PixSys PA Series Manu (Word98) - OSU Array Tech and …microarray.okstate.edu/files/pixsys.pdf · PixSys PA Series Workstation and AxSys Control Software. Although applicable to most

PixSys PA Series

System and SoftwareOperating Manual

Version 2.0April 1999

Cartesian Technologies, Inc.17781 Sky Park Circle

Irvine, CA 92614TEL: (949) 440-3680 FAX: (949) 440-3694

Table of Contents

1. QUICKSTART ................................................................................................................................32. PURPOSE .........................................................................................................................................43. PRODUCT DESCRIPTION.........................................................................................................44. GLOSSARY......................................................................................................................................4

Version 2.0 PixSys PA Series Operating Manual Page 2 of 53

Cartesian Technologies, Inc

5. CONVENTIONS ............................................................................................................................66. PRECAUTIONS..............................................................................................................................77. INSTALLATION............................................................................................................................9

7.1. Tools Needed................................................................................................................................97.2. Basic Unit Assembly...................................................................................................................97.3. Preparing the Wash Station and Slide Hold-Down. ...............................................................117.4. Installing AxSys Software........................................................................................................13

8. OPERATION.................................................................................................................................149. CALIBRATION.............................................................................................................................1610. PRACTICAL CONSIDERATIONS........................................................................................18

10.1. Theory of Array Printing with Quill Pins.............................................................................1810.2. Array Layout Considerations .................................................................................................1810.3. Sample Loading.......................................................................................................................2110.4. Pre-Printing ............................................................................................................................23

11. USING THE AXSYS SOFTWARE..........................................................................................2511.1. Installing AxSys Software......................................................................................................2511.2. The Program List ....................................................................................................................2611.3. Tools.........................................................................................................................................2611.4. Programming Commands.......................................................................................................2811.5. Manual Mode..........................................................................................................................3911.6. Function Libraries...................................................................................................................4111.7. Foundation Functions.............................................................................................................42

12. EXAMPLE PROGRAMS...........................................................................................................4612.1. Editing Commands Unique to Each Example Program........................................................46

13. MAINTENANCE........................................................................................................................4713.1. Cleaning the PixSys................................................................................................................4713.2. Care and Cleaning of the Telechem ChipMakerTM Pins ........................................................47

14. TROUBLESHOOTING.............................................................................................................4815. EQIUPMENT SPECIFICATIONS ..........................................................................................50

15.1. PixSys™ PA series General Specifications............................................................................5015.2. PixSys 5000 Specifications.....................................................................................................5015.3. PixSys 5200 Specifications.....................................................................................................5115.4. PixSys 5500 Specifications.....................................................................................................5215.5. ArrayIt Chipmaker™ Pin Specifications ...............................................................................52

16. REPLACEMENT PARTS..........................................................................................................53



1. QUICKSTART

The following is an abbreviated procedure for quickly getting started (for advancedcustomers only):

1. Place the PixSys unit in a suitable place (Note: the stage moves approximately 50cmto the left and 25cm to the right (facing unit) during full travel).

2. Connect the power, communication, and P1 cables.

3. Install the peristaltic pumps and connect the vacuum and wash tubing to the PixSysunit. Be sure all tubing accommodates the PixSys unit’s full range of motion.Provide a source of clean wash solution, a receptacle for used wash solution, avacuum source for the slide hold-down, and a vacuum source for the wash station.

4. Install the AxSys Software on the computer that will be used with the PixSys unit.

5. Place an empty microwell plate of the kind that you will be using on the PixSys unit.Place microscope slides of the thickness you will be using in positions A1 and A10 ofthe slide nest.

6. Calibrate the PixSys unit using the AxSys program entitled “Calibration”. Use asingle calibration pin in the front, left slot of the print head. Refer to the section onunit calibration if necessary.

7. Export the Function Library from the “Calibration” program and import it into theexample program of your choice.

8. Adjust the example program by editing the “Move” command values that instructthe unit to move to the pre-printing area, the first slide on which an array will beprinted, and the source plate.

9. Execute a dry run of the calibrated example program using a calibration pin toensure the accuracy of the programmed movements.

10. The unit is now ready for arraying using the calibrated example program. Use tapeor adhesive film to cover the vacuum plate openings that will not be used. Placepins in the appropriate positions of the print head. Place a microwell plate withsamples the source position. Place slides on the slide nest and engage the vacuumhold-down. Execute the program.



2. PURPOSE

This manual serves as both a tutorial and reference for the Cartesian TechnologiesPixSys PA Series Workstation and AxSys Control Software. Although applicable tomost of the units in the series, most of the hardware descriptions apply to the PixSys5500. The manual covers all of the important areas of equipment use: installation, setup,array design, programming, maintenance and troubleshooting. It is strongly suggestedthat the user read this manual in its entirety before operating the equipment. Specificsections should be referred to as necessary.

3. PRODUCT DESCRIPTION

The Cartesian Technologies PixSys PA series array printing system consists of adispense platform with a wash station, one or more ArrayIt Chipmaker™ pins, avacuum source(s) for the wash station and slide hold-down, peristaltic pumps for thewash station, the AxSys software, and optional hardware or software adaptations.

The AxSys software is used to control any Cartesian Technologies robot, including thearraying units of the PixSys PA series. The software is used to instruct the PixSys unit tomove the ArrayIt Chipmaker™ pins about the dispense platform.

The ArrayIt Chipmaker™ pins function to transfer sample obtained at a microwell plateto a solid substrate, such as a glass slide or membrane. The pins dispense a fixedvolume of sample onto a solid substrate. When properly used the pins are able to printa minimum of 150 spots per sample.

The PA Series includes the larger PixSys™ PA 5500 and 5200 platforms or the smallerPixSys PA 5000™ platform. The system is controlled by Windows software, which isaccessed via a user provided PC. The operator may use Windows 95, Windows 98 orWindows NT to operate this system. The ArrayIt Chipmaker™ pins are capable ofdispensing sub-nanoliter volumes into arrays with centers as close as 150 microns.

4. GLOSSARY

Array: A population of reagent samples that have been attached to a solid support andarranged into a pattern, usually a grid.

Arraying : The process of transferring samples from a source to an array.

Command : A unit of information used by the AxSys software to instruct the unit tocomplete a simple task. The user supplies the parameters of a command.

Dispense Platform : The portion of the PixSys unit that can be reached by the print head.A sliding stage that may include positions for a wash station, microwell plates, glassslides, membranes, or other printing substrates.

Dispensing: The act of applying liquid material (reagents or other chemistries) to a solidsupport in an array.

Calibration Pins : These inexpensive pins are used for calibrating the unit and for testingmethods. They are scored to allow easy bending or breakage should the encounter



resistance from hard parts. They are can not be used for making arrays. They aresometimes referred to as “practice pins” or “break-away pins”.

Foundation Function : A function that is used in all methods. These include (but are notlimited to) functions that carry out such operations as pin washing and moving the pinsup and down for spot printing.

Function : A user determined list of commands that can be used as a macro whenprogramming. Often, simpler functions are used to create more complex functions.

Function Library : Each method contains a list of functions that are available for usewithin the method. This library of functions exists independently of any functions thatmay be found in other methods, regardless of name or other identifiers. Libraries maybe exported from and imported into methods.

Method : A set of commands created with the AxSys software that performs a specificseries of tasks. They exist as individual AxSys files. This term is used interchangeablywith the term “program”.

Pre-printing : The process of transferring sample to a substrate prior to transfer to thearray. This typically consists of a series of spots printed on a glass slide that will bediscarded. Pre-printing is done in order to ensure the quality of spots printed in thearray.

Print Head: The brass head on the robotic which holds the ChipMaker pins.

Program : see “Method”

Program List : The operator determined list of commands that the computer will followin a program. The operator can add, remove, or edit commands as necessary.

Set Up : Preparing the equipment to transfer samples into arrays.

Source Plate : The microwell plate containing sample that is to be transferred to arrays.

Wash Station : An assembly consisting of a wash solution bath and vacuum dryingapparatus for cleaning the ChipMaker Pins™.



5. CONVENTIONS

• When facing the PixSys 5500 unit the robotic arm that holds the print head is at theback of the unit, on the left side.

• The five columns of the slide nest are designated with the letters A through E, fromleft to right. The rows are designated with the numbers 1 through 10, from front toback. This means that slide A1 is in the front, left corner of the nest and that slideE10 is in the back, right corner.

• Well A1 of a microwell plate is in the front, left corner of the unit.

• The frosted edges of the microscope slides are oriented towards the right side(PixSys 5200 and 5500) or the front (PixSys 5000) of the unit.

• The demonstration programs are constructed so that the arrays are printed within anarea that is a maximum of 22mm x 60mm. As such, arrays are typically begun at apoint 1.5mm from the front edge and 1mm from the left edge of the slides.

• Slides A1 and A2 are typically reserved for pre-printing.

• Pre-printing requires maximal use of the slide area and is typically begun at aposition 0.5mm from the front, left corner of the slide. It is best to use a slide that isnot frosted for pre-printing.



• The first slide on which an array is printed is typically slide A3.

• Pins placed in the print head should always include a pin in the front, left slot. If asingle pin is used, the front left slot is used. Refer to the Figure below:

6. PRECAUTIONS

• Do not drop or expose the equipment to severe mechanical shock. The PixSys PAcomponents are assembled with delicate precision and should be treated with care.

• Do not expose any portion of the unit to strong acids, strong bases, aromaticalcohols, ketones, esters, or chlorinated hydrocarbons.

• Always wash reagents from the ArrayIt Chipmaker™ pins after use or prior tostorage. Leaving reagents in contact with ArrayIt Chipmaker™ pins for prolongedperiods of time can cause clogging or changes in the pin surface and will diminishperformance.

• Clean all reagents from the PixSys unit should any be spilled or otherwise broughtinto contact with the unit.

• Always use pre-filtered reagents to prevent particulate matter from degradingperformance. We suggest using a 10-micron filter or finer pore size.



• Be sure ample clearances exist between the ArrayIt Chipmaker™ pins and itemsmounted to the platform when the X or Y axes are active. Damage to both itemsmounted to the platform and the ArrayIt Chipmaker™ pins can occur if thisprecaution is not observed. Damage can be avoided by carefully ensuring that the Zaxis is properly controlled to move the ArrayIt Chipmaker™ pins to a safe travelheight before all moves in the X and Y dimension. All programs should be testedwith the calibration pins provided with the unit before being used with ArrayItChipmaker™ pins for array printing.

• Do not attempt to disassemble any defective platform components beyond thatdescribed in this manual. This will void all warranty obligations and may renderthe unit unserviceable.



7. INSTALLATION

This section describes the actions necessary to prepare the equipment for operation.After completing this section, the unit will be ready for programming using the AxSyssoftware.

When unpacking, confirm that all parts, tools, and accessories are accounted for. A listof all included items accompanies the PixSys unit.

7.1. Tools NeededA set of hex Allen wrenches is provided with the unitTweezers are provided with the unit

7.2. Basic Unit AssemblyPlace the unit on a stable workbench or table where it will be operated. Leave enoughroom around the unit to allow for full travel of the slides. (refer to the section on PixSysUnit Specifications for your model). Be aware that the X, Y, and Z slides can beprogrammed to make quick movements.

Verify that the main power switch on the right side of the rear panel, is in the “OFF”position. Install power cord and plug into power source. Please refer to Figure 7.1 onthe next page.

Install the included COM port adapter (serial to phone jack) into the COM port of thecomputer that will be used with the PixSys unit. Connect the computer to the portlabeled “TERMINAL” on the rear panel of the PixSys unit via the included curly phonecable. . Please refer to Figure 7.1 on the next page.

Connect the P1 cable assembly to the P1 connector on the rear panel. This port islabeled “P1”. Connect the free end of the P1 cable assembly with the 2 connectorsexiting the Y axis slide assembly. Note the label identification for proper matching ofinterconnections. The connectors will have letters labeling them and should beconnected to the mate with the same letter. For example, connector “C” would go to theother connector “C”. Please note that there may not be mates for every connector on thefree end of the P1 cable assembly. Please refer to Figure 7.1 on the next page.



Figure 7.1:Rear View of the PixSys™5500

Install the print head to the mounting plate located on the front of the arm of the PixSysunit as shown if Figure 7.2 below. The mounting plate has a series of closely spacedholes as well as holes at the edges of the plate. The print head should be mounted in thevery leftmost of the series of holes, but not in the holes set apart at the left edge of themounting plate (the position of the Allen screw shown below). Remove the Allen screwfrom the bracket, push the print head and bracket assembly into the correct position,replace the Allen screw in the center hole and tighten the screw.

Figure 7.2: Print Head Mounting Plate with Correct Mounting Position Shown

P1

COM port adapter(to computer COM: port)

Y-slideconnectors



7.3. Preparing the Wash Station and Slide Hold-Down.

The wash station is located in the back, left corner of the dispense platform.

Install the two peristaltic pumps on the right side of the platform. Please note that thereare two types of pump bodies used: an outboard body with no extension shaft and aninboard body that has mounting provisions on the front surface. The pump body ismounted by first locating the mounting tab on the rear surface in the respectivemounting slots to which it is affixed and then rotating the pump body clockwise until anaudible click is heard.

Open the peristaltic pumps located on the right side of the platform by lifting the topportion of the pump straight up by grasping from both sides. This rotates the pumpingchamber into the open position.

Install one end of the larger of the two furnished lengths of tubing into the larger of thetwo wash station ports (the port towards the front of the unit). Route this tubingthrough the harness at the rear of the dispense platform. The harness clips must beaffixed so that the loop is in upward position above the screw that holds it in place. Thetubing should be held close to the dispense platform but remain free of kinks,constrictions, or pinches that might impede the flow of liquid. This larger diametertubing must then be routed through the front of one of the peristaltic pumps. The largerdiameter tubing should exit the back of the pump and terminate at the receptacle forused washing solution. Refer to Figures 7.3 and 7.4 on the following page.

The smaller diameter tubing should be connected to the smaller of the two wash stationports (the port towards the back of the unit). Route this tubing through the harness atthe rear of the dispense platform. The harness clips must be affixed so that the loop is inupward position above the screw that holds it in place. The tubing should be held closeto the dispense platform but remain free of kinks, constrictions, or pinches that mightimpede the flow of liquid. The smaller diameter tubing should carry clean washsolution to the front of the pump. Please take note of the dispense platform’s full rangeof motion in the X dimension and be sure ample tubing length is provided betweenperistaltic pumps and the dispense platform. Refer to Figures 7.3 and 7.4 on thefollowing page.



Figure 7.3:Tubing Connections to Wash Station

Figure 7.4:Direction of Flow Through Peristaltic

Pump

Close the pump tops by rotating them downward. The pump tubing should exit thepump on either side and be held firmly in place.

When first operating the system, check that the amount of liquid exiting the WashStation exceeds the amount entering. Adjustments can be made on the pump body toadjust flow at the point the tubing emerges by rotating the thumb wheels.

Attach a length stiff black tubing to the vacuum fitting on the wash station and route itthrough the tubing harness on the back of the dispense platform. The vacuum fittingswill release the tubing when the fitting collar is pushed in while the tubing is beingpulled out. The remaining end of the tubing should be attached to one of the vacuumsources. If the two vacuum sources vary in strength, the stronger of the two should beused with the wash station. Please take note of the dispense platform’s full range ofmotion in the X dimension and be sure ample tubing length is provided between thevacuum source and the dispense platform.

Attach the remaining length of stiff black tubing to the vacuum fitting at the front, leftcorner of the dispense platform. This is the slide hold-down vacuum port. Route thistubing along the left edge of the dispense platform and then through the tubing harness.Tighten the screws on the harness so that the harness loops are held in an uprightposition above the screws. The remaining end of this tubing should be attached to avacuum source. Please take note of the dispense platform’s full range of motion in the Xdimension and be sure ample tubing length is provided between the vacuum source andthe dispense platform.



7.4. Installing AxSys Software

Insert disk 1 into your floppy disk drive.

Click the Windows 95/98/NT “Start” button. Select the control panel option, thenchoose “Add/Remove Programs”. Next select Install and follow the Windows prompts.

Before opening the AxSys software program, be sure your PixSys unit is connected toyour computer. The black curly phone cable should be connected to the TERMINALport, located on the back of the PixSys unit. The other end of the phone cable fits into anadapter, which can then be plugged into one of the computer’s COM ports. This isnormally COM 1, however you may use any of the computer’s COM port. Make surethe connector is screwed in completely.

Turn on the unit.

Open the AxSys software program. The software will search for the unit connection onCOM port 1. If there is a connection, then the software will display a green connectionin the lower right hand corner of the screen. If it is not connected you will receive anerror message. Select “ R etry” and choose the COM port into which the phone cable isconnected. Select the COM port by clicking on the arrow next to the blank labeled“Switch To:” within the “Communications Settings” window. The software will try toconnect again. If you receive an error message again, close the AxSys software programand turn off your PixSys unit. Wait 10 seconds, turn on your PixSys unit, wait anadditional five seconds, and run AxSys. If a connection can not be established, check allof your connections again and try again.



8. OPERATION

After installation, the following instructions are used to operate the PixSys unit to makearrays:

1. Be sure the PixSys unit is clean and dry.

2. Turn the PixSys power switch to the “ON” position. Turn on the vacuum pumps sothat supply vacuum to the wash station and slide hold-down.

3. Open the AxSys software on your computer. Make sure the computer connects tothe PixSys unit. If you have difficulties connecting see the Troubleshooting section.

4. Close the peristaltic wash pumps (they should be left open when the PixSys is not inuse). Using the manual mode, turn the wash pump on and make sure the fountain isoperating normally and that the liquid is being scavenged at a rate greater than thestation supply flow.

5. Open the method file that you wish to use for printing the array(s). If the methodhas not been tested using a calibration pin, do so before use with arraying pins andreagents. If any changes have been made to the program or hardware (includingslide and source plate type) since the PixSys was last used, the program should betested using a calibration pin.

6. Carefully place pins in the print head in the desired configuration. There should bea pin in the front, left slot of the print head (refer to section 5 Conventions). Be surethat the tips of the pins do not strike any hard surfaces while they are being handled.The pins and brass print head must be clean and dry before use.

7. Seal the unused holes in the vacuum plate of the wash station with laboratory film orremovable tape. Be sure that all holes that correspond to pins place in the print headare open.

8. Fill the source microwell plate with the samples to be transferred to the array. Besure to avoid overloading the wells with sample (see section 10.3 Sample Loading).Place the source plate on the dispense platform with well A1 of the plate in the front,left corner of the platform. Check to make sure the plate is seated properly andsnugly within the spring clips.

9. Place the target slides on the dispensing platform. Carefully push each slide into theback, left corner of the nest position it is in. Engage the vacuum hold-down (Note:Each lever provides vacuum to each column of slides with the exception of columnA where the left lever supplies vacuum to slide A1 only and the right lever to slidesA2 through A10). Listen for leaks and test a slide in each column to make sure theslides are immobilized.



10. Begin your arraying application by clicking on the “Go” icon in the AxSys software.

11. Upon completion, clean the unit of all reagents using the procedures outlined in theMaintenance section. Be sure that the pins are free of any residual sample, debris, orother substance.

12. Open the peristaltic pumps (to prevent deformation of the tube), switch the PixSysand vacuum pumps “OFF”, and exit the AxSys software.



9. CALIBRATION

When a PixSys unit is first received, the XYZ positioning stage must be calibrated usingthe AxSys software. The program entitled Calibration is included with the AxSyssoftware. You will need a single, straight calibration pin to calibrate the unit. Do notinsert the pin into the printing head yet. The user will also need to place glassmicroscope slides of the thickness that will be used for making arrays in positions A1and A10 of the slide nest.

Calibration Procedure

1. Run AxSys and open the file called Calibration . This method sequentially calls aseries of move functions and pauses at each position to permit checking ofalignment. Alignment is done by exiting the method, aligning the print head usingmanual control, and entering the new values into the method.

2. Run the calibration method without a calibration pin to see the different positions(Click the [Yes] button to proceed to the next position).

3. Insert the calibration pin into the front, left slot of the print head (See Figure 2 in the“Conventions” section), and run the Calibration method again and stop the methodat the first position.

4. Carefully read Section 11.5 Manual Mode before proceeding.

5. Enter the manual mode and manually move the pin to the position that is describedby the function in question. Record the coordinates of the position in the chartprovided on the following page.

6. Edit the “Move” command of the function in question and enter the new values intothe move command. Be sure to enter values into only the windows that alreadycontain data. For instance, one should not enter values into the Z-axis window ifthere aren’t already values in the window or if the movement type window is set to“None”.

7. After editing a function, run the program again, letting it continue until it reaches afunction that has not been calibrated. Repeat steps 5 and 6 until each position hasbeen calibrated (The functions calls with an asterisk (*) need to be edited).

8. All of the moves to the slides are intended to move to a point 1.5mm from the frontedge and 1mm from the left edge of the slide. There is no pause following moves toslides A3 and B1. The values for these positions can be calculated by adding 86mmto each slide position in the X dimension and 26mm to each slide position in the Ydimension. Be sure to edit these functions. After all of the functions have beenedited, run the program a final time to double-check the positions.



9 . After the calibration has been completed the user should export this library offunctions, saving it as Foundation Library . If a file with this title already exists itshould be overwritten with the one you have modified using the calibration method.This function library with the calibrated positions can now be imported into otherexisting programs as well as new programs (See Section 11 Using the AxSysSoftware for more information on importing and exporting libraries).

Move Values for Calibration/Foundation Functions

Function Title Axis Axis Date Initials Notes

Move to Vac Dry* X= Y=

Pins Up--Wash Station* Z=

Pins Down--Vac Dry* Z=

Move to Rinse Tank* X= Y=

Pins Down--Rinse Tank* Z=

Move to Source Plate* X= Y=

Pins Up--Well* Z=

Pins Down--Well* Z=

Move to Slide A1 for Pre-prints* X= Y=

Pins Up--Slide* Z=

Pins Down--Slide* Z=

Move to Slide A3 forArraying* X= Y=

Move to Slide B1* X= Y=



10. PRACTICAL CONSIDERATIONS

This section outlines the practical considerations of using a quill-type pin for printingarrays onto glass slides.

10.1. Theory of Array Printing with Quill PinsThe pins are moved to a microwell plate where sample is loaded into the pins bydipping into the sample. Prior to printing spots in the array it is necessary to dispense aseries of pre-print spots. These pre-prints are used to blot sample from the exterior ofthe pin and are necessary to insure spot quality in the array. The pins are then moved toindividual slides where sample is accurately dispensed into the array. This dispensingprocess is shown schematically in figure below:

The wetted tip of the pin is gently touched to surface, leaving a small spot (tapping thepin to the surface leaves a larger spot). To prevent cross-contamination the pins arethoroughly cleaned at the wash station after each sample. The process is repeated untilthe desired array is manufactured.

10.2. Array Layout Considerations

10.2.1. Print AreaDNA samples must be spotted or printed in an area defined by the area detectable by aslide reader. For example, a General Scanning ScanArray 3000 confocal scanner candetect an area 22 mm x 60 mm, as shown below:

60 mm

22.0 mm



10.2.2. Print HeadThe print head of the PixSys PA Series Workstation is either a ChipMaker 2 print headwhich holds up to 32 Chipmaker pins in a 4 x 8 array or a ChipMaker 3 print headwhich holds up to 48 pins in a 4 x12 array. The pins are held on 4.5 mm centers, asshown in the following schematic:

A B C D E F G H

1

2

3

4

I J K

ChipMaker 2 (4 x 8)

ChipMaker 3 (4 x 12)

L

4.5 mm

4.5 mm

The print head is oriented relative to the microwell plate on the PixSys platform suchthat pin A1 corresponds to well A1 of a microwell plate (row 1 on the print head andplate are located at the front of the unit). For a 384 well plate, pins may be placed in anyof the positions for printing since the 4.5 mm pin spacing matches the 4.5 mm wellspacing. For a 96-well plate (9 mm well spacing), every other position can be used (A1,A3, C1, C3, …). In order to maintain convention among AxSys programs, it isrecommended that position A1 of the print head always contain a pin. Other pinsshould be placed in the print head using the pin in postion A1 as a reference point oranchor positon.

10.2.3. Single Pin PrintingPrinting arrays with single pins is the most straightforward type of printing but is themost time consuming. With single pin printing, a source plate can be directly mappedinto an array. In other words, 384 samples from a source plate can be spotted as a 16x24array such that the spots A1, B1, C1, etc. in the array correspond to wellsA1,B1, C1, etc.of the source plate. This makes post-hybridization analysis trivial. Using this approachand 0.25 mm spot-to-spot spacing, 21,120 spots (55-384 well plates) can be placed on aslide.

10.2.4. Multiple Pin PrintingPrinting with more than one pin is faster than with a single pin. It also requires moreplanning for array layout and more sophisticated sample tracking. To spot withmultiple pins, the pins are dipped into the sample wells to load the pins and thentouched in unison onto the surface to create different spots. For example, pins drawingsample from a 384 well plate will be held at 4.5 mm spacing. The first round of sampletransfer produces spots on 4.5 mm spacing. The next rounds are done by spotting witha small offset (200 to 400 um) from the previous location. This permits maximum spotdensity but requires deconvolution at the analysis stage to identify the sample spot. Thefollowing are a few possible pin configurations which may be used with the PixSys PASeries.



1 x 8 Pin Configuration (ChipMaker 2) . If 8 pins are placed in row 1 of the ChipMaker2 print head (4.5 mm centers) and used to sample column-wise from a 384 well plate,then the following array pattern will be generated (showing only the first two of eightpins spotting left to right) :

Pin A1 Pin A2

4.5 mm

A1 A2 A3 A4 B1 B2 B3 B4Sample

As one proceeds with the arraying process, eventually, spots from Pin A1 will approachspots which were spotted by Pin A2 (sample B1). With the appropriate choice of spot-to-spot spacing, a desirable number of spots can be printed between pins. For example,if spots are placed on 0.28 mm centers, then 16 spots can be placed between adjacentpins (16 x 0.28 ≈ 4.5). Sixteen is desirable since it is an even multiple of a 96 or 384 wellplate. Using the 1 x 8 array, the maximum number of spots that can be printed on aslide is 9,984 spots (26-384 well plates). The area of the array is shown in the followingschematic:

78 x 128

As can be seen, the array does not fully utilize the detectable area of the slide.

1 x 12 Configuration (ChipMaker 3 ). If 12 pins are placed in row 1 of a ChipMaker 3print head and used to sample row-wise from a 384 well plate, then more of the slidecan be printed with spots. Using the same spot spacing as 1 x 8 configuration, thenumber of spots increases to 14,976 (78 x 192 array or 39-384 well plates).

4 x 1 Pin Configuration for 384 Well Plates . Another alternative is to place 4 pins incolumn A of a ChipMaker 2 or 3 print head. With this configuration, 64 spots can beplaced in a column along the short edge of the slide. Continuing to spot in a left to rightfashion with 0.28 mm offsets, a total of 240 columns can be printed to fill the entire printarea. The resulting 64 x 240 array contains 15, 360 spots (40-384 well plates).

4 x 8 or 4 x 12 Configuration . For maximum throughput, the ChipMaker 2 or 3 printhead can be used with the maximum number of pins, 32 and 48 respectively. In doingso, the spots from one pin approach the other pins two dimensions. In other words,each pin can only print a 22 x 22 array (assuming the 0.20mm spot spacing usingChipmaker 2 pins). Thus the total number of spots for the 4 x 8 configuration is 8,064and for the 4 x 12 configuration is 12, 288.



Pin Configurations for 96 Well Plates . For 96 well plates, a similar strategy is used butpin density in the print head is reduced by four since these plates have 9 mm wellspacing.

10.2.5. SummaryThe following table gives a summary of maximum spots for each configuration for0.20mm spot spacing (rounded to the nearest entire microwell plate):

ConfigurationMax. Spotsper Slide96 well

Max. Spotsper Slide384 well

#96 well /#384 well

Single pin 32,928 32,640 343 / 851 x 2 29,664 29,568 309 / 772 x 2 24,288 24,192 253 / 632 x 4 16,128 24,192 168 / 634 x 1 NA 26,880 NA / 701 x 8 NA 19,584 NA / 511 x 12 NA 29,568 NA / 774 x 8 NA 16,128 NA / 424 x 12 NA 24,192 NA / 63

10.3. Sample Loading

The DNA sample to be spotted on the slides is usually placed in a 96 or 384 well plate.For best results, a flat bottom plate is used with enough sample to cover _ to _ of thequill slot when the pin is touched to the plate bottom, as shown schematically below:

The actual volume required will vary with microwell plate type and manufacturer. It isbest to use the minimum amount of sample necessary to cover the bottom of the well.Often, surface tension will cause small volumes of sample well to stick in the corner of a



microwell plate well. Gently tapping the bottom of the plate squarely onto a flat surfacea few times will cause the sample to spread across the bottom of the plate. Ideally, themicrowell plate will be centrifuged to induce sample spreading.

This technique uses minimum volume of sample in each well. Dipping the pin into alarger volume of sample results in an increased number of pre-print spots necessarybefore the spots become consistent (see next section).



10.4. Pre-PrintingAfter loading the pin(s) with sample, they must be spotted a number of times onto aslide to create consistent spots. In the example shown below, three pins were loadedand spotted a number times to show the change in spot size as spotting progresses:

Spot Variation, 10uL Source Volume

0

2000000

4000000

6000000

8000000

10000000

12000000

0 10 20 30 40 50 60 70 80 90 100

Spot Number

Flu

ore

sen

ce In

ten

sity

Pin 1Pin 2

Pin 3

Experimental Details: Cy3-labeled 31-mer in spotting solution; 10 µL in384 well plate (manufactured by Greiner); pin touched to the bottom ofplate for loading; spotting on CSS slides using PixSys 5500, scanned withScanArray 3000

As the data shows, after 10 to 20 pre-print spots, the spotting becomes more consistent.



Loading the pin with a larger volume of sample (i.e. dipping the pin deep into thesample solution) results in more pre-prints required before consistent spotting can beachieved, as shown in the figure below:

Spot Variation, 35uL Source Volume

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

20000000

0 10 20 30 40 50 60 70 80 90 100

Spot Number

FluorescenceIntensity

Pin 1

Pin 2

Pin 3

Experimental Details: Cy3-labeled 31-mer in spotting solution; 35 µL in384 well plate (manufactured by Greiner); pin touched to the bottom ofplate for loading; spotting on CSS slides using PixSys 5500, scanned withScanArray 3000

It appears from the data that when drawing sample from a large volume, the samplesolution drains down the pin, making a large number of pre-print spots necessary forconsistent spotting.



11. USING THE AXSYS SOFTWARE

This section covers the basic items found within the AxSys software program. Afterreading this section, the operator will understand the process of programming thePixSys unit via the PC and Windows Software for arraying.

11.1. Installing AxSys Software

1. Insert disk 1 into your disk drive.

2. Click the Windows 95/98/NT “Start” button. Select the control panel option, thenchoose “Add/Remove Programs”. Next select Install and follow the Windowsprompts.

3. Before opening the AxSys software program, be sure your PixSys unit is connectedto your computer. The black curly phone cable should be connected to theTERMINAL port, located on the back of the PixSys unit. The other end of the phonecable fits into an adapter, which can then be plugged into one of the computer’sCOM ports. This is normally COM 1, however you may use any of the computer’sCOM port. Make sure the connector is screwed in completely.

4. Turn on the unit.

5. Open the AxSys software program. The software will search for the unit connectionon COM port 1. If there is a connection, then the software will display a greenconnection in the lower right hand corner of the screen. If it is not connected youwill receive an error message. Select “ R etry” and choose the COM port into whichthe phone cable is connected. Select the COM port by clicking on the arrow next tothe blank labeled “Switch To:” within the “Communications Settings” window. Thesoftware will try to connect again. If you receive an error message again, close theAxSys software program and turn off your PixSys unit. Wait 10 seconds, turn onyour PixSys unit, wait an additional five seconds, and run AxSys. If a connectioncan not be established, check all of your connections again and try again.



11.2. The Program ListThe Program List is where the list of operator determined commands resides. Theseinstructions are carried out in sequential order. Below is a graphic of a Program List:

11.3. ToolsThe following toolbar tools are available in AxSys. Most of them function in a mannertypical of Windows based applications.

New Save Copy Delete About Stop

Open Cut Paste Print Go

11.3.1. NewSelecting NEW allows the user to create a new program. A blank page will appear andonly the END function will be listed. The user can now begin inserting instructions.

11.3.2. OpenThis icon opens a list of programs that the user has programmed and saved.

11.3.3. SaveSelecting “Save” will save the program to the location instructed by the user.

11.3.4. CutThis button cuts out any instruction that has been highlighted using the mouse. Tohighlight instructions, click the left mouse button and drag it over the instructions youwish to cut out. First highlight your selection and then click the CUT button. Theinformation will be removed from the list once the CUT button has been selected. Cutitems are stored in the “clipboard” and can be Pasted (see following).



11.3.5. CopyThis button copies any information that has been highlighted using the mouse, to theclipboard. To highlight information click the left mouse button and drag it over theinstructions you wish to copy. First highlight your selection and then move the pointerso that it clicks on the COPY button. The selected information will now be copied ontothe clipboard. This copied information can be Pasted (see following).

11.3.6. PasteSelecting this button will paste any information on the clipboard to the current locationin the information list. If an instruction is highlighted then the item on the clipboard willtake its place. If you do not wish to remove the instruction yet wish to put the pastebelow the instruction, click to the side of the instruction. The instruction should nowhave a small dotted box around it. You will not lose the old instruction and the newinformation pasted will be inserted below it.

11.3.7. DeleteThis button deletes any highlighted information in the list of instructions. To deletemore than one instruction, hold down the left mouse button and drag it until all of theinstructions to be deleted are highlighted. Lift up on the left button and place thepointer on the Delete button. Now click the left mouse button and the information willbe erased completely. Note: There is no “Undo” button.

11.3.8. PrintThis icon will print the information within the program list. The information printed isa condensed version and all details regarding each instruction will not be printed.

11.3.9. AboutThis button displays the current software version.

11.3.10. GoThis button begins the programmed list of instruction. Once this button is clicked theinstructions will begin in the order that they are listed.

11.3.11. StopThe “Stop” icon appears in the tool bar as a bright red stop sign. This commandterminates the program when clicked. The stop icon will only need to be clicked once.The program can not be restarted from where it was stopped.



11.4. Programming CommandsCommands are inserted into the Program List by clicking their icons in the toolbar orselecting them from the “Insert” menu. Below is a description of the function of eachicon displayed on the toolbar. An inserted command can be edited by double-clickingits icon.

11.4.1. Axes SpeedThis instruction sets the speeds of the X, Y and Z axes. The “AxisSpeed” icon appears in the toolbar as a red automobile. The following information canbe edited in the submenu: XY Axes Profile, Z Axis Profile, and Notes. Values for the startspeed, slew speed, and acceleration can be edited in the sub-menus XY Axes Profile andZ Axis Profile. The minimum and maximum values are listed for the operator. To movefrom the XY Axes Profile to the Z Axis Profile in this sub-menu, simply click the tab usingthe left mouse button. It is possible to adjust values for the XY axes and Z axisindependently. When the “ignore” feature is checked for the value in question, theprogram will refer to the most recent previous value programmed for that axis

The X, Y, and Z axes operate at their optimum with a very slow start speed, <1.0mm/sec, particularly when a high acceleration is used. The slew speed controls howfast the axes move. This value will depend on what the program needs to accomplish.Set the slew speed to a low value, around 40 mm/sec, when first programming. Werecommend that during initial programming, the scored practice pins be used tooptimize movements. When programming is complete, it is best to run a few practiceruns to ensure that the Telechem ChipMaker pins do not strike anything. Slew speedsof more than 100 mm/sec will cause the platform to move very quickly. Fast slewspeeds also require a slow start speed, <1.0 mm/sec. If the slew speed is too high thestepper motor will stall and the platform will not move. We recommend 175 mm/sec asa maximum slew speed. Keep in mind that any fluid in the Wash Station may splash iffast slew speeds are programmed.



The acceleration rate controls how fast the axes accelerate. This value will also dependon what the program needs to accomplish. Set the acceleration to a low value, 100mm/sec2, when first programming. Again, use the practice pins until programming iscomplete, and then make a few trial runs. We recommend a maximum acceleration of1000 mm/sec2. Much slower acceleration speeds, <100 mm/sec2 may be desired fordelicate tasks such as printing.

If the operator does not set the axes speeds then the original speed values used to homethe platform will be used. It is suggested that each “Move” command be preceded byan “Axis Speed” command. Doing so will help to maximize the operating efficiency ofthe PixSys. Otherwise, all movements will be carried out according to the last “AxisSpeed” command encountered in the program.

The Notes tab provides the operator with an area to record any textual informationregarding the “Axis Speed” command.

When finished editing the “Axis Speed” command, click the “OK” button. Thisinformation can be adjusted by double clicking the “Axis Speed” icon within thefunction.



11.4.2. MoveThis instruction moves the axes. Clicking this button will add it to the list ofinstructions. To edit this information, place the pointer on top of the move symbol inthe list of instructions then double click the left mouse button. The Axis Speedcommand (see above) must exist somewhere in the program prior to a Move command.All Move commands will be executed according to the most recently encountered AxisSpeed command in the program.

The move icon appears in the toolbar as a cross with arrows. The following informationcan be edited within the sub-menu: Move, Iterate X-Axis, Iterate Y-Axis, and Notes.

The Move tab lists the movement type, minimum allowable value, actual value andmaximum allowable value for each axis. The movement can be one of the followingtypes: None, Home, (+)Home, Absolute, or Relative. Selecting “None” causes the axis tostay in the current position. Selecting “Home” will cause the axis to move to the 0 orminimum position of the platform. Selecting “(+)Home” will cause the axis to move tothe maximum position on the platform. For the X-axis the home position is at the far leftof the platform. For the Y-axis the home position is at the front of the platform. The Z-axis home is at the highest position for the printing head.

“Absolute” movements cause the axis to move to an exact position relative to home andindependent of the current position of the axis. For example, if a value of 100 is enteredfor an absolute “Move” on the X-axis, then the platform will move to 100mm to the rightof home. “Relative” movements cause the axis to move relative to its current location.For example, if the X-axis absolute “Move” mentioned above is followed by a relative“Move” of 50mm, the platform will move to a position 150mm to the right of home.Note that the method will not allow relative movements that extend an axis beyond itslimit. The method will stop at the point where an improper relative “Move” isencountered.



The Move tab lists minimum and maximum values for the axes. These are the limits ofthe platform. The actual movement values are entered under the “Value” column. TheNotes area allows the operator to insert information regarding the “Move” command.

The Iterate X Axis and Iterate Y Axis tabs allow the operator to make secondarymovements offset from the original location when the “Move” command is used inconcert with a “Loop” command. “Loop” commands are discussed below. To enableiterations for an axis, click the box named “Enable Iterations” on the appropriate tab. Ifyou wish the position of the move to be tracked throughout the method then the boxreading “Global” should be checked. If the “Global” box is not checked the move willreset to it’s initial value every time the it is encountered in either a “Loop” or “Call”command in the program (see below for explanations of these commands).

To iterate a move, enter the value that you wish to be added to the original “Move”value. This value will be added after a number of passes specified by the operator in thespace labeled “after every # of pass(es)”. This tells the unit when to make the offset. Forexample, if an increment of 10 mm was programmed with “after every # of passes” setto 2 within the Iterate X Axis tab, then the unit will complete the original movement andthen offset an additional 10 mm every two passes. After the fourth pass the X axiswould be offset by a total of 20 mm from the original value. It is important to keep inmind that the offset is added after each pass. The 20 mm total offset would be seen onthe 5th and 6th pass.

Under the Iteration tab the operator may also set the number of passes after which thevalue of the move is reset. The operator must click in the box labeled “Enable Reset”and then enter a number of passes after which the reset will occur. The AxSys softwarewill reset the move value to the initial value by default. In the unusual instance that theuser would wish to reset the move value to something other than the initial value the“To initial value” box should be unchecked and the desired value entered. It is possiblethen to use a single iterated move command to instruct the unit to perform a complextask, such as sequentially taking samples from every well in a source plate.



The use of iterated relative moves is also valuable for accomplishing complex tasks withthe unit. To create multiple levels of structure within an array an iterated absolute moveis followed by one or more iterated relative moves. This, in effect, allows a nesting ofiterative moves to be associated with the absolute move. To do this the “Move” valuefor the relative move must be set to 0.00 mm for the axis of interest. The iterationsshould be set to the desired values and the reset value to 0.00 mm (!) after a desirednumber of passes. This reset affects only the value of the relative move and does notcorrespond to any absolute coordinate or absolute move command. Using relativemove commands in this fashion is particularly useful when programming methodsusing multiple pins.

The Notes tab provides the operator an area to record any textual information regardingthe instruction.

When finished editing a move, click on the “OK” button. The move may be edited atany time by double clicking on the icon within the function list.

11.4.3. Calls and FunctionsThe “Call” command inserts a function, or pre-programmed list of commands. The“Call” icon appears as a set of black parentheses in the toolbar. A “Call” instructioninserts a “Function”. A function is a list of commands that are grouped together toperform a task. It is suggested that even very simple tasks, those that may involve onlyone or two commands, be pre-programmed as functions. This can become particularlyimportant for “Move” commands that are to be iterated. Complex functions can becreated with a series of “Call” commands for simpler functions. Designing programsusing functions as a hierarchical set of sub-routines is efficient and effective.

Functions are created in the Function Editor, which may be accessed by selecting“EDIT”, followed by “FUNCTIONS” from the toolbar. Clicking the “New” button willplace a new function in the Function List. The new function will be named “Function 1”until renamed by the operator. Double clicking on the function title will allow theoperator to modify the list of commands that compose the function. New commandsmay be inserted into the function list by clicking on “Insert” on the toolbar within the



Function Editor window. Unlike the main program list, there is no command toolbarwithin the Function Editor. A Notes tab in the function editor provides an area for theoperator to record any textual information regarding the function.

If a function is edited, the changes made are reflected throughout the method. In anyplace that the function in question is called within a method the newly changedinformation will be executed. It is important to note that these changes are reflectedonly within the method where the function is edited and not in any other method wherethe same function may be in use. For additional information related to thisphenomenon see the section entitled, “Function Libraries”.

At least one function must be created before using the “Call” command. To select afunction to call, double-click on the “Call” command. Click on the arrow at the rightside of the blank labeled “Select function to call:” and a pull down menu of functionswill appear. Highlight the function you wish to call.

Clicking the “Edit” button will allow the operator to modify the function contained inthe “Select function to call:” area. The function will be displayed in a Function Editorwindow. Any modifications made to a function by editing it within the “Call”command will be reflected in all other “Call” commands for the same function



The Notes tab provides the operator with an area to record any textual informationregarding the “Call” command.

When finished editing the “Call” command, click the “OK” button. This informationcan be adjusted by double clicking the “Call” icon within the function.



11.4.4. Loops The “Loop” icon appears as a circular, blue arrow. The “Loop” command allows afunction to be repeated any number of times. Remember that iterated “Moves” thatexist within a looped function will be reset to it’s initial values every time the function iscalled of looped unless the “Global” box is checked under the iteration tab for the“Move” command.

The Notes tab provides the operator with an area to record any textual informationregarding the “Loop” command.

When finished editing the “Loop” command, click the “OK” button. This informationcan be adjusted by double clicking the “Loop” icon within the function.



11.4.4.1. Loops as SwitchesLoops may also be used as switches within a method to allow for flexibility. When a“loop” is used instead of a “call” command, a function can be included or excluded inthe method by setting the loop number to 1 or 0, respectively.



11.4.5. Pause This command pauses the program. The “Pause” icon appears as a red exclamationpoint within a yellow triangle on the toolbar. This instruction will insert a timed delayor operator message into the program list. The Delay and Notes may be edited withinthis command.

The Delay tab gives the operator several choices on how the pause will operate. Thepause can stop the program for a specified length of time by clicking on the “FixedDuration” box and then entering a value, measured in milliseconds, in the spaceprovided. The operator may also program the “Pause” instruction to display textualmessages when the “Pause” instruction is reached within the method. Click within the"Operator Message" box and type the text. The operator can also have the computersignal with an audible beep when it has reached this point. To enable the beep, click onthe “Beep” box.

The Notes tab provides the operator with an area to record any textual informationregarding the “Pause” command.

When finished editing the “Pause” command, click the “OK” button. This informationmay be adjusted by double clicking the “Pause” icon within the function.



11.4.6. Wash Pump The “Wash Pump” icon appears as a gasoline-dispensing pump in the toolbar. Thisinstruction controls the peristaltic wash pump that is located on the right panel of thePixSys unit. The Wash Pump and Notes can be edited within this command. Click the“Wash Pump” icon to insert this command into a function.

The Wash Pump tab controls the state of the wash pump: “on” or “off”. Individualcommands must be inserted turn the pump on and then off. The pump will remain onunless a command is given to turn it off. Typically, the wash pump is operated onlyduring the washing of pin(s).

The Notes tab provides the operator with an area to record any textual informationregarding the “Wash Pump” command.

When finished editing the “Wash Pump” command, click the “OK” button. Thisinformation may be adjusted by double clicking the “Wash Pump” icon within thefunction.

11.4.7. End The “End” instruction remains as a constant icon on the program list. This tells theprogram where the end of the application is. This command is automatically insertedby the AxSys software and can not be removed from the program list.

The Notes tab provided with the “End” icon is a good place to record general notesabout the program.



11.5. Manual ModeThe manual mode can be utilized by selecting “Tools” from the toolbar, then “ManualMode”. The following commands can be manipulated using the manual mode: AxisSpeed, Move, and Wash Pump.

Manual mode is useful in calibrating moves to specific positions on the stage, such asthe vac wash station, source plate, and slides. To do this, select the Move tab undermanual mode. There are nine buttons in the “Manual Move” box. The buttons witharrows move the unit in the X and Y dimensions when pressed. The center button has a“Z” on it. When the center button is activated, the up and down arrows are highlightedand the Pin Array head can be moved in the Z dimension. Above the “Manual Mode”box is a small window labeled “Profile”. This window contains a value that expressesthe percent of the Axis Speed value that the operate wishes to use to make moves in themanual mode. Setting the “Profile” value to 1% will allow the user to make very fineadjustments. The speed at which the platform moves can be slowed even further byentering low values for the slew speeds under the Axis Speed tab. After adjusting theAxis Speeds, the “Apply” button must be pushed.



Once the desired position is achieved in the manual mode, a “Move” commandcontaining the coordinate values may be inserted into the program list. To do this, clickthe “Active” button in the “Enter Move” box of the Move tab in manual mode. Thenpress the “Insert” button at the bottom of the screen. Once the “Move” commandappears in the program list it can be cut and pasted into the appropriate function.

The manual mode can also be used to turn the wash pump on and off. To do this, selectthe Wash Pump tab, then select either the “On” or “Off” button and press the “Apply”button.



11.6. Function LibrariesA function library is a list of the functions that are associated with a method. Librariesof functions may be imported and exported from method to method. It is stronglysuggested that a library of commonly used basic functions (e.g. pin washing and spotprinting) be created and used to program all methods. A program titled Calibration isincluded with the AxSys software and can be used to generate this library. Explanationsof the functions in this library are made later.

11.6.1. Exporting and ImportingTo export a function library click on the “File” tab and then on “Export Library…”.Name the library and click on the “Save” button.

To import a function library click on the “File” tab and then on “Import Library…”.Select a library from the directory and click on the “Open” button. The user will beasked if duplicate functions are to be overwritten. Decide which option is desirable andclick on the appropriate button.

11.6.2. Duplicate FunctionsEach time a function is newly created in the function library it is assigned a number thatis used as a global identifier. After creation, a function may be edited or it’s namechanged but the global identification number remains constant. When a function libraryis imported into a new or existing program, or when a program is saved under a newname there exist multiple methods that include in their function library functionshaving the same global identification number. Each method and its associated functionsare independent. Though these functions share the same global identification number,they may contain different information because they are stored independently withineach program. It is possible then to export a function library from one program andimport it into another program that already contains functions with the same globalidentification number. These functions may or may not have the same name or containan identical set of commands but they appear as identical entities to the AxSys software.These are considered duplicate functions by the AxSys software.

When libraries are imported the user may choose either to overwrite existing duplicatefunctions by the functions being imported or to exclude duplicate functions fromimport, thereby preserving the existing duplicate functions intact.

It is advisable to minimize the degree to which existing functions are modified. Thiswill allow the user to avoid confusion when deciding whether to overwrite a function ornot. It is a good idea to create a new function for new tasks. Commands may be copiedfrom old functions and pasted into new ones. Modified commands pasted into a newfunction is often preferable to modifying the commands in an existing function.



11.6.3. Importing Portions or Subsets of a LibraryThere are instances in which a user may wish to selectively import a subset of functionsinto a program. The best way to do this is to open the AxSys method from which thefunctions are to be exported and delete from the function list all those that the user doesnot wish to import. The user may then export that subset of functions as a library andimport them into the desired method. Be sure not to save the method from which thefunctions have been deleted after exporting the library.

11.7. Foundation FunctionsThis section contains a list of the functions provided in the Foundation Library , a fileincluded with the AxSys software. An explanation of each function is provided. Thesefunctions are intended to serve as a demonstration, foundation and guideline for thecreation of a customized set of basic functions that an operator will use to createmethods.

A few of the functions in the list are found in many programs but are not included in theFoundation Library. These few functions consist primarily of an iterated “Move”command that provides information unique to the individual arraying patterns of eachmethod. While the structure of the functions remains the same, the array patterndifferences will require unique “Move” parameters within the function. Since theFoundation Library consists of functions that will remain unchanged among programs,these variable functions are not included. These functions will need to be individuallyedited within each program that they are used. A description of these functions isincluded because it is useful to understand how they are used in creating programs.They are denoted the diamond symbol: ♦.

Each PixSys unit is likely to have a slightly different home position for each axis. It isimportant that the operator calibrate the “Move” functions that correspond to placessuch as the wash station, microwell plate, and slides to account for these differencesbetween units. An asterisk (*) after the function title denotes functions in whichcalibrations will need to be made. A description of how this is done is found in the“Calibration” section that follows this list of functions.

Begin Method : Sets the initial Axis Speed, homes the dispense platform, andwashes the pins.

End Method : Washes the pins and moves the Pin Array head to the rear of the platform.

Fill Pins : Moves the pins down to the bottom of the microwell plate well and then backout again.



♦ Move to Pre-Print Slides *: An initial “Move” to the front left corner of slide A1 withsubsequent moves using portions of slide A2. This function is iterated so that aclean portion of the Pre-Print slides is used for each sample. Using this functionin conjunction with the function “Relative Move for Pre-Prints” allows theoperator to maximize the use of pre-print slide space when multiple pins areused.

Move to Rinse Tank *: Positions the Pin Array head over the rinse tank, allowing pins inall slots to be immersed.

Move to Slide A1 *: A “Move” to a position 1.5mm from the front edge and 1mm fromthe left edge of the slide. (This position is the front left corner of the imagingarea for the General Scanning ScanArray 3000). The “Move” may be iterated tofacilitate the printing of arrays. This function may be used in conjunction withthe function “Relative Move for Arraying on Slides” to facilitate the printing ofmore complex arrays.

♦ Move to Slides *: Moves to first spot in array area of a slide. Typically the move isiterated to generate the array sub-structure.

♦ Move to Slides for Arraying *: see “Move to Slides”

Move to Vac Dry *: Positions the front left pin of the Pin Array head over the front leftopening in the vac wash plate.

Move to Source Plate *: Moves to a position above the front, leftmost well of themicrowell plate.

Pins Down—Rinse Tank *: Z axis "Move" into the rinse tank. The pins should not touchthe bottom of the tank.

Pins Down— Slide *: Z axis "Move" to the slide and then a Pause. The pins touch theslide and slip upward through the slot in the Pin Array head. A short pauseafter contacting the slide may improve spot quality.

Pins Down—Vac Dry *: Z axis “Move” down and into the orifices of the vacuum plate atthe wash station. The pins should be inserted approximately 10mm into thevacuum orifices.

Pins Down— Well *: Moves the pins down to gently touch the bottom of a well in themicrowell plate.

Pins Up— Slide *: Z axis "Move" to a position slightly above the slide.

Pins Up—Wash Station *: Z axis "Move" to a position above the surface of the washstation.

Pins Up— Well *: Moves the pins to a position above the microwell plate.

♦ Pre-Print Spots : This is a complex function designed to print a series of spots after thepins collect sample and before arrays are printed. It is intended to ensureuniformity among spots printed in arrays.



Print Additional Slides : This is a more complex function, the purpose of which is toreplicate the desired array on any number of the remaining slides, after the firstslide is printed. It utilizes a series of “Loop” commands as switches to select thecolumns of slides on which the arrays are to be reproduced. These “Loop”commands should never be set to any value greater than 1. Additionally, allslides in a column must be printed before slides in the next column are printed.Using the functions that are contained within the “Print Additional Slides”function, it is possible to select only a portion of a column of slides to be printed.For example, if the operator wishes to print a total of 25 slides then the “Loop”switches for only columns A, B, and C will be set to 1, while the “Loop” value forcolumns D and E will be set to 0. See “Slides--A Column, B Column, etc.” for adescription of how to set the number of slides within each column that will beprinted.



Print Next Slide in Column : This function is used to print a spot in the same position inan array as was printed in the previous slide of a column of slides. By loopingthis function from 1 to 9 times, any number of slides beyond that in position oneof a column may be printed. Commands to print on the first slide of a columnmust be supplied external to this function.

Print Spot : The functions “Pin Down—Slide” and “Pin Up—Slide”.

♦ Relative Move for Arraying on Slides : if needed, this function must be used inconjunction with an “Absolute Move”. It serves as a second tier of iterativeoffsets for an “Absolute Move” within a function.

♦ Relative Move for Pre-Prints : If needed, this function must be used in conjunction withthe “Move to Pre-Print Slides” function. It serves as a second tier of iterativeoffsets for the “Absolute Move” of the “Move to Pre-Print Slides” function.

Rinse Pins : A set of functions that move the pin(s) to and into the rinse tank, stir, andthen move back out of the rinse tank.

Slides--A Column, B Column , etc.: There are separate functions for each column of slideson the platform, A-E. These functions are used within the “Print AdditionalSlides” function. All of these functions utilize a “Loop” of the “Print Next Slidein Column” function to set the number of slides to be printed within eachcolumn. For each time the function “Print Next Slide in Column” is “Looped”,an additional slide after the first one will be printed. The first slide in eachcolumn is printed independently of the “Loop” command. For example, if theoperator wished to print 5 slides in a column, the “Loop” value would need to beset at 4. The commands needed to print the first slide in the column arecontained within the functions “Slides—B Column”, “Slides—C Column”,“Slides—D Column”, and “Slides—E Column”. The commands needed to printthe first slide in column A are external to the function “Slides—A Column”.

Speed --: There are three different speed functions: Fast, Medium, and Slow. Each one issimply an “Axis Speed” command. They are intended to simplify the process ofoptimizing the speed at which various movements are made. The “Slow” speedis typically used when the pins come into contact with a solid surface and “Fast”for movements that do not involve pin contact. The intermediate speed is alsouseful at times.

Shake Pins : This is a function that is used while the pins are in the rinse tank. It is aseries of commands that rapidly vibrates the pins. This activity improves thewashing effiency. This function includes a unique “Axis Speed” command inwhich the start speeds are set to very high values. This is done in order togenerate turbulence. This is the only time or place when such high values in an“Axis Speed” command are indicated.



Vac Dry Pins : A set of functions that move the pins to, into, and back out of the vacwash chamber.

Wash Pins : This is a complex function that encompasses all of the tasks necessary toclean the pins. Among the functions within the “Wash Pin” function is a “Loop”that uses the “Wash One Time” function. Usually a minimum of two cycles inthis “Loop” will be needed to thoroughly clean the pins, while more may beused for samples that persist in the pin or for additional assurance that the pinsare cleaned completely.

Wash One Time : The “Rinse Pins” and “Vac Dry Pins” functions are combined toproduce one washing cycle.

12. EXAMPLE PROGRAMS

Several AxSys programs have been provided. These programs are intended to serve asa demonstration of the techniques that the operator will use to program task specificmethods for the PixSys 5500 series instruments. After carefully reading this document,calibrating the unit using the Calibration program, updating the example programs withthe new Foundation Library, and properly editing the moves specific to each exampleprogram (described below) the operator may run these programs. Observation of theunit carrying out the instructions contained in the programs, combined with directstudy of the command set, should yield an understanding of the programmingtechniques.

12.1. Editing Commands Unique to Each Example ProgramThere are move commands that are unique to the individual programs (that are not apart of the Foundation Library). These commands pertain to the moves to the pre-printslides and to the slides for array making. The values for these moves should be editedusing the values from the functions included in the Foundation Library as referencepoints. If properly calibrated, the “Move to Slide ##” functions within the FoundationLibrary should be 1.5mm from the front edge of the slides and 1mm from the left edgeof the slide. Moves for arraying on a slide should begin within the area accommodatedby the scanner that will be used to analyze hybridized arrays. Most arrays begin in thefront, left corner of the scan area of the slide. Some programs begin making the arraycloser to the back of the slide and work towards the front.

These programs will be identified with a negative iteration value within the “Move”command that specifies the position where arraying is to begin. “Moves” for arrays thatwork from back to front should be given initial values that allow for space in front of theinitial position. The pre-print slide move should begin about 0.5mm from the front, leftcorner of slide A1.



MaintenanceThis section will help the user understand how to perform preventative maintenanceand cleaning procedures on the PixSys unit and ArrayIt ChipMaker™ pins.

12.2. Cleaning the PixSysIt is recommended that the routine cleaning procedure listed below be followed aftereach period of use.

Examine the PixSys for any spilled reagents or accumulations of dust. Using a moist,lint free cloth remove any dust or spilled reagents. If necessary, use isopropanol toremove spilled reagents. Let the PixSys unit air dry before further use.

12.3. Care and Cleaning of the Telechem ChipMakerTM PinsThe pins should be stored dry between printing experiments. Storing the ChipMakerpins in a wet environment can cause corrosion and permanent damage.

Never subject the ChipMaker pins to extremes in temperature or pH and avoid the useof strong solvents as these may damage the pin surface chemistry.

To clean the pins, carefully remove them from the print head. The pins can be cleanedby sonication in a bath containing ArrayItTM Micro-Cleaning Solution, followed byextensive rinsing with distilled water and drying with forced air.

During sonication, the pins must never come in direct contact with the sonicator.Direct contact between the pins and the sonicator can lead to permanent damage of thepins.

The pins should never be left in Micro-Cleaning Solution for longer than 10 minutes.Storing the pins in Micro-Cleaning Solution for periods in excess of 10 min can lead topermanent damage.

For more information contact Telechem: (408) 744-1331, [email protected].



13. TROUBLESHOOTINGThis section will help the user understand how to identify and clear most errors orproblems. If further assistance is required please telephone Cartesian Technologies at(919) 572-6888 or e-mail [email protected] .

No Power or Software Unable to Connect with Computer COM PORT:

• Check the power cord and switch setting.

• Check COM PORT settings.

• Make sure curly cable is plugged into “Terminal” Port of PixSys Unit

• Exit AxSys software, turn unit “OFF” . Wait 5 seconds then turn the unit “ON”.Enter AxSys software again.

• Check the fuse. Replace if necessary.

Platform (X,Y, or Z axis) does not move or makes a loud humming noise.

• Occasionally the stepper motors that drive the axes will create a louder than normalhum, this is due to harmonic interactions between two motors or when a singlemotor is operated at certain speeds. These are normal noises and should not affectthe performance of the PixSys. If the noise in question is not similar to what isdescribed here then proceed with the trouble shooting steps that follow.

• An “Axis Speed” instruction has not been placed in the program list.

• “Axis Speed” profile parameters are inappropriate for the platform. Set the platformstart speed to a low value (<1). Change the slew speed to a range of 50-175. Realizethat slew speeds of 100 or greater produce quick table movements, make sure theplatform’s path is clear. Decrease the acceleration to a range of 10-1000.

• The unit needs to be homed. If the unit is failing to properly home and the pointsabove have been covered, it is possible that one of the platform sensors is notoperating correctly. Contact Cartesian Technologies.

The Wash Station Overflows

• It is probably that the tubing in the peristaltic pumps has become stuck together.Open the peristaltic pumps and massage the tubing to open it. Shift the tubing sothat a new portion of the tubing is contained by the pump. To avoid this problemleave the pumps in the open position when the PixSys is not in use.

• Observe the wash station and tubing to make sure there are no blockages, kinks orcrimps that might impede the flow of wash fluid.



Printing Problems

• Examine pins to make sure they are not bent or clogged. Use of a dissectingmicroscope or magnifying loop will allow the user to assess the condition of thepins. If a pin is bent or the quill tips have been pinched together the pin is ruinedand can no longer be used to make quality arrays. If the pins are clogged or dirtyclean them (refer to the section titled “Maintenance”).

• Make sure that the array support (usually a glass slide) is clean and free of dust andlint.

• Check to be sure there is sample in the source plate. Observe the PixSys in operationand make sure the pins are contacting the bottom of the source plate wells. This isparticularly critical with source plates that do not have flat bottom wells. Make surethat the pins are contacting the bottom of the wells at the lowest point (the center) ofthe wells. The user should be able to see the pins float up in the print head whencontact is made. The user may also check for pin contact by lightly placing a fingeron top of the pins in the print head. Slight movement will be felt on the finger ifcontact is made. If pin contact is checked by touching the print head be sure to touchthe print head very gently, do not apply any force to the PixSys while it isoperating. If the pins are not contacting the bottom of the well then the programmust be edited. The Z-axis move value responsible for moving the pin down ontothe bottom of the well must be increased.

• It is sometimes useful to allow the pins to remain in the source for a few extramilliseconds. To do this, introduce a pause command with a value of 500-1500ms inbetween the move command responsible for lowering the pins to the bottom of thewell and the move command responsible for raising the pins out of the well.

• Observe the PixSys in operation to see if the pins are contacting the surface of thearray support (usually a glass slide). The user should be able to see the pins float upin the print head when contact is made. The user may also check for pin contact bylightly placing a finger on top of the pins in the print head. Slight movement will befelt on the finger if contact is made. If pin contact is checked by touching the printhead be sure to touch the print head very gently, do not apply any force to thePixSys while it is operating. If the pins are not contacting the array support thenthe program must be edited. The Z-axis move value responsible for moving the pindown onto the array support must be increased.



• Check to make sure that the vacuum drying station is drying the pins adequately.First make sure that the vacuum is functioning by sealing the openings in thevacuum plate with your fingers. You should be able to feel a pull on the skin of yourfingers. If you have a gauge on your vacuum pump, check for an increase inpressure when the vacuum is sealed. Check the vacuum for possible leaks. Finally,run a method on the PixSys and stop it right after the end of a pin cleaning cycle.Remove the pin that is not printing and check to see if it is dry. Using a dissectingmicroscope or a magnifying loop the user will be able to see if there is fluid in thequill or not. If there is fluid in the quill, the vacuum drying station is not functioningadequately.

14. EQIUPMENT SPECIFICATIONS

14.1. PixSys™ PA series General Specifications

Source Plate Compatibility96, 384, and 1536 microwell plates

Dispenser AccommodationsUp to 32 Pins in a 4x8 configuration with 4.5mm spacing

Spotting Precision±15 microns

Speedup to 25 features per second

Transfer Time15 minute printing time to transfer 384 samples to 50 slides

Power Requirements110/220 VAC, 50/60 Hz, 2A

Platform Motion controlsX, Y, and Z axis stepper motor control

Platform Positioning RepeatabilityX and Y axis: 0.002” (0.05mm)

Platform SpeedX, Y and Z axis: 0.0 to 330.0 mm/second

14.2. PixSys 5000 SpecificationsSize16” (35 cm) high x 16.7” (42.5 cm) wide x 32.3” (82 cm) deep

Operating Width with Slide Travel



32” (80 cm) wide

Weight100 lbs. (45.5 kg)

Platform MotionX slide: 0-355 mmY slide: 0-70 mmZ slide (motorized): 0-60 mm

Slide CapacityFour Slides

14.3. PixSys 5200 SpecificationsSize16” (35 cm) high x 16.7” (42.5 cm) wide x 32.3” (82 cm) deep

Operating Width with Slide Travel32” (80 cm) wide


Platform MotionX slide: 0-355 mmY slide: 0-70 mmZ slide: 0-60 mm

Slide Capacity32 Slides



14.4. PixSys 5500 Specifications

Size16” (35 cm) high x 16.7” (42.5 cm) wide x 32.3” (82 cm) deep

Operating Width with Slide Travel32” (80 cm) wide


Platform MotionX slide: 0-355 mmY slide: 0-70 mmZ slide: 0-60 mm

Slide Capacity50 Slides

14.5. ArrayIt Chipmaker™ Pin SpecificationsGiven the fine structure of the pin, extreme care must be used in handling the pins.Although they are robust when tapped onto the surface in the Z-direction, anymovement of the pin across the surface will cause the tip to bend. Also, touching the tipof the pin can result in contamination or clogging of the pin. In the event that a pinbecomes clogged, it can be cleaned with an ultrasonic bath (Note: TeleChem does notrecommend extensive cleaning in an ultrasonic bath since this may damage the tip).

Power RequirementsNone, derived from dispensing platform

Uptake Volume200 nL

Dispense VolumeApproximately 1.0nL per spot with the ArrayIt Chipmaker2™ pins.Approximately 0.5nL per spot with the ArrayIt Chipmaker3™ pins.

(depending on sample and substrate)

Spot DiameterTypically 140-170 microns for ArrayIt Chipmaker2™ pins.Typically 110-130 microns for ArrayIt Chipmaker3™ pins.

Dispense Repeatability*<15% CV per spot volume across multiple pins, samples, and slides.

*Determined by use with the PixSys 5500 unit.



REPLACEMENT PARTSThe following replacement parts are available from the sources indicated below:

DESCRIPTION PART NO.

ChipMaker 2 Replacement Pin CMP-2ChipMaker 3 Replacement Pin CMP-3Test Pin B35002Vacuum Inserts on Wash-Station B35004Vacuum Tubing _" TIUB007Vacuum Tubing 3/8" TIUB11Vacuum Elbow _" 51025K268Vacuum Elbow 3/8" 511K26Toggle Switch at Vacuum Table H-11-20-16_" Tube to Wash-Station 011227FA25P3/16" Tube to Wash-Station 01127FA15PWash-Station Tubing _" 51135K28Wash-Station Tubing 3/8" 5111K26Spring for Source Plate ZR22029

Options:

ArrayIt Microspotting Solution, 50ml MSS-1ArrayIt Cleaning Solution, 500 ml MCS-1Genomic Starter Kit SKM-105

Documents

PixSys PA Series Manu (Word98) - OSU Array Tech and …microarray.okstate.edu/files/pixsys.pdf · PixSys PA Series Workstation and AxSys Control Software. Although applicable to most