Patent Publication Number: US-6901819-B2

Title: Probe tip alignment for precision liquid handler

Description:
This a continuation of U.S. patent application Ser. No. 10/202,040, filed on Jul. 24, 2002 now U.S Pat. No. 6,666,065, which claims priority to U.S. application Ser. No. 09/769,569; filed on Jan. 24, 2001, now U.S. Pat. No. 6,474,181, the entire disclosures of which are incorporated herein by reference for all purposes. 

   FIELD OF THE INVENTION 
   The present invention relates to aligning the probe tips of a precision automated liquid handler. 
   DESCRIPTION OF THE PRIOR ART 
   In pharmaceutical, genomic and proteomic research and drug development laboratories, and other biotechnology applications, automated liquid handlers are used for handling laboratory samples in a variety of laboratory procedures. For example, liquid handlers are used for biotechnological and pharmaceutical liquid assay procedures, sample preparation, compound distribution, microarray manufacturing and the like. An automated liquid handler has a work bed that supports an array of sample receptacles. One-piece sample containing plates having an integral array of many sample containing receptacles or wells are widely used. The liquid handler has an array of multiple probes that are moved into alignment with one or more sample containing wells to carry out liquid handling operations such as adding liquid to the wells. 
   It is desirable to decrease the volumes of samples treated with automated liquid handlers. Sample containing plates with a footprint of about three and one-half by five inches and having an X-Y array of 96 wells in an eight by twelve well pattern have been widely used. In order to increase throughput and to reduce consumption of sample constituents, these plates are being superceded by microplates of the same footprint but having an array of smaller wells, for example 384 wells in a sixteen by twenty-four array. This trend is continuing, and there is a need for an automated liquid handler able to accommodate microtiter plates having a very dense array of a very large number of very small volume wells with volumes in the nanoliter range. High density microplates presently in use, with the same footprint as previously used plates, have 1,536 wells in a thirty-two by forty-eight well array. 
   Microtiter plates with a dense array of small, closely spaced wells present serious problems for an automated liquid handler. In operation, the handler must be precise enough to place every probe of a multiple probe array into alignment with a corresponding number of sample containing wells. As well size and spacing decreases, it becomes more difficult for an automated handler to reliably place the liquid handling probes directly over selected sample containing wells. 
   The margin for error in positioning the probes relative to the plates and wells decreases as well array density increases. One aspect of the problem is the precise location and alignment of the probe tips. If the group of probes is misaligned, or if individual probes of the group are out of position relative to other probes of the group, then it may not be possible to locate each probe of the group directly over a sample well of the plate. It is time consuming and difficult manually to check and reposition the probes to be sure they are properly positioned and aligned. Even if the probes are initially set up correctly, they can become displaced from their intended positions after a period of use. It would be desirable to provide an automated system for quickly and accurately checking and correcting probe tip positioning and alignment without substantial operator time and skill. 
   SUMMARY OF THE INVENTION 
   A principal object of the present invention is to provide an improved method for aligning probe tips of a precision liquid handler. Other objects are to provide a probe tip locating method using an electrical sensing capability that may preexist in the liquid handler; to provide a probe tip alignment method for detecting skew of a multiple probe array; to provide a probe tip alignment method for detecting misaligned probes and for bending a misaligned probe into an aligned position; to provide a probe tip alignment method that detects locator bed skew; to provide a probe tip alignment method that determines a center of probe scatter for use as a correction factor for a probe drive system; and to provide a probe tip alignment method that is automated and does not require operator time and skill. 
   In brief, in accordance with the invention, there is provided a probe tip alignment method for a precision liquid handler having a probe array moved by a probe drive system relative to a locator bed holding sample wells. The method includes sequentially inserting the probe tips of the probe array with the probe drive system into a locator well at a known position on the locator bed, then sequentially sensing the position of each probe tip in the locator well, and then mapping the positions of the probe tips. 

   
     BRIEF DESCRIPTION OF THE DRAWING 
     The present invention together with the above and other objects and advantages may best be understood from the following detailed description of the preferred embodiment of the invention illustrated in the drawings, wherein: 
       FIG. 1  is a simplified, diagrammatic, isometric view of a representative precision automated liquid handler with which the method of the present invention can be performed; 
       FIG. 2  is an enlarged, fragmentary front view of the probe carrier and multiple probe array of the precision liquid handler of  FIG. 1 , showing the probes in registration with wells of a high density microplate; 
       FIG. 3  is a top plan view of the locator bed of the precision automated liquid handler of  FIG. 1 ; 
       FIG. 4  is a fragmentary, enlarged, cross sectional view of the locator bed of  FIG. 3 , taken along the line  4 — 4  of  FIG. 3 , together with a schematic block diagram of other components of the precision automated liquid handler of  FIG. 1 ; 
       FIG. 5  is an enlarged sectional view of a locator well, including a diagrammatic illustration of a routine for finding the offset of a probe tip from a nominal or ideal aligned position in a probe array; 
       FIG. 6  is a flow chart of steps in carrying out the routine shown diagrammatically in  FIG. 5 ; 
       FIG. 7  is a flow chart of a wall finding subroutine used in the routine of  FIG. 6 ; 
       FIG. 8  is a diagram showing the measured probe tip offsets used for detecting probe holder skew; 
       FIG. 9  is a scatter chart showing a probe tip cluster with one misaligned probe tip; 
       FIG. 10  is a view like  FIG. 9  showing correction of the probe tip misalignment, and showing the offset of the cluster center from the nominal center; and 
       FIG. 11  is a view like  FIG. 10  showing correction for the cluster center offset using a global correction factor. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
   Having reference now to the drawings, and initially to  FIG. 1 , there is shown in simplified, diagrammatic form an example of an automated precision liquid handler generally designated as  20 . The liquid handler  20  includes a table or work bed  22  below an X-Y-Z probe drive system  24  carrying a probe holder  26 . A locator bed  28  is supported on the surface of the work bed  22 . The locator bed  28  supports a number of high density sample containing microplates  32 . The probe holder  26  supports a multiple probe array  30  of individual probes  34  each having a probe tip  36 . In the preferred embodiment of the invention, the array  30  includes twelve probes  34  in a common plane, although other arrays and different numbers of probes could be used. The preset invention is concerned with aligning the probe tips  36  in predetermined positions along a straight line oriented relative to the locator bed so that the probe tips are moved by the drive system  24  into accurate registration with the high density microplates  32 . 
   The X-Y-Z probe drive system  24  moves the probe holder  26  above the work bed  22  and positions it with great precision in predetermined positions relative to the work bed  22 . The system  24  includes an X drive assembly  38  mounted above and to the rear of the work bed  22  by suitable supports  40 . An X drive motor  42 , with an encoder  44 , operates a mechanism within an X arm  46  to move a Y arm  48  from side to side in the X direction. A Y drive motor  50 , with an encoder  52 , of a Y drive assembly  54  operates a mechanism within the Y arm  48  to move a Z arm  56  forward and back in the Y direction. A Z drive motor  58 , with an encoder  60 , of a Z drive assembly  62  operates a mechanism within the Z arm  56  to move the probe holder  26  up and down in the Z direction. Linear encoders may be used in place of the illustrated encoders  44 ,  52  and  60 . 
   The liquid handler  20  includes a programmable controller  64  connected to the motors  42 ,  50  and  58  and to the encoders  44 ,  52  and  60  or other encoders. Controller  64  includes a microprocessor and an operating system capable of controlling the motion of the probe holder  26  in accordance with programmed instructions saved in memory of the controller and/or communicated to the controller from a remote source. Controller  64  using position feedback signals from the X, Y and Z encoders is able to position the probe holder  26  accurately precisely, within a very small margin of error in the microns range. 
   Each microplate  32  includes an array of many individual sample containing wells  66 . The plate  32  has a footprint of about three and one-half by five inches, and known plates may have an X-Y array of 96 wells in an eight by twelve well pattern, or an array of 384 smaller wells in a sixteen by twenty-four array, or a high density array of 1,536 nanoliter volume wells in a thirty-two by forty-eight well pattern. The method of the present invention is particularly advantageous when the liquid handler is used to dispense nanoliter volumes into small sample wells of high density microplates and arrays. 
   A cross sectional view of a high density microplate  32  of is seen in  FIG. 2 , along with the probe holder  26  carrying twelve individual probes  34 . The microplate  32  includes thirty-two rows extending in the X direction, each having forty-eight sample wells  66 . One row  64  is seen in FIG.  2 . Each well  66  has a length and a width of 1.2 millimeters and the center to center well spacing is 2.25 millimeters. The probes  34  are on 9 millimeter centers (spanning five wells  66 ) and the diameter of each probe tip  36  is 1.1 millimeters. 
   Each probe tip  36  can discharge liquid in a droplet size of 0.2 millimeter. The probe holder  26  is moved to the location seen in  FIG. 2  to distribute liquid to the twelve wells  66  that are aligned under the probe tips  36 . The probe holder  26  is then moved by the X-Y-Z probe drive system  24  to align the probe tips  36  with another set of wells  66 . In this manner some or all of the wells  66  of the plate  32 , and of some or all of the plates  32 , can be supplied with nanoliter volumes of liquid. Because of the small well size and spacing, and the small probe size and spacing, great precision is required. In order to assure that ejected droplets are dispensed into the intended sample wells  66 , and to assure that the droplets fall cleanly into the sample wells  66 , the probe tips  36  must be precisely aligned, and accurate probe tip position information must be available for use by the controller  64 . 
     FIG. 3  shows the locator bed  28 , preferably a thick, stable plate of metal such as aluminum supported upon the work bed of  32  the liquid handler  20 . Three leveling and locating points  68  permit the location and orientation of the locator bed  28  to be precisely adjusted and fixed on the work bed  22 . The locator bed  28  includes a probe rinse station  70  and a system of posts  72  for positioning and holding an array of twelve microplates  32  in precisely determined positions upon the locator bed  28 . The plates  32  have a consistent, known configuration, and are held by the posts  72  in precisely fixed positions on the locator bed  28 . Therefore, if the locator plate is correctly positioned, without skew, on the work bed  22 , and if the probe tips  36  are properly aligned and positioned, the probe drive system  24  can position the probe tips  36  in precise registration with a selected group of sample wells  66 . 
   In accordance with the invention, the locator bed  28  includes a probe locator station generally designated as  74 . The locator station  74  includes three probe tip locator wells  76 ,  78  and  80  aligned in a straight line in the X direction along the rear portion of the locator bed  28 . The locator wells  76 ,  78  and  80  are preferably equidistant from one another and are spaced apart by a distance greater than the length of the probe array  30  (FIG.  4 ). Each locator well  76 ,  78  and  30  includes a metal, electrically conductive post  82  carried by an insulating bushing  84  received in a hole extending vertically through the locator bed  28 . An electrical terminal  86  is connected to the bottom of each post  82  beneath the locator bed  28 . A well  76 ,  78  or  80  is formed as an axially aligned opening in the top of each post  82 . Each well has a diameter of about 8 millimeters and a depth of about 6 millimeters and is surrounded by a continuous, circular cylindrical side wall  88  with a thickness of about one millimeter. Recessed clearance areas  90  are provided between the wells  76  and  78  and between the wells  76  and  80 . 
   In performing the probe tip alignment method of the present invention, the probe tips  36  are sequentially inserted into the locator well  76  by the drive system  24  and probe holder  26 . The area of the target provided by the well  76  is far larger than a probe tip  36  and is large enough to receive a probe tip  36  even if it is misaligned, for example by bending of the corresponding probe  34  or variations in the mounting of the corresponding probe  34 . After each probe tip  36  is inserted into the locator well  76 , the position of the probe tip  36  is detected and its offset from an ideal or nominal aligned position in the array  30  is recorded. When the position and offset information is obtained for each of the probe tips  36  of the probe array  30 , this information is used, if necessary, to correct the position of any seriously misaligned probe tip  36 , to correct skew of the probe array  30  and to permit the controller  64  to correct for probe tip cluster offset in operating the drive system  24 . 
   A routine for detecting probe tip position and offset is illustrated in  FIGS. 5-7 . This routine is carried out in accordance with programmed instructions implemented by the controller  64 . As seen in  FIG. 5 , in order to insert a probe tip  36  into the locator well  76 , the controller  64  operates the drive system  24  to place the probe tip  36  at a position that would be at the center of the well  76  if the probe tip  36  were precisely aligned at its nominal position in the probe array  30 . However, the probe tip  36  normally is offset at some distance from the ideal position. As seen in the example of  FIG. 5 , the probe tip  36  is initially located at the position designated as A. The routine of  FIG. 6  is then performed to measure the probe position and offset. 
   The probe measuring routine commences at start block  92  and at block  94  this initial position A is recorded for subsequent calculation in the course of the routine. Then, as indicated at blocks  96  and  98 , the probe tip  36  is moved in the negative Y direction (upward as seen in  FIG. 5 ) until the probe tip contacts the side wall  88  of the locator well  76 . This contact is sensed electrically. More specifically, the controller  64  is connected to both the conductive probe holder  26  and each conductive probe  34 , and is also connected to the electrical terminal  86  of the locator well  76 . A small do voltage, for example four volts, is applied to the locator well  76  and the probes  34  are at ground potential. When the probe tip  36  contacts the wall  88 , the resulting electrical signal is used by the controller  64  to detect the contact. An advantage of this sensing approach is that the liquid handler  20  may include preexisting electrical sensing capability for use in liquid level detection in applications where the probes  34  can descend into larger wells of less dense plates. The point of contact resulting from movement in the negative Y direction is designated as B in FIG.  5 . In block  100  this location is stored for further use. 
   The preferred subroutine called in block  98  for finding the wall  88  is illustrated in detail in FIG.  7 . Before the find wall subroutine is called, an increment of probe tip movement, delta, is set in block  96 . For movement in the negative Y direction, delta is set to negative 0.1 mm in the Y direction. The subroutine of  FIG. 7  commences at block  102  where the probe tip  36  is moved 0.1 mm in the negative Y direction. At the end of this motion, at block  104 , the probe tip is moved up and down in the Z direction. The purpose of this motion is to establish a good electrical contact between the probe tip  36  and the wall  88  if the probe tip  36  has reached the wall  88 . The presence or absence of this contact is tested in decision block  106 . If there is no contact, the subroutine returns to block  104 , and continues to loop, moving the probe tip  36  in increments of delta until contact is sensed between the probe tip  36  and the wall  88  at point B. 
   This portion of the  FIG. 7  subroutine locates point B with an accuracy limited by the size of the initial delta, 0.1 mm. Any overtravel of the probe tip  36  after initial contact against the wall  88  at the maximum delta value is well within the elastic limit of the probe  34  and does not cause permanent deformation. To increase the measuring resolution and achieve a more accurate measurement, at block  108  and block  110  the probe tip  36  is moved in the reverse direction, back away from the wall  88 . Then at block  112 , delta is halved, and the subroutine returns to block  102  described above. When contact again occurs, at block  108  the present value of delta is compared with a minimum increment to providing the desired accuracy. For example, the minimum delta value may be in the order of microns, consistent with the positional accuracy of the probe drive system  24 . If delta is larger than the stored minimum, the subroutine returns again to blocks  110 ,  112  and  102  and the value of delta is again decreased. This loop continues until contact is sensed at a resolution determined by the minimum delta value. At this point the routine returns to block  100  of  FIG. 6  where the resulting value of position B is stored. 
   The next step is to move the probe tip  36  in the positive Y direction (down as seen in  FIG. 5 ) to find another point of contact with wall  88  aligned in the Y direction. This point is designated as C in FIG.  5 . In block  114  of  FIG. 6 , delta is set to 0.1 mm in the positive Y direction and the find wall subroutine of  FIG. 7  is called in block  116 . The position of location C is returned and stored at block  118 . 
   The center of a line between points B and C is roughly on a Y diameter of the circular wall  88 . In block  120  this point, designated as D in  FIG. 5 , is calculated by averaging the values of positions B and C, and the probe tip  36  is moved to this point D. Then the probe tip  36  is moved in the transverse X direction to find opposed points of contact E and F along the X axis. Delta is set to the negative X direction in block  122  and the find wall subroutine is called in block  124 . The location of point E is returned and stored at block  126 . Similarly, delta is set to the positive X direction in block  128  and the find wall subroutine is called in block  130 . The location of point F is returned and stored at block  132 . 
   The center of a line between points E and F is on an X diameter of the circular wall  88 . In block  134  this point, designated as G in  FIG. 5 , is calculated by averaging the values of positions E and F, and the probe tip  36  is moved to this point G. Because the point D can be determined by non perpendicular contact of the probe tip  36  with the wall  88 , and because the line B-C may be substantially offset from the X diameter of the wall  88 , the probe tip  36  is moved again in the Y direction to find opposed points of contact H and I along the Y diameter to obtain an accurate measurement in the Y direction. Delta is set to the negative Y direction in block  136  and the find wall subroutine is called in block  138 . The location of point H is returned and stored at block  140 . Similarly, delta is set to the positive Y direction in block  142  and the find wall subroutine is called in block  144 . The location of point I is returned and stored at block  146 . 
   The Y coordinate of the center point G is recalculated in block  148  by averaging points H and I in the Y direction. The offset of the probe tip  36  at point A in  FIG. 5  from the center point G is indicated by the line A-G. This offset is calculated at block  150  by subtracting the coordinates of point A from the coordinates of point G, and the offset is stored for subsequent use in the probe tip alignment method. The routine terminates at stop block  152 . 
   The probe position and offset routine of  FIGS. 5-7  is repeated for each of the twelve probe tips  36  in sequence until offset coordinates are stored for each of the probes. These stored offsets are used for determining whether or not the probe holder  26  and the probe array  30  are aligned with the X axis.  FIG. 8  illustrates this step. On the grid in  FIG. 8  the X axis base line  154  is intersected by 12 lines extending in the Y direction. The twelve intersections are the twelve nominal probe tip positions. The offsets of each of pobes numbered  112  are plotted on the grid. These are indicated by the circles in  FIG. 8. A  least squares fit line  156  is calculated for the offset points, and the slope, or skew, designated by angle  158  is determined and compared with a maximum tolerance angle close to zero degrees. If the skew of the probe carrier  26  is excessive, the angle  158  is larger than the minimum tolerance angle, and the controller  64  provides an error indication including the amount of skew to be corrected. The operator then corrects the skew condition by adjusting the mounting of the probe carrier  26 , bringing the least square fit line into alignment with the X direction. 
   If probe carrier deskewing is needed, then after the skew condition is corrected, the probe tip position and offset measuring routine of  FIGS. 5-7  is repeated for all probe tips, and the skew is checked again. If the skew angle  158  is now smaller than the minimum tolerance angle, then the method of the present invention proceeds with the correction of X-Y group scatter error. The probe tip position offsets as stored in block  150  ( FIG. 6 ) for the twelve probe tips  36  are numbered 1-12 and are seen in the form of a scatter chart in the example of  FIG. 9. A  maximum range of X offset is indicated by the points  160  and  162  on the nominal or ideal X position line  164 , and a maximum range of Y offset is indicated by the points  166  and  168  on the nominal or ideal Y position line  170 . As seen in the example of  FIG. 9 , the offsets for probes  1 - 9 ,  11  and  12  are within the maximum X and Y bounds, However, the offset for probe  10  is beyond the maximum offset boundary in the positive X direction. This offset is unacceptable because it makes it impossible for the probe carrier  26  to reliably align all twelve probe tips  36  of the probe array  30  with targeted sample wells  66 , 
   In accordance with the present invention, the probe drive system  24  is used by the controller  64  to correct this measured probe tip misalignment. The drive system  24  again inserts the misaligned probe tip  36  into the locator well  76 , and then moves the probe tip in the direction of the detected excessive offset. In the example of  FIG. 9 , the probe tip numbered  10  is inserted into the locator well  76  and moved in the positive X direction against the wall  88 . The movement is large enough to exceed the limit of elastic deformation of the probe  34 , and the probe  34  is deformed and bent so that the probe tip  36  is moved in the negative X direction relative to the other probe tips of the probe array  30 . After his bending motion, the probe tip position and offset measurement routine of  FIGS. 5-7  is repeated for the realigned probe tip  36 , and, if necessary, the probe deformation process is repeated until the misaligned probe tip is within the boundaries of maximum offset. This corrected position of probe numbered  10  can be seen in FIG.  10 . 
   When all the twelve probes are in an acceptable, tight cluster inside the maximum offset ranges  160 ,  162 ,  166  and  168  of the scatter chart (FIG.  9 ), then a global correction factor is calculated for use by the controller  64  in operating the probe drive system  24 .  FIG. 10  shows the twelve offset points before correction. The maximum and the minimum X offsets (probes  6  and  12 ) are averaged, and the maximum and minimum Y offsets (probes  9  and  11 ) are averaged to provide X and Y offset coordinates for the center of the scattered cluster group. In the example of  FIG. 10 , the center is at point  172 , and this center is offset from the nominal or ideal center  174  by offset line  176 . Rather than attempting to physically move or reposition the probe tips  36  to center the clustered probe array  30 , the offset  176  is stored by the controller  64  as a global correction factor. When the controller  64  moves the probe holder  24  to a desired position over the locator bed  28 , the target X and Y coordinates are modified by the global correction factor  176 . As a result the scattered cluster is effectively repositioned to a corrected position indicated graphically in  FIG. 11  where the nominal center  174  and the cluster center  172  of  FIG. 10  are seen to coincide at the point  178 . 
   The stored probe tip offset information is also used to check the alignment of the locator bed  28  on the work bed  22  of the liquid handler  20 . The left most probe tip  36  ( FIG. 4 ) is inserted into the locator well  80 , and the position measuring routine of  FIGS. 5-7  is carried out to obtain offset coordinates for the left probe in the locator well  80 . The right most probe tip  36  ( FIG. 4 ) is inserted into the locator well  78 , and the position measuring routine of  FIGS. 5-7  is again carried out to obtain offset coordinates for the right probe in the locator well  78 . Because the actual positions of the left and right probes are known relative to the central locator well  76 , the Y offset coordinates of the probe tips  36  in the laterally spaced locator wells  78  and  80  are compared with the Y offset coordinates of the same probe tips  36  in the well  76 . If a discrepancy is detected, a determination is made that the locator bed  28  is skewed upon the work bed  22 . The controller  64  provides an error message including the information needed for the operator to readjust the position of the locator bed  28  and correct the locator bed skew condition. 
   While the present invention has been described with reference to the details of the embodiment of the invention shown in the drawing, these details are not intended to limit the scope of the invention as claimed in the appended claims.