excess solution on the dispense tips to be removed prior to the solution being spotted onto slides 220(1)–220 (140). This is achieved by repeatedly pressing the dispense tips onto the pre-spot slide until the excess solution is removed.

[0108] To pre-spot solution onto pre-spot slide 206, linear actuator 217 moves linear actuator 214 along the y-axis so that dispense head 202 is positioned above pre-spot slide holder 205 having pre-spot slide 206, as shown in FIGS. 45A and 45B. Linear actuator 214 then lowers mounting plate 213. As previously stated, dispense head 202 is mounted to mounting plate 213. Linear actuator lowers mounting plate 213 with dispense head 202 until dispense tips 208 come into contact with the surface of pre-spot slide 206, as shown in FIG. 45C. The act of dispense tips 208 pressing against the surface of pre-spot slide 206 causes excess solution on the sides of the dispense tips to come off the dispense tips. The third preferred embodiment is programmed so that linear actuator 214 raises and lowers dispense head 202 in a repetitive fashion so that dispense tips 208 are pressed against pre-spot slide 206 repetitively until the desired amount of solution is removed. The amount of times dispense tips 208 should be pressed varies depending on the properties of the solution being deposited and can be modified by the user by inputting instructions into computer 280 (FIG. 42).

[0109] After the solution has been pre-spotted onto pre-spot slide 206, the solution can then spotted onto selected microplates 220(1)–220 (140) in a fashion similar to that described above in reference to the first and second preferred embodiments. For example, to spot solution onto slide 220(1), linear actuator 219 moves platform 218 along the x-axis and linear actuator 217 moves linear actuator 214 along the y-axis so that dispense head 202 is positioned over slide 220(1), as shown in FIG. 46. The solution is then spotted in a fashion similar to that described for the earlier preferred embodiments. Camera 215 with strobe 216 is focused so as to permit recording of the deposition process and functions to permit verification of slide identification information, permit verification of proper deposition of solution on the slides, and to verify slide alignment. As explained above, slide image data is transferred via camera 215 to a PC control system where the data is analyzed. The results of the analysis are then available for improving the spotting of the solution onto the slides. For example, spots that have failed to meet the threshold limits can be reworked. Also, the computer can automatically make adjustments to the relative positions of the slides and dispense tips based on the slide alignment analysis. Periodically, during the cycle, the dispense tips are cleaned in sonic cleaner 231, then rinsed in the rinse fountain 230 and dried in vacuum manifold 232.

[0110] FIG. 43 shows robotic plate loader 252 positioned between microarrayers 250. Microarrayers 250 and robotic plate loader 252 are controlled by programmable computer 280.

[0111] As shown in FIG. 43, microplate staging area 254 has 4 microplate storage racks 255(A)–255(D). Each storage rack is capable of holding up to 15 microplates. Robotic plate loader 252 is capable of removing the microplates from each of the storage racks. Robotic plate loader 252 can pivot so that arm 257 can be extended towards a desired storage rack. Robotic gripper 256 can then grip the desired plate and remove it from the storage rack. The microplate can then be placed on either of the platforms 218(A)–(B) (as shown) or at pre-staging area 258. Microplate lids 259 can then be removed and placed at lid holding areas 260 and 261.

[0112] By storing microplates in an incubator, the temperature of the solution inside the microplates can be kept at a controlled level. FIGS. 47 and 48 show another preferred embodiment in which microplates stored in incubator 270 are automatically loaded via robotic arm 271 onto platform 218. FIG. 48 shows a top view and FIG. 47 shows a perspective view of incubator 270.

[0113] In FIG. 48, incubator access door 278 has been raised permitting outside access to incubator 270. Fifteen microplates are stored inside storage racks 274(A)–274(D). Robotic pivot arm 275 of robot 272 inside incubator 270 is capable of pivoting about axis 273 so that it can reach storage racks 274(A)–274(D).

[0114] FIG. 48 shows robotic pivot arm 275 in dotted line positioned so that gripper 276 is in front of storage rack 274(A). Robotic arm 275 is shown in solid line in a vertical position placing microplate 274(A)(13) on access plate 277.

[0115] In FIG. 47, robotic arm 271 has maneuvered gripper 279 so that it was able to remove microplate 274(A)(13) from access plate 277, rotate it 90 degrees, and place it on platform 218 as shown.

[0116] In the third preferred embodiment, microarrayer 250 can be programmed to remove solution from multiple microplates and spot the solution onto multiple slides 220 (1–140). FIG. 52A shows a top view of platform 218. Microplates 204(A), 204(B) and 204(C) are on platform 218. Microplates 204(D–O) are in storage rack 255(A). Microplates 285(A–O) are in storage rack 255(B). Microplates 286(A–O) are in storage rack 255(C) and microplates 287(A–O) are in storage rack 255(D), as shown in FIG. 52AB.

[0117] Robotic plate loader 252 is positioned between platform 218 and storage racks 255(A–C) and is capable of accessing the positions occupied by microplates 204(A-C) and any of the microplate storage positions in storage racks 255(A–D). Dispense head 202 (see also FIG. 42) is capable of being positioned over microplates 204(A–C) via microarrayer 250 and is capable of removing solution from microplates 204(A–C) and spotting that solution on any of the slides 220(1–140). Robotic plate loader 252 can move microplates 204(A–C) and store them back in storage rack 255(A) and then place other microplates onto platform 218. For example, a microarrayer 250 could be programmed to place microplates 204(A–C) back into storage rack 255(A) and then place microplates 285(A), 286(D), and 287(H) onto platform 218.

[0118] In the preferred embodiment, a user of microarrayer 250 (FIG. 42) is able to customize the manner in which spots are deposited on slides 220(1–140).

[0119] FIG. 49A shows a top view of 384 well microplate 204A. In the preferred embodiment, microplate 204A is divided into 8 sections 204A1–204A8, as shown in FIG. 49B. Each section 204A1–204A8 covers a 4×12 array of wells. In one preferred embodiment, a unique solution is deposited in each section 204A1–204A8.

[0120] FIG. 50 shows a top view of 4×12 pin dispense head 202 next to microplate 204(A). Dispense tips P1–P48 are shown as “+” signs and are arranged as shown. The 48 dispense tips on dispense head 202 are spaced to fit into the wells of each 4×12 well section A1–A8 of microplate 204A (FIGS. 51 and 49B). FIG. 49C shows dispense head 202 positioned over section 204A2 and FIG. 49D shows dispense head 202 positioned over section 204A5.

[0121] FIG. 53 shows a top view of slide 220(1). Dispense head 202 having dispense tips P1–P48 dispenses the spots onto slide 220(1). Each dispense tip P1–P48 is responsible for dispensing the spots in its own 8×8 array. For example, dispense tip PI dispenses all the spots in array 283(1) and dispense tip P4 dispenses all the spots in array 283(4), (FIG. 53).

[0122] In the preferred embodiment, a user can program computer 280 so that dispense head 202 will remove solution from section A1–A8 of microplate 204A and deposit the solution in a customized fashion on slide 220(1).

[0123] For example, in a preferred embodiment a user will use computer 280 to control microarrayer 250 and will see on monitor 281 a screen image similar to that depicted in FIG. 54. At the upper left corner of the screen the user inputs the number of microplates for spotting. In the example shown, the user has indicated eight plates. Eight microplates will provide solution to fill one slide with 3072 spots. For example, as shown in FIG. 53 slide 220(1) holds forty-eight 8×8 arrays of spots. Each 8×8 array has 64 spots. Since there are 48 arrays on slide 220(1), there are a total of 48×64=3072 spots on slide 220(1).

[0124] Each microplate has eight 4×12 sections from which dispense head 202 can remove solution. Since there are eight microplates from which dispense head 202 can remove solution and each microplate has eight sections, there are a total of 8×8=64 sections from which solution can be removed. The total number of sections is displayed in the upper left corner of the screen in FIG. 54.

[0125] In mapping the forty-eight 8×8 arrays to be deposited on slide 220(1) it is only necessary for the user to focus on how one of the forty-eight arrays will appear. This is because each array 283(1–48) is identical to the other. For example, as shown in FIG. 53, while dispense tip P1 of dispense head 202 (FIG. 50) is depositing solution to position Al of array 283(1), dispense tip P2 and P28 are depositing solution from the same microplate section to positions A1 of array 283(2) and A1 of array 283(28), respectively. Moreover, while dispense tip P1 is depositing solution to position C6 of array 283(1), dispense tip P32 is depositing solution from the same microplate section to position C6 of array 283(32). Therefore, after all 64 spots have been deposited by P1 onto array 283(1), dispense tips P2–P48 have simultaneously deposited 64 spots to each array 283(2–48) for a total of 3072 spots.

[0126] To map an 8×8 array, the user selects from which plates he wants to remove solution by entering the plate numbers into boxes 300, as shown in FIG. 54. In the present example the user wants to remove solution from each section of microplates 204(A), 204(B), 204(C), 204(D), 285(C), 286(D), 286(H), and 287(B). To select which microplate he wants to first map, the user mouse clicks on one of the eight microplates at the bottom of the screen.

[0127] In FIG. 55, the user has selected microplate 204(A) to map. Microplate 204(A) appears in the upper center of the screen under the heading “Current Plate”. The user then mouse clicks on a section that he wants to map. In the present example the user has mouse clicked on section 204A1 under the “Current Plate” heading. This causes the 204A1 section to darken. The user then mouse clicks on position A1 of spot array map 301. The numerals 204A1 then appear in the Al position of the array to indicate that the A1 position has been mapped and section 204A1 of microplate 204(A) at the left bottom corner of the screen has darkened to indicate that the 204A1 section has been mapped.

[0128] In FIG. 56, the user has mouse clicked on section 204A2 under the “Current Plate” heading. This causes the 204A2 section to darken. Then the user mouse clicks on C4 of spot array map 301. The numerals 204A2 then appear in the C4 position of the array to indicate that the C4 position has been mapped and section 204A2 of microplate 204(A) at the left bottom corner of the screen has darkened to indicate that the 204A2 section has been mapped.

[0129] In a similar fashion, the user can map the rest of microplate 204(A) onto spot array map 301. FIG. 57 shows microplate 204(A) completely mapped onto spot array map 301.

[0130] The user can continue to map the rest of the microplates by following the above-described procedures with respect to each of the seven other microplates. For example, FIG. 58 shows spot map array 301 completely mapped.

[0131] After having filled in spot map array 301, the user saves his customized mapping and runs microarrayer 250 (FIG. 42). Microarrayer 250 will remove solution from microplates 204(A–C) first because they are positioned on platform 218 so that they can be accessed by dispense head 202. After solution has been removed from each of the eight sections of a microplate, robotic plate loader 252 (FIG. 52A) will return the microplate to its appropriate storage rack. The robotic plate loader will then place the next microplate at the position vacated by the previous microplate on platform 218. For example, after solution has been removed from microplate 204(A), robotic plate loader 252 will return microplate 204(A) to its slot in storage rack 255(A) (FIGS. 52A and 52B). Robotic plate loader 52 will then place microplate 204(D) at the spot vacated by microplate 204(A). Microarrayer will continue to spot slide 220(1) until it has been completed spotted, as shown in FIG. 53.

[0132] If the user would like to spot more slides with the pattern shown in spot map array 301 in FIG. 58, he inputs into computer 280 the number of slides he would like to spot. For example, if he would like to spot all of the slides 220(1–140) the user inputs that information into the computer and microarrayer 250 will spot slides 220(1–140). Each slide will be spotted in the same pattern as the other slide. For example, each slide will be spotted in accordance with the pattern shown in spot map array 301 in FIG. 58. Similarly, if the user just wants to spot slides 220(1–10), he inputs that information into computer 280 and slides 220(1–10) will be spotted in accordance with the pattern shown in spot map array 301 in FIG. 58.

[0133] As stated previously, camera 215 with strobe 216 is focused so as to permit recording of the deposition process and spot quality and functions to permit verification of slide identification information, permit verification of proper deposition of solution on the slides, and to verify slide alignment. Spot data covering the qualities of each spot on the slides is transferred via camera 215 to a computer 280 system where the data is analyzed and stored. In a preferred embodiment, this information is displayed in table format to the user via monitor 281.

[0134] FIG. 59 shows a preferred spot inspection data table. Information such as spot size and spot offset is reported. Status indicating the spot as either good or bad is reported as well as whether rework was required for the spot.

[0135] FIG. 60 shows a preferred table reporting spot inspection statistics. In this table, the user can quickly ascertain the quality of dispense tips P1–P48 on dispense head 202. For example, by referring to FIG. 60, the user can see that dispense tip P1 deposited 20 spots and they were all good spots and rework was not required. In contrast, the user can see that dispense tip P2 also deposited 20 spots but that only 15 of them were good. Rework was required for 5 spots. The user can then determine that dispense tip P1 is operating effectively, but that dispense tip P2 might need to be cleaned, repaired or replaced. Preferably, the table shown in FIG. 60 also reports information such as spot diameter statistics for each dispense tip and spot offset statistics for each dispense tip.

[0136] In a preferred embodiment, it is possible to export to another computer the data associated with the spotting process. For example, as discussed above under the headings “Flexible Mapping” and “Customized Mapping of the Arrays” the user can customize the mapping process in accordance with his wishes. The mapping information can then be saved by computer 280 and matched with the appropriate slide for later reference. For example, if slide 220(1) was mapped in accordance with spot map array 301 shown in FIG. 58, then an individual later working with slide 220(1) could refer to the saved mapping information and immediately know the source microplates for each spot on the slide. Likewise, it is also possible to save and export the spot inspection data and spot inspection statistics discussed above for later reference.

[0137] In one preferred embodiment, the saved information is exported via a computer network to another computer on the computer network. In another preferred embodiment, the saved information is saved to a computer disc and the computer disc is physically transferred to another computer where it can be accessed.

[0138] Preferably, microarrayer 250 (FIG. 42) includes calibration target 303 (see also FIGS. 52 and 61). As shown in FIG. 61, calibration target 303 includes array 304. Array 304 has a known dimension. Also, the size of the dots of the array is known as well as the distance between the dots. The color of calibration target 303 is also known. To calibrate camera 215, the user positions it over calibration target 303. Camera 215 is focused onto array 304 and measurements are taken. The measurements are compared to the known value. If there are discrepancies, then adjustments can be made to camera 215 to calibrate it. For example, if there is a discrepancy between the known values and the size of array 204, or the size of the dots, or the spacing of the dots, then zoom lens of the camera can be adjusted. Or, if there is a discrepancy between the known color of the calibration target and the reported color (i.e., too light or too dark), the iris or the aperture of the camera can be adjusted.

[0139] In the preferred embodiment, microarrayer 250 includes two vacuum stations 232 and two sonic cleaning stations 231 (FIG. 52A). By having two sets of stations, the user is better assured of thoroughly cleaning dispense tips P1–P48. Preferably, one sonic station 231 contains water and the other sonic station 231 contains alcohol.

[0140] In a preferred embodiment, the user is able to adjust parameters relating to spot quality via computer 280 (FIG. 42). On monitor 281, the user will see a screen image similar to that shown in FIG. 62. The user can adjust load parameters and dispense parameters by entering values into boxes 305. The definitions of the adjustable parameters are given below in Table 1:

[0141]

TABLE 1
Load Dwell	The amount of time the dispense tips are immersed in the
	well solution before lifting up.
Load Offset	The distance (in microns) from the bottom of the wells
	that the dispense tips stop while immersed in the well
	solution. (Ex: −500 microns means that the dispense tips
	stop 500 microns above the bottom of the well.)
Load Speed	The speed of the dispense tips as they move up and down
	over the microplate wells.
Load Repeat	The number of times the loading of the dispense tips is
	repeated as they are positioned over the wells.
Load Repeat	The amount of time in the up position before the loading
Delay	of the dispense tips is repeated.
Dispense/Load	The number of times the dispense tips dispense solution
	before they are loaded again.
Dispense	The amount of time the dispense tips are in contact with
Dwell	the slide before lifting up.
Dispense	The distance (in microns) the springs of the dispense tips
Offset	compress before lifting up.
Dispense	The speed of the dispense tips as they move up and down
Speed	over the slide.
Dispense	The number of times the dispensing of solution
Repeat	(i.e., spotting) is repeated as the dispense tips are
	positioned over the slide.
Disp Repeat	The amount of time in the up position before the
Delay	dispensing of solution is repeated.

[0142] While the above description contains many specifications, the reader should not construe these as limitations on the scope of the invention, but merely as exemplifications of preferred embodiments thereof. Those skilled in the art will envision many other possible variations are within its scope. For example, although the above sequence described a dispensing process utilizing slides that have 96 dispense positions, those of ordinary skill in the art will recognize that it is possible to use other slides as well. For example, 384 or 1536 position slides could be used. It is also possible to use a blank microscope slide with no pre-etched dispense positions. Accordingly, the location of the different dispense positions will vary depending on the type of slide being used. The spacing and orientation of the slide can be selected by an operator through the maintenance menu on the computer interface. Also, the previous embodiments disclosed using a strobe light to illuminate the slide below the camera. One of ordinary skill in the art will recognize that it is possible to illuminate the slide with other light sources besides a strobe light. For example, the slide could be illuminated with a camera flash, a constant bright light, or a fluorescence device, such as a fluorescent LED. Additionally, the slide could be illuminated from the side opposite the camera. If a fluorescence device is used to illuminate the slide, those of ordinary skill in the art will recognize that it is possible to add a fluorescent dye to the solution being spotted to achieve more in depth characterizations. For example, by using a fluorescent LED and adding fluorescent dye to the solution, greater volume determination can be achieved based on fluorescent intensity of the spot. Also, in the preferred embodiment, it was mentioned that dispense tips 7 and 42 were quill type dispense tips, it would be obvious to substitute other types of dispense tips. For example, piezo type dispense tips could also be used. The present invention was taught showing the microarrayer depositing solution onto a plurality of slides. One of ordinary skill in the art would recognize that the microarrayer could be used to deposit solution onto receiving surfaces that include a variety of slide types and surfaces other than slides. For example, the microarrayer could deposit solution onto a glass slide, or a slide whose surface has been treated to enhance the spotting or the experimental results, or slides that have porous or semi-porous surfaces, membrane slides, gold coated slides, slides made of porous silicon, or any other surface that may be beneficially printed upon. Additionally, while the present invention was taught using substantially flat slides, it would be obvious to use surfaces that are slightly to moderately curved to print upon. Accordingly the reader is requested to determine the scope of the invention by the appended claims and their legal equivalents, and not by the examples which have been given.